ResearchArticle - Université Laval Julian... · ResearchArticle A DNA Barcode-Based Evaluation of...

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Research Article A DNA Barcode-Based Evaluation of the Southeast Asian Catfish Genus Hemibagrus Bleeker, 1862 (Teleostei: Siluriformes; Bagridae) Julian J. Dodson 1 and Frédéric Lecomte 2 1 epartement de Biologie, Universit´ e Laval, 1045 Avenue de la M´ edecine, Qu´ ebec, QC, Canada G1V 0A6 2 Direction G´ en´ erale de l’Expertise sur la Faune et ses Habitats-Secteur de la Faune, Minist` ere des Forˆ ets, de la Faune et des Parcs, 880 chemin Ste-Foy, 2e ´ etage, Qu´ ebec, QC, Canada G1S 4X4 Correspondence should be addressed to Julian J. Dodson; [email protected] Received 25 July 2014; Revised 1 December 2014; Accepted 22 December 2014 Academic Editor: Bernd Schierwater Copyright © 2015 J. J. Dodson and F. Lecomte. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Species of the genus Hemibagrus are large river catfishes found throughout South-east Asia. e complexity of the region’s bio- geographical history and the lack of well-defined morphological characters render the taxonomy and phylogenetic reconstruction of Hemibagrus problematical. Early molecular studies of the H. nemurus species group revealed extensive genetic subdivisions, the taxonomic status of which remained unclear. A recent, morphologically-based, revision of the genus provides an opportunity to clarify the taxonomic status of these lineages. We employ a DNA barcode derived from the mitochondrial cytochrome b gene to expand our genetic analyses of the genus and to test the congruence of morphologically and genetically based taxonomies. Secondly, we evaluate phylogenetic relationships among taxa. irdly, we describe the phylogeography of Hemibagrus in South- east Asia. e species groups and nominal species proposed in the morphology-based revision generally reflect a hierarchy of monophyletic groups based on phenetic and maximum likelihood reconstructions of mtDNA phylogenies. e most notable exception involves the definition of a morphologically cryptic group from North Borneo. H. nemurus from West Java appears to be a regional population of H. capitulum. e phylogeography of the genus has been principally influenced by the formation of North Borneo and the emergence of the Sunda Islands. 1. Introduction Southeast Asia represents a unique biogeographical situation, combining complex geological dynamics with extraordinary species richness [1]. e Sunda continental shelf unites mainland Asia and the ai-Malay Peninsula with the Greater Sunda Islands (Sumatra, Java, and Borneo). e shelf is no more than 100 meters in depth and most of it is much shallower. During the early and middle Miocene, from 24 to 13 Ma ago, sea levels were typically 100–150 m above present day sea levels [2]. During these 11 Ma of generally high sea levels, there were 3 significant marine regressions, 2 of about 100 ka and one of 1.0 Ma duration. In the early Pliocene, sea levels rose 90–100 m and stayed there for nearly 1.4 Ma, from 5.5 to 4.2 Ma ago [2]. ese early, periodic marine transgressions would have been sufficient to isolate Borneo from the ai-Malay Peninsula and isolate the peninsula from the mainland, providing many opportunities for clado- genesis and allopatric speciation. As these periods of marine transgressions were followed by periods of regressions, there were also opportunities for dispersal and secondary contact among species and lineages. During the more recent Pleis- tocene glaciations, it is generally accepted that sea level was between 100 and 200 m lower than today [3]. Great areas of the Sunda Shelf were thus converted to land traversed by rivers during periods of glacial maxima, thus facilitating relatively recent exchanges of the freshwater fish fauna [4, 5]. e contemporary distribution patterns of species have thus been shaped largely by complex pre-Pleistocene dispersal and vicariance events, whereas more recent changes in the connectivity of islands and the mainland have influenced the partitioning of intraspecific variation [1]. Hindawi Publishing Corporation Advances in Evolutionary Biology Volume 2015, Article ID 490158, 21 pages http://dx.doi.org/10.1155/2015/490158

Transcript of ResearchArticle - Université Laval Julian... · ResearchArticle A DNA Barcode-Based Evaluation of...

Page 1: ResearchArticle - Université Laval Julian... · ResearchArticle A DNA Barcode-Based Evaluation of the Southeast Asian Catfish Genus Hemibagrus Bleeker, 1862 (Teleostei: Siluriformes;

Research ArticleA DNA Barcode-Based Evaluation ofthe Southeast Asian Catfish Genus Hemibagrus Bleeker1862 (Teleostei Siluriformes Bagridae)

Julian J Dodson1 and Freacutedeacuteric Lecomte2

1Departement de Biologie Universite Laval 1045 Avenue de la Medecine Quebec QC Canada G1V 0A62Direction Generale de lrsquoExpertise sur la Faune et ses Habitats-Secteur de la Faune Ministere des Forets de la Faune et des Parcs880 chemin Ste-Foy 2e etage Quebec QC Canada G1S 4X4

Correspondence should be addressed to Julian J Dodson juliandodsonbioulavalca

Received 25 July 2014 Revised 1 December 2014 Accepted 22 December 2014

Academic Editor Bernd Schierwater

Copyright copy 2015 J J Dodson and F Lecomte This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Species of the genus Hemibagrus are large river catfishes found throughout South-east Asia The complexity of the regionrsquos bio-geographical history and the lack of well-defined morphological characters render the taxonomy and phylogenetic reconstructionof Hemibagrus problematical Early molecular studies of the H nemurus species group revealed extensive genetic subdivisionsthe taxonomic status of which remained unclear A recent morphologically-based revision of the genus provides an opportunityto clarify the taxonomic status of these lineages We employ a DNA barcode derived from the mitochondrial cytochrome b geneto expand our genetic analyses of the genus and to test the congruence of morphologically and genetically based taxonomiesSecondly we evaluate phylogenetic relationships among taxa Thirdly we describe the phylogeography of Hemibagrus in South-east Asia The species groups and nominal species proposed in the morphology-based revision generally reflect a hierarchy ofmonophyletic groups based on phenetic and maximum likelihood reconstructions of mtDNA phylogenies The most notableexception involves the definition of a morphologically cryptic group from North Borneo H nemurus from West Java appearsto be a regional population of H capitulum The phylogeography of the genus has been principally influenced by the formation ofNorth Borneo and the emergence of the Sunda Islands

1 Introduction

Southeast Asia represents a unique biogeographical situationcombining complex geological dynamics with extraordinaryspecies richness [1] The Sunda continental shelf unitesmainlandAsia and theThai-Malay Peninsulawith theGreaterSunda Islands (Sumatra Java and Borneo) The shelf is nomore than 100 meters in depth and most of it is muchshallower During the early and middle Miocene from 24 to13Ma ago sea levels were typically 100ndash150m above presentday sea levels [2] During these 11Ma of generally high sealevels there were 3 significant marine regressions 2 of about100 ka and one of 10Ma duration In the early Pliocenesea levels rose 90ndash100m and stayed there for nearly 14Mafrom 55 to 42Ma ago [2] These early periodic marinetransgressions would have been sufficient to isolate Borneo

from the Thai-Malay Peninsula and isolate the peninsulafrom the mainland providing many opportunities for clado-genesis and allopatric speciation As these periods of marinetransgressions were followed by periods of regressions therewere also opportunities for dispersal and secondary contactamong species and lineages During the more recent Pleis-tocene glaciations it is generally accepted that sea level wasbetween 100 and 200m lower than today [3] Great areasof the Sunda Shelf were thus converted to land traversedby rivers during periods of glacial maxima thus facilitatingrelatively recent exchanges of the freshwater fish fauna [4 5]The contemporary distribution patterns of species have thusbeen shaped largely by complex pre-Pleistocene dispersaland vicariance events whereas more recent changes in theconnectivity of islands and the mainland have influenced thepartitioning of intraspecific variation [1]

Hindawi Publishing CorporationAdvances in Evolutionary BiologyVolume 2015 Article ID 490158 21 pageshttpdxdoiorg1011552015490158

2 Advances in Evolutionary Biology

Species of the genus Hemibagrus [6] are large catfishesfound in rivers east from Indiarsquos Godavari River and southfrom the Yangtze in China They occupy a wide range ofhabitats ranging from near estuarine to high altitude fast-flowing headstreams The genus reaches its greatest diversityin Southeast Asia [7] Throughout its geographical rangeand particularly in Southeast Asia species of Hemibagrusare valuable food fishes and species identification is ofparamount importance to sustainably exploit this speciescomplex However the taxonomy ofHemibagrus is confusingand the validity of many nominal species is unclear Morsquos [6]rehabilitation of the genusHemibagrus was not accompaniedby a comprehensive examination of types of all the nomi-nal species The complexity of the regionrsquos biogeographicalhistory coupled with a lack of well-defined morphologicalcharacters and considerable plasticity in phenotypic traitscommonly used for diagnosing species in other catfish groupsrenders the identification and phylogenetic reconstruction ofHemibagrus problematical [7] P K L Ng and H H Ng [8]divided the SundaicHemibagrus into four species groups butsubsequent work by Ng and coworkers revealed that themor-phological characters they used may not always distinguishthe species reliably nor do they allow easy assignment ofmany of the species to any of the groups [7]This situation ledto the publication of a revision of the genus byNg andKottelat[7] The revision is based on morphological criteria andrecognizes eight species groups H baramensis H guttatusH menoda H nemurus H olyroides H planiceps Hpluriradiatus and H wyckii species groups A species groupas defined in Tan and Kottelat [9] is ldquoan assemblage ofspecies sharing a set of diagnostic characters which mayor may not be a monophyletic lineagerdquo Ng and Kottelat [7]employed various morphometric and meristic characters todefine species and species groups including various bodydimensions fin lengths head dimensions eye diameter andvarious fin ray counts (see [7 Figure 1] for a complete listof morphological variables used by Ng and Kottelat) Thetaxonomic revision of Ng and Kottelat [7] was based onthe diagnosis of unique combinations of characters ratherthan discrete autapomorphies Thirty-two valid species arerecognized in the revision three of which are new [7]

Early molecular phylogenetic and phylogeographic stud-ies of the putativeH nemurus species group in Southeast Asiarevealed extensive genetic subdivision of the group [10 11]The occurrence of genetically divergent groups in sympatryin widely separated locations supported the proposition thatlow sea levels promoted the dispersal and mingling of somegenetic groups [10]These studies also revealed very divergentlineageswithinHemibagrus predating the Pleistocene [10 11]However in the absence of a comprehensive examination oftypes of all the nominal species in the genus the taxonomicstatus of these genetic lineages remained unclearThe revisionof the genus proposed by Ng and Kottelat [7] provides abasis by which we may clarify the taxonomic status andthe biogeography of independent genetic lineages withinHemibagrus

The analysis of single-locus data from genomic regionssuch as short sequences of mitochondrial genes used asDNA barcodes [12] represents a useful tool for species

identification and evaluating taxonomic diagnoses based onmorphological characters (eg [13 14]) Given the phe-notypic plasticity inherent in many of the morphologicalvariables used by Ng and Kottelat in their revision [7] and thepossibility that species groups so defined may not representtrue monophyletic lineages we here employ a DNA barcodederived from the mitochondrial cytochrome 119887 gene (Cyt b)to expand our genetic analyses of the genus in Southeast Asiaand to evaluate the revision of the genus Hemibagrus pro-posed by Ng and Kottelat [7] by testing the congruence of themorphologically and genetically based taxonomiesWe chosethis marker to be consistent with earlier work done on thegenus in Southeast Asia [10 11] Our principal objective wasto test the hypothesis that species groups and nominal speciesdefined with morphological criteria by Ng and Kottelat [7]reflect a hierarchical arrangement of monophyletic groupsbased on the similarity of mtDNA sequences Our secondobjectivewas to provide a tentative evaluation of phylogeneticrelationships among taxa Our third objective was to describethe geographical distribution of taxa and to relate majorcladogenetic events with major geological events that mayhave contributed to the radiation ofHemibagrus in SoutheastAsia We pay particular attention to the phylogeography ofthe widespread H nemurus species group

2 Materials and Methods

A total of 452 hemibagrid catfish particularly those of thepresumedH nemurus species group were sampled through-out their distribution range in Southeast Asia and successfullysequenced encompassing 67 sampling sites located on 37river systems (Figure 1 Table 1) Sampling of the other speciesgroups defined byNg andKottelat [7] was sparser thus not allspecies groups were represented in the genetic analysis Fishwere principally obtained from local fishermen Many fishwere placed in the Zoological Reference Collection (ZRC)of the Raffles Museum of Biodiversity Research Universityof Singapore where they received a ZRC accession number(see Table S1 in Supplementary Material available onlineat httpdxdoiorg1011552015490158) Ethanol-preservedtissue samples of many of the specimens identified in TableS1 are freely available upon request from JJD

Genetic Analysis Total genomic DNA was extracted from100 120583g of tissue (fresh or 95 ethanol-preserved) with astandard phenol-chloroform-isoamyl protocol [15] precipi-tated with cold 95 ethanol and suspended in 200120583L TEbuffer (10 nMTris-HCl pH 8 1mMEDTA) A 605 bp portionfrom the 51015840-end of cytochrome 119887 was amplified (including 8-bp located before the start codon) using derivatives of theuniversal primers of Kocher et al [16] P1 51015840-AAAACC-ACCGTTGTTATTCAACTACA-31015840 (light strand) and P251015840-GGGTTGTTTGATCCTGTTTCGTG-31015840 (heavy strand)Polymerase chain reaction (PCR) conditions were carriedout in 50 120583L total volumes containing (final concentrations)200120583M of each of dATP dGTP dCTP and dTTP 400 nM ofeach primer 1ndash25Uof TaqDNApolymerase 25mMMgCl

2

10mMTris-HCl (pH 83) 50mMKCl and between 50 and200 ng (3ndash5 120583L) of template (purified mtDNA) DNA was

Advances in Evolutionary Biology 3

Table 1 Rivers river code (as presented in Figure 1) sampling sites latitudelongitude and distance upstream from riverrsquos mouth of samplingsites and the number of specimens of Hemibagrus spp sequenced

River code Site code River Sample site Latituded∘ mm1015840 ss10158401015840

Longituded∘ mm1015840 ss10158401015840

Distance upstreamfrom river mouth

(km)

Number ofspecimens

1a 1 Mekong Bungkan 18 21 54 N 103 39 04 E 1285 41b 2 Mekong Bannan 17 58 00 N 104 14 00 E 1200 21c 3 Mekong That Phanom 17 24 00 N 104 48 00 E 1100 21d 4 Mekong Warinchamrap 15 12 00 N 104 53 00 E 885 31e 5 Mekong Mun River 15 17 00 N 105 04 00 E 800 131f 6 Mekong Phibum 15 14 00 N 105 14 00 E 845 41g 7 Mekong Nomh Penh 11 33 00 N 104 55 00 E 250 42a 8 Chao Phraya Market 14 35 00 N 100 27 00 E 100 82b 9 Chao Phraya Nakhom Sawan 15 41 00 N 100 07 00 E 275 53a 10 Chanthaburi 12 48 00 N 102 09 00 E 60 64a 11 Mea Khlong Kanchanaburi 14 01 00 N 99 32 00 E 100 55a 12 Thai Pen Phatthalung 7 37 00 N 100 05 00 E 90 16a 13 Kelantan 6 00 00 N 102 10 00 E 50 127a 14 Terengganu Kuala Berang 5 04 00 N 103 01 00 E 40 147b 15 Terengganu Sekayu 4 58 00 N 103 01 00 E 80 3

8a 16 Pahang KampongMengkarak 3 19 00 N 102 27 00 E 100 9

9a 17 Endau Park 2 40 00 N 103 15 00 E 100 69b 18 Endau Kahang 2 18 00 N 103 35 00 E 50 19c 19 Endau Ambat 2 11 00 N 103 51 00 E 70 29d 20 Endau Mersing 2 25 00 N 103 56 00 E 5 1010a 21 Johor Kota Tingi 1 4 00 N 103 54 00 E 45 311a 22 Perak 5 12 00 N 101 02 00 E 180 1311b 23 Perak Chenderoh 4 58 00 N 100 57 00 E 160 611c 24 Perak Gerik 5 25 00 N 101 08 00 E 200 1212a 25 Bernam Sabak 3 46 00 N 100 59 00 E 50 113a 26 Muar Selabu 2 9 00 N 102 36 00 E 20 613b 27 Muar Market 2 14 00 N 102 46 00 E 40 413c 28 Muar Kundang Ulu 2 14 00 N 102 46 00 E 40 213d 29 Muar Panchor 2 10 00 N 102 43 00 E 35 414a 30 Batang Hari Jambi 1 36 00 S 103 37 00 E 100 2714b 31 Batang Hari Kerinci 2 05 00 S 101 23 00 E 800 715a 32 Musi Palembang 2 55 00 S 103 37 00 E 70 1516a 33 Ci Liwong Sawagangan 6 24 00 S 106 46 00 E 45 217a 34 Ci Sadane Bogor 6 35 00 S 106 47 00 E 60 118a 35 Ci Tarum Cirata 6 33 00 S 107 18 00 E 90 1819a 36 Ci Manuk Garut 7 13 00 S 107 54 00 E 150 1420a 37 Kali Progo Mayudan 7 45 00 S 110 11 00 E 30 221a 38 Brantas River Surabaya 7 28 00 S 112 26 00 E 50 622a 39 Mahakam Samarinda 0 24 00 S 116 58 00 E 50 1023a 40 Barito Banjarmassin 3 5 00 S 114 38 00 E 50 924a 41 Kapuas Pontianak 0 17 00 S 109 19 00 E 20 1224b 42 Kapuas Sanggau 0 08 00 N 110 36 00 E 120 224c 43 Kapuas Nanga Pinoh 0 20 00 S 111 44 00 E 320 3

4 Advances in Evolutionary Biology

Table 1 Continued

River code Site code River Sample site Latituded∘ mm1015840 ss10158401015840

Longituded∘ mm1015840 ss10158401015840

Distance upstreamfrom river mouth

(km)

Number ofspecimens

24d 44 Kapuas Sintang 0 04 00 N 111 30 00 E 270 324e 45 Kapuas Danau Sentarum 0 51 00 N 112 6 00 E 550 224f 46 Kapuas Putussibau 0 50 00 N 112 56 00 E 600 224g 47 Kapuas Sibau 0 55 00 N 112 57 00 E 750 224h 48 Kapuas Mendalam 1 00 00 N 113 16 00 E 800 625a 49 Sadong Serian 1 10 00 N 110 34 00 E 50 2525b 50 Sadong Balai Ringin 1 03 00 N 110 45 00 E 70 1126a 51 Rajang Kapit 2 01 00 N 112 56 00 E 210 726b 52 Rajang Sibu 2 18 00 N 111 49 00 E 80 1026c 53 Rajang Katibas 1 43 00 N 112 33 00 E 170 1227a 54 Baram Mulu 3 30 00 N 115 30 00 E 380 327b 55 Baram Bakung 4 17 00 N 114 14 00 E 60 627c 56 Baram Marudi 4 11 00 N 114 19 00 E 80 1528a 57 Tutong Merimbun 4 36 00 N 114 40 00 E 40 329a 58 Belait Ingei 4 10 00 N 114 43 00 E 100 330a 59 Padas Tenom 5 08 00 N 115 57 00 E 70 931a 60 Segaliud Sandakan 5 45 00 N 117 52 00 E 50 132a 61 Segama Danum Valley 5 01 00 N 117 47 00 E 170 1632b 62 Segama Lahad Datu 5 02 00 N 118 19 00 E 90 633a 63 Kinabatangan Batu Putih 5 25 00 N 117 55 00 E 100 634a 64 Umas-Umas Tawau 4 22 00 N 117 44 00 E 15 235 65 Salween Myanmar mdash mdash mdash 236 66 Red Hanoi mdash mdash mdash 237 67 Pearl Guangdong mdash mdash mdash 8

Total 452mdash exact origin unknown

amplified in a programmable thermal cycler using the follow-ing profile one single preliminary denaturation at 95∘C for5min 40 cycles of amplification with denaturation at 94∘Cfor 60 s primer annealing at 45∘C for 60 s primer extensionat 72∘C for 90 s and a final single extension step performed at72∘C for 5min Total 50 120583L PCR products were purified on a2 agarose gel After electrophoresis at 90V for 2 hours theamplified fragments were excised from the gel and extractedand purified using the QIAquick Gel Extraction Kit Protocol(using a microcentrifuge) fromQIAGEN to a final volume of15 to 50120583L (in EB buffer 10mMTris Cl pH 75)

Double stranded purified PCR products were sequencedin both directions (complementary strand was sequenced inall cases) using the ABI PRISM BigDye Terminator CycleSequencing Ready Reaction Kit (Applied Biosystems Inc)Sequencing reactions were carried out in 20120583L total volumecontaining 32 picomoles of the sequencing primer 30 to60 ng of purified PCR product 6 120583L of 25X dilution buffer(CSA seq Buffer Perkin Elmer) and 2 120583L of ABI PRISMBigDye Terminator Ready Reaction Mix Reactions wereperformed in thermal cycler GeneAmp PCR system 9600using the following profile 10 s at 96∘C 5 s at 50∘C and

4min at 60∘C for 25 cycles Sequences were separated byelectrophoresis at 1680V (150W 500mA) for 5 to 7 h on ABIPRISM 377 DNA Sequencer (Applied Biosystems Inc)

Sequences were extracted from the gel image using ABIPRISM Sequencing Analysis 30 program The sequences ofthe complementary strands were then imported in SequenceNavigator program for comparative alignment and correctionof ambiguities (Applied Biosystems Inc) Sequences werealigned using the BioEdit sequence alignment editor [17]Each sequence was identified by a unique code designatingthe origin of the sample (river name) the cumulativenumber of the sequences found in that river and thenumber of individuals characterized by that particularsequence For example the code MEK1502 designates the15th sequence found in the Mekong River and occurring intwo specimens Following the comparison of morphologicaland genetic designations mtDNA codes were preceded bythe putative species designation (eg SPMEK1502 indicatesthat specimens bearing sequence MEK1502 were assignedto Hemibagrus spilopterus) Sequences found in multiplerivers were identified by river name and the total number ofspecimens Sequences were deposited in GenBank [18] with

Advances in Evolutionary Biology 5

(1) Mekong(2) Chao Phraya(3) Chanthaburi(4) Mea Khlong(5) Phatthalung(6) Kelatan(7) Terengannu(8) Pahang(9) Endau(10) Johor(11) Perak(12) Bernam

(13) Muar(14) Batang Hari(15) Musi(16) Ci Liwong(17) Ci Sadane(18) Ci Tarum(19) Ci Manuk(20) Kali Progo(21) Brantas(22) Mahakam(23) Barito(24) Kapuas(25) Sadong(26) Rajang

(27) Baram(28) Tutong(29) Belait(30) Padas(31) Segaliud(32) Segama(33) Kinabatangan(34) Umas-Umas(35) Salween(36) Red(37) Pearl

0 250 500 750

(km)

0 250 500

(mi)100

∘110

∘120

0∘

10∘

Siam RiverNor

th Su

nda R

iver

East Sunda River

35 36

37

1

5

11

12

17

8 9

10

14

16

17

18

19

25

24

23

20

21

22

27

26

28

29 3031

32

33

34

15

6

13

2

3

4

Shelf minus75mShelf minus120 m

Figure 1 Location map of Southeast Asia illustrating the 37 rivers sampled in this study Black dots along rivers represent the sampling sitesas identified in Table 1 and Table S1 Dark grey shading illustrates the extent of the emerging Sunda Shelf at sea levels 75m below present daylevels light grey denotes emerging shelf at sea levels 120m below present day levels (as illustrated by Voris 2000) Rivers illustrated on theSunda Shelf represent drowned Pleistocene river drainage systems according to Voris (2000)

accession numbers corresponding to sequence codes andreported in Supplementary Material Table S1

Data Analysis We first used the Neighbor-Joining (NJ)algorithm in MEGA 52 (Tamura et al 2011) to develop ataxon identification phenogram ofHemibagrusThe Tamura-Nei distance matrix considering invariable site contribution

(+119868) was selected according to the model selection algo-rithm implemented in MEGA 52 [19] The rate variationamong sites was modeled with a gamma distribution (shapeparameter = 1) Recognizing the limitations of single-locusanalyses in phylogeny reconstruction and the objectionsof cladists to distance-based methodology we neverthelesssought to provide a tentative evaluation of phylogenetic

6 Advances in Evolutionary Biology

relationships among taxa To do so the Cyt 119887 sequence of theChinese longsnout catfish (Leiocassis longirostris) obtainedfrom GenBank (accession number DQ3217551) was usedas an outgroup to root the phenogram Chinese speciescurrently assigned to Leiocassismay be considered membersof the genus Pseudobagrus [20] Furthermore we employeda maximum likelihood (ML) phylogenetic reconstructionbased on the comparison of nucleotides within sites to assessthe congruence of the taxon identification phenogram andthe ML phylogenetic reconstruction In the latter case theevolutionary model used was the Hasegawa-Kishino-Yanomodel (HKY)with gammadistribution (+119866 = 13290) Initialtree(s) for the heuristic search were obtained by applyingthe Neighbor-Joining (NJ) method to a matrix of pairwisedistances estimated using the maximum composite likeli-hood (MCL) approach The rate-variation model allowedfor some sites to be evolutionarily invariable (+119868 4644of sites) The ML tree was rooted with the Cyt 119887 sequenceof the Chinese longsnout catfish (Leiocassis longirostris) andconstructed using MEGA52 [19]

TheNJ phenogram classifiedmtDNA sequences based onoverall similarity Ng and Kottelat [7] also defined speciesgroups based on overall similarity but according to mor-phological criteria Of the 8 species groups defined by Ngand Kottelat [7] two groups (H olyroides (1 species) and Hpluriradiatus (5 species)) were not included in our samplesWe thus evaluated the congruence of the two methods indefining the remaining six species groups (H baramensisH guttatus H menoda H nemurus H planiceps and Hwyckii species groups) When possible mtDNA sequenceswere linked to ZRC accession numbers identifying specimensexamined by Ng and Kottelat [7] in their morphologicalrevision of the genus (Table S1)

To provide time estimates for the major phylogeneticevents we present a calibrated linearized tree obtained fromour ML reconstruction (MEGA 52) Time of divergencebetween Hemibagrus lineages has been approximated byapplying a rate of between 10 and 15 genetic divergence permillion years as reported for bagrid fish by Yang and He [21]However the justification for this rate is not clear We thusapplied the more widely used rate of 2 sequence divergenceper million years [22] As there is undoubtedly a wide errorassociated with any such molecular clock calibrations esti-mated divergence times are used to discriminate more recentfrom more ancient cladogenetic events while providing veryapproximate dates for such events

Genetic divergence estimates using 119901-distances (119901) wereused in the present study to identify the different taxonomiclevels observed among the Hemibagrus spp analyzed Sincesuch information is readily available for thousands of animalgroups [23] we used 119901-distance (119901) as a benchmark for taxo-nomic levelsThe survey of 20 731 vertebrate and invertebratespecies reported by Kartavtsev [23] revealed increasing levelsof 119901-distances () of the mtDNA Cyt 119887 gene with taxonomiclevel populations within species 119901 = 138 plusmn 030 (SD)subspecies or sibling species119901 = 510plusmn091 morphologicallydistinct species within genera 119901 = 1031 plusmn 093 generawithin a family 119901 = 1786 plusmn 136 and families within anorder 119901 = 2636 plusmn 388 Although heterogeneity in the

gene evolution rate differs amongdifferent taxonomic groupsthese estimates provide an order of magnitude evaluationof intraspecific versus interspecific divergence and permittedus to discriminate between population- and species-leveldivergences However as 119901-distances underestimate truegenetic distance (eg site saturation and back-mutation)divergence times based on119901-distances greater than about 10are generally underestimated We calculated 119901-distances inMEGA 52 and report them as percentages (standard error)

3 Results

The 452 samples yielded a total of 118 distinct sequences(not including the outgroup taxon) The Neighbor-Joiningphenogram clustered the 119 sequences into a large numberof hierarchically organized monophyletic clades To facilitatepresentation we first present a compressed NJ tree andidentify 11 major mtDNA clades (Figure 2) One unidentifiedfish from the Rajang River was characterized by a genotypethat clustered with the outgroup taxon (XXRAJ0604GenBank accession number KP322872 Figures 2 and3) A second fish from the Rajang was characterized byan isolated genotype (XXRAJ0301 GenBank accessionnumber KP322869) of unknown affiliation (Figures 2and 3) To assess the congruence of the species groupsdefined by Ng and Kottelat ([7] hereafter referred toas NampK) and the mtDNA groupings revealed by thepresent analysis we provide a direct comparison of thetwo methods following the order determined by the taxonidentification phenogram (Figure 2 Table S1) Table S1 alsoprovides additional information about the geographicaldistribution of sequences Gene trees of individual cladesincluding all sequences are presented at each step in theanalysis along with their geographical distributions We alsopresent a compressed maximum likelihood phylogenetictree (Figure 3) The phenogram and the ML tree providedcongruent phylogenetic reconstructions However both treesfailed to resolve the phylogenetic relationships of H wyckiiH guttatus and H menoda species groups most likelydue to undersampling of these lineages The relationshipsamong an unspecified species group H spilopterus and Hfortis are also not well resolved Their position in the NJtree receives weak bootstrap support and they appear as amultifurcation along with a clade composed of H capitulumand H hoevenii in the ML phylogenetic tree (Figures 2 and3) To facilitate comparisons with the taxonomic scheme ofNampK we follow the sequence of taxa as identified in Figure 2

mtDNAClade 1The H wyckii Species GroupThe first clade isclearly consistent with theH wyckii species group (Figure 4)This species group is composed of 4 valid speciesHmaydelliH microphthalmus H wyckii andH wyckioides (NampK)Thelatter species is known from the Mekong and Chao Phrayadrainage Only specimens of H wyckioides obtained fromthe Mekong and the Chao Phraya were available for DNAanalysis Three specimens from the Chao Phraya bearingsequence WYCHP0203 (Table S1) were identified as Hwyckioides (H H Ng personal communication) although

Advances in Evolutionary Biology 7

8811

10

9

8

7

6

5

4

3

1

2

66

85

44

32

98

91

81

8432

57

99

99

99

49

002

34

15

43

86

99

96

H capitulum (Indochinese clade)

H capitulum (Sundaic clade)

H hoeveniiH fortis

H spilopterus

H baramensis species group

H planiceps species groupH menoda species group

H guttatus species group

H wyckii species group

XXRAJ0604

PESAL0102

XXRAJ0301Leiocassis longirostris

mtDNAclade

H nemurusspecies group

Unspecified species group

Figure 2 The taxon identification phenogram of Hemibagrus inferred using the Neighbor-Joining method in MEGA 52 The optimal treewith the sumof branch length = 186668598 is shown Bootstrap values are indicated on each node of the tree (500 replicates) Bar indicates thenumber of substitutions changes estimated using Tamura-Nei distance considering invariable site contribution (+I)The analysis involved 119nucleotide sequences All positions containing gaps and missing data were eliminatedThere were a total of 597 positions in the final dataset

mtDNAclade

H capitulum (Indochinese clade) 11

93

97H capitulum (Sundaic clade)H hoevenii

9999

84H fortis 8

9

10

H spilopterus 773 92

Unspecified species group 678

XXRAJ060454 34

90 H baramensis species group 5

49 H planiceps species group 4

H menoda species groupPESAL0102 3

H wyckii species group 199

99

99H guttatus species group 2

XXRAJ030149

47

Leiocassis longirostris

005

H nemurusspecies group

94

72

Figure 3 Evolutionary history of Hemibagrus inferred by using the maximum likelihood method based on the Hasegawa-Kishino-YanomodelThe tree with the highest log likelihood (minus56774825) is shown Bootstrap values are indicated on each node of the tree (500 replicates)Bar indicates the number of substitution changes estimated using Hasegawa-Kishino-Yano model (HKY) with gamma distribution (+119866 =13290) The analysis involved 119 nucleotide sequences All positions containing gaps and missing data were eliminated There were a totalof 597 positions in the final dataset

their ZRC numbers do not appear in the analysis of NampK Asno specimens of the other 3 species comprising the H wyckiispecies group were available for analysis we cannot confirmif this species group is monophyletic

mtDNA Clade 2 The H guttatus Species Group The secondclade of the Hemibagrus gene tree (Figure 2) is composed oftwo reciprocally monophyletic subclades clearly reflectingthe H guttatus species group as defined by NampK (Figure 4)

8 Advances in Evolutionary Biology

100∘

10∘

110∘

0∘

120∘

0

0 250

250

500

500

750(km)

(mi)

002

98

43

84

34

99

99

79

96

51

59

99

78

49

H peguensis

H guttatus

H macropterus

H wyckioides

PESAL0102

XXRAJ0301Leiocassis longirostris

MAPEA0201MAPEA0302MAPEA0101

GUPEA0501GURED0102

GUPEA0401GUPEA0602

WYCHP0401WYMEK1101

WYCHP0203

See Figures 7ndash12

See Figures 5 and 6

Shelf minus75mShelf minus120 m

Figure 4 Phylogenetic relationships of the individual sequences comprising four species H guttatus H macropterus (together forming theH guttatus species group) H wyckioides and H peguensisThe subtree is extracted from the taxon identification phenogram inferred usingthe Neighbor-Joining method (see Figure 2) The percentage of replicate trees in which the associated sequences clustered together in thebootstrap test (500 replicates) is shown next to the branches The distribution of these sequences is illustrated in the right panel

The group comprises three valid species H guttatus Hmacropterus andH vietnamicus (NampK) Specimens availablefor DNA analysis were obtained from the Pearl River andRed River (11 fish 7 sequences Table 1 and Table S1) Thetwo reciprocally monophyletic subclades comprising theH guttatus species group represent H guttatus and Hmacropterus (Figure 4) Although no ZRC specimens wereexamined by NampK species identification was confirmed(H H Ng personal communication) No H vietnamicusspecimens were available for DNA analysis The twospecies composing the group are closely related (119901 = 51(SE = 08))

mtDNA ldquoCladerdquo 3 The H menoda Species Group Thisisolated genotype (Figure 4) most likely identifies a cladeencompassing the H menoda species group although the

single sequence (Table S1) is shared by only 2 specimensof H peguensis from the Salween River Myanmar alsoincluded in the analysis of NampK (where its provenance wasidentified as the neighboring Irrawaddy River) The otherspecies comprising this group (H menoda H caveatusand H punctatus) were not sampled for DNA analysis Themonophyletic nature of this species group thus remains to bedemonstrated

31 The H planiceps + H baramensis Group The remainderof the gene tree is split into two large sister groups withmarginal bootstrap support (73 in ML and 43 in NJ)the H planiceps + H baramensis group and the H nemurusspecies group (Figures 2 and 3) The H planiceps + Hbaramensis group is strongly supported in both NJ and MLanalyses (84 and 90 bootstrap support respectively

Advances in Evolutionary Biology 9

110∘

47

53

69

81

69

5199

52

95

97

78

001

H bongan

H velox

H gracilis

H planiceps

H divaricatus

BOKAP1801

BOKAP1601BOKAP1701

BOKAP1402BOKAP1503

VEBAH0907GREND0206

PLCIM0114PLCIS0101

DIPER0401DIPER0301

DIPER0201

100∘

10∘

0∘

120∘

0

0 250 500

250 500

750(km)

(mi)

Shelf minus75mShelf minus120 m

Figure 5 Phylogenetic relationships of the individual sequences and species comprising theH planiceps species groupH bongan H veloxH gracilis H planiceps and H divaricatus The subtree is extracted from the taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicate trees in which the associated sequences clustered together in the bootstrap test(500 replicates) is shown next to the branches The distribution of these sequences is illustrated in the right panel

Figures 2 and 3) The two groups are genetically similarwith a genetic divergence estimate of 119901 = 121 (SE = 09)Although theH planiceps species group is strongly supportedin both trees (99) bootstrap support of the H baramensisspecies group is weak (Figure 2 32 Figure 3 34) Thegeographical distribution of the two groups differs Membersof the H planiceps species group are found throughout theGreater Sunda Islands and Peninsular Malaysia but they areisolated in the upper reaches of large rivers (Figure 5) Incontrast members of theH baramensis species group appearto be restricted to Sabah North Borneo (Figure 6)

mtDNA Clade 4 The H planiceps Species Group The Hplaniceps species group (Figure 5) is comprised of 5 closelyrelated but genetically distinct species (mean within-groupdivergence 119901 = 20 (SD = 033) H planiceps (Java)H gracilis (eastern Peninsular Malaysia) H divaricatus(western Peninsular Malaysia) H bongan (Borneo) andH velox (Sumatra)) H lacustrinus the sixth species of thisgroup (NampK) was not sampled in the present study WithintheH planiceps species groupH planiceps formed a distinctspecies (Figure 5) comprising two sequences One sequence(PLCIM0114 Table S1) characterized 14 specimens obtainedfrom Ci Manuk West Java 9 of which were identified as Hplaniceps by NampK (Table S1) A second sequence (PLCIS0101

Table S1) was obtained from one specimen sampled atCi Sadane West Java that we classified as H planicepsH bongan is represented by 5 sequences obtained from 8specimens sampled in the Kapuas River (Figure 5) Two ofthese specimens (ZRC 43013 and 43015) were identified asH bongan by NampK (Table S1) H divaricatus is composedof 3 sequences from three specimens obtained at Gerik onthe Perak River (western Peninsular Malaysia) One of thesespecimens (ZRC 41151) is the holotype of this new species(NampK) H gracilis is represented by only one sequence(GREND0206 Table S1) shared by 6 specimens sampledat Ulu Endau Jasin River eastern Peninsular Malaysiafour of which were identified by NampK (Table S1) ZRC21484 comprises the holotype and ZRC 21482 21486 and21487 comprise the paratypes (NampK) of this species (TableS1) Finally H velox is represented by only one sequence(VEBAH0907) from 7 specimens sampled at Kerinci BatangHari River Sumatra Three of these specimens (ZRC 41503Table S1) represent 3 paratypes of the species (NampK)

mtDNA Clade 5 The H baramensis Species Group The threespecies comprising the H baramensis species group (Figures2 and 3) form three monophyletic subclades (mean within-group divergence 119901 = 106 (08) Figure 6) H semotus iscomposed of two sequences (SEPAD0101 SEPAD0202) that

10 Advances in Evolutionary Biology

110∘

002

H sabanus

H semotus

H baramensis

SAKIN0204SASGM0706

BASGM0402BASGM0301

BASGM0201BASGM0104

SESGM0502SEUMA0101

SEPAD0202SEPAD0101

See Figure 5

100∘

10∘

0∘

120∘

0

0 250 500

250 500

750(km)

(mi)

99

57

32

83

99

72

99

56

66

54

99

Shelf minus75mShelf minus120 m

Figure 6 Phylogenetic relationships of the individual sequences and three species comprising the H baramensis species group H semotusH sabanus and H baramensis (furcatus) The subtree is extracted from the taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicate trees in which the associated sequences clustered together in the bootstrap test(500 replicates) is shown next to the branches The distribution of these sequences is illustrated in the right panel

characterize fish that are the holotype and paratypes of Hsemotus (ZRC 46122 Table S1) along with two somewhatmore divergent sequences (Figure 6) One of these sequences(SGM0502) was found in the Segama River (Danum Valley)and identified as H baramensis by NampK (ZRC 40525)(Table S1) The second sequence (UMA0101) was from afish sampled at Tawau Umas Umas River (ZRC 46123) alsoidentified as H baramensis by NampK These sequences mostlikely reflect distinct lineages within H semotus inhabitingdifferent rivers as the mean intraspecies sequence divergence(119901 = 16) indicates population-level divergence

H sabanus and H furcatus (synonymized with H bara-mensis by NampK) cluster together to form a distinct subcladebut clearly diverge at the species level (Figure 6) H sabanuscomprises 2 sequences that are shared respectively by 6specimens (ZRC 42741) from the Segama River Sabah and4 specimens (part of ZRC 42743) from the KinabatanganRiver Sabah (Table S1) All of these specimenswere identifiedas H sabanus by NampK (Table S1) Finally H baramensisforms a distant subclade (147 mean sequence divergencebetween H baramensis and H sabanus) composed of 4sequences (Figure 6) shared by 8 specimens all sampled inthe Segama River Danum Valley Conservation Area SabahSix of these specimens (ZRC 40523 (2 specimens) ZRC40527 (2 specimens) and ZRC 40526 (2 specimens) Table S1)comprise the paratypes of H baramensis (furcatus) (NampK)

32 The H nemurus Species Group In both NJ and ML trees(Figures 2 and 3) the H nemurus species group is strongly

supported (bootstrap support 98 in NJ and 99 in ML)indicating that it forms a distinct and widespread geneticentity within the genus Hemibagrus However relationshipswithin the group are not so clear In general the H nemurusspecies group is composed of 4 clades The first clade may beconsidered as a species group nested within the H nemurusspecies group (85 and 93 bootstrap support in NJ andML resp) encompassing three clades H hoevenii (clade9) the H capitulum Sundaic clade (clade 10 including Hnemurus) and the H capitulum Indochinese clade (clade 11)(Figures 2 and 3) An additional three clades (Figures 2 and3) are formed by an unidentified species group (clade 6) Hspilopterus (clade 7) andH fortis (clade 8)These three latterclades are distinct at the congeneric species level (unspecifiedversus H spilopterus 119901 = 77 (07) unspecified versus Hfortis 119901 = 85 (07) and H spilopterus versus H fortis119901 = 75 (08)) The distribution of these three clades issomewhat disparate H fortis and the unspecified speciesgroup are principally found in Borneo whereasH spilopterusis widely distributed on the Indochinese mainland

mtDNA Clade 6 Unspecified Species Group This geneticallydiverse group encompassing at least 3 subclades (H spp AB and C Figure 7 within-group mean sequence divergence119901 = 68(06)) is confoundedwith theH baramensis speciesgroup byNampK It is composed of sequences fromfish sampledin Sarawak Sabah and eastern PeninsularMalaysia Subclade119867 spp A (Figure 7) is particularly interesting as it contains2 sequences (CHSARA20 CHRAJ0401) that characterize

Advances in Evolutionary Biology 11

110∘

XXPAD0303XXPAD0601

XXPAD0401XXBEL0202

XXBRM0202XXRAJ0511

XXPAD0501

XXEND0410CHSARA20CHRAJ0401

XXBEL0101XXSEG0101

100∘

10∘

0∘

120∘

0

0 250 500

250 500

750(km)

(mi)

91

86

83

99

98

47

81

001

91

72

99

80

H spp C

H spp B

H spp A

Shelf minus75mShelf minus120 m

Figure 7 Phylogenetic relationships of the individual sequences comprising the unspecified species group containing three subcladesidentified as H spp A B and C The subtree is extracted from the taxon identification phenogram inferred using the Neighbor-Joiningmethod (see Figure 2) The percentage of replicate trees in which the associated sequences clustered together in the bootstrap test (500replicates) is shown next to the branches The distribution of these sequences is illustrated in the right panel

H chrysops a new species identified by Ng and Dodson [11]However specimens of H chrysops from the Rajang Riverwere subsequently synonymized by NampK with H capitulumof the H nemurus species group (ZRC 23008ndash23014 TableS1) However an additional 14 fish from the Sadong Riverat Serian bearing genotype SARA20 (Table S1) and thusgenetically identical to the Rajang H chrysops samples wereidentified by NampK as H spilopterus (ZRC 29583ndash96 TableS1) These specimens however were erroneously identifiedin NampK as originating in the Chao Phraya River (Thailand)Subclade A also contains one sequence obtained from 10 fishsampled on the Endau River (Mersing) in eastern PeninsularMalaysia These fish were not morphologically examinedFinally subclade A also contains one sequence from oneunidentified fish sampled in the Segaliud River Sabah andanother unidentified fish sampled in the Belait River BruneiSubclade H spp B encompasses 2 sequences one of whichcomes from a fish sampled in the Padas River (XXPAD0501)and part of the ZRC 46122 fish lot identified as H semotusby NampK (Table S1) The second sequence (XXRAJ0511) wasshared by 11 unidentified fish sampled at Katibas on theRajang River Sarawak (Table S1) Finally subclade 119867 sppC encompasses 5 sequences Three sequences (XXPAD0303XXPAD0401 and XXPAD0601) were obtained from thePadas River and identified byNampKasH semotus (ZRC46122Table S1) The fourth sequence (XXBRM0202) was obtainedfrom 2 fish sampled in the Baram River and was identified

as H baramensis by NampK (ZRC 42742) The 5th sequencecomposing this group was obtained from 2 unidentified fishsampled in the Belait River Brunei (Table S1)

mtDNA clade 6 is thus problematical within the contextof the revision of the genus Hemibagrus Two highlydivergent mtDNA clades (clade 5 and clade 6 Figures 2and 3) occur in sympatry principally in the Padas River ofSabah North Borneo (Figures 6 and 7) Specimens of theunspecified species group are morphologically similar tothe H baramensis species group where they are assigned inthe analysis of NampK However the mean genetic distancebetween the unspecified species group and theH baramensisspecies group is 119901 = 193 (28) close to genus leveldivergence Furthermore subclade 119867 spp A (Figure 7)contains not only H chrysops but also an unidentifiedpopulation from eastern Peninsular Malaysia (XXEND0410Table S1) and two additional Sabah sequences On theother hand the unspecified species group clusters with allother members of the H nemurus species group (meangenetic divergence of unspecified group versus H capitulum(Sundaic clade) 119901 = 76 (07) (Figure 2)) with strongbootstrap support Such genetic clustering may justify theinclusion of H chrysops and associated sequences in theH nemurus species group but its synonymization with Hcapitulum (NampK) is difficult to defend on genetic groundsWe tentatively conclude that H chrysops is a valid speciesbased on genetic identity [11] forming part of an unknown

12 Advances in Evolutionary Biology

110∘

SPCPCHMK12

SPMEK0202SPMEK0401

SPMEK0301SPMEK0103

SPMEK1401SPMEK0501

SPMEK1201SPMEK1301

SPMEK1601SPMEK1502

SPMEK0701SPMEK0801SPMEK1701

SPCHP0501SPCHA0201

SPCHA0304SPPAT0101

100∘

10∘

0∘

120∘

0

0 250 500

250 500

750(km)

(mi)

000499

99 98

60

47

48

54

96

7091

38

86

87

99

80

58

H spilopterus ldquoCrdquo

H spilopterus ldquoBrdquo

H spilopterus ldquoArdquo

Shelf minus75mShelf minus120 m

Figure 8 Phylogenetic relationships within H spilopterus comprised of 3 subclades identified as A B and C The subtree is extracted fromthe taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicate trees in whichthe associated sequences clustered together in the bootstrap test (500 replicates) is shown next to the branches The distribution of thesesequences is illustrated in the right panel

species group found throughout North Borneo (and toa lesser degree in eastern Peninsular Malaysia) that maybe considered as part of the H nemurus species groupHowever the morphological confusion of the unspecifiedspecies group with the sympatric highly divergent Hbaramensis species group suggests the existence of a crypticspecies assemblage (see Section 4)

mtDNA Clade 7 Clade 7 (Figures 2 and 3) is composed of atleast 3 distinct subclades that appear to reflect 3 geneticallydivergent populations ofH spilopterus Subclade A (Figure 8)encompasses 7 sequences obtained from 8 fish sampled inthe Mekong River Four fish bearing 3 of these sequenceswere identified as paratypes (ZRC 43324) of H spilopterusby NampK (Table S1) One additional fish was identified asH spilopterus by NampK (ZRC 46157 Table S1) Subclade Bencompasses 6 sequences obtained from 9 fish all sampledfrom the Mekong River (Table S1) The fish sampled at ThatPhanom (ZRC 46012) was identified as H spilopterus inthe analysis of HampK (Table S1) Subclade C encompasses 5sequences obtained from 19 fish sampled in the Chao PhrayaMae Khlong and Chanthaburi Rivers Thailand Onesequence (CPCHMK12) was found in all of these drainages(Table S1) A closely allied sequence was found in one fishsampled at Phatthalung just south of the Isthmus of Kra onthe Thai-Malay Peninsula None of these fish were examined

by NampK and thus we are unable to definitively identifythe species However given the widespread occurrence ofH spilopterus in the Chao Phraya and Mae Klong Riversreported by NampK we conclude that these fish are part of awidespread H spilopterus population complex The meanwithin-group (clade 7) genetic divergence (119901 = 29 (04))suggests intraspecific population-level divergence

mtDNA Clade 8 Clade 8 encompasses 3 sequencesobtained from 9 fish sampled in east Sabah specifically theKinabatangan Segama and Umas Umas Rivers (Figure 9)The Kinabatangan sequence is somewhat divergent withinthe clade and the specimen was not identified by NampK Onesequence (SGM0606) characterized 6 fish from the SegamaRiver 2 of which were identified as H fortis (ZEC 4052140522 Table S1) by NampK (Table S1) Finally one fish bearinga closely related genotype from the Umas Umas River wasidentified as H baramensis by NampK (Table S1)

mtDNA Clade 9 Clade 9 is a sister group to the remaining2 clades composing the H nemurus species group Thiswell-defined clade (Figure 10) clusters 6 sequences from42 fish that are clearly identified as H hoevenii (NampK)Some of these haplotypes are geographically widespread onesequence (JOMSKA11 Table S1) was found in the Johor River(East Peninsular Malaysia) the Musi River (Sumatra) and

Advances in Evolutionary Biology 13

100∘ 110∘

0∘

120∘

00 250

250500

500750

(km)

(mi)

FOKIN0102FOSGM0606

FOUMA0201H fortis

10∘

99

86

001

Shelf minus75mShelf minus120 m

Figure 9 Phylogenetic relationships within H fortis comprised of 3 sequences The subtree is extracted from the taxon identificationphenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicate trees in which the associated sequencesclustered together in the bootstrap test (500 replicates) is shown next to the branches The distribution of these sequences is illustrated in theright panel

the Kapuas River (West Borneo) and a second sequence(HMUBHKABM23 Table S1) was found inMuarRiver (WestPeninsular Malaysia) and the Batang Hari (Sumatra) theKapuas (Sarawak) and the Baram Rivers (Sarawak) Many ofthese fish were identified as H hoevenii by NampK (Table S1)The existence of twodistinct but very closely related subclades(Figure 10 119901 = 06 (03)) that are not geographicallysegregated suggests some recent isolation event followed bywidespread dispersal and secondary contact

33 The H capitulum Group The final two monophyleticgroups of the Hemibagrus gene tree are approximatelyequivalent to the remaining species composing the Hnemurus species group as defined by NampK Dodson etal [10] and Ng and Dodson [11] have previously referredto clade 10 as the Sundaic clade as most sequences wereobtained from fish sampled on the Greater Sunda Islandsand clade 11 as the Indochinese clade asmany sequences wereobtained from fish sampled on the Indochinese mainland(Figure 2 Table S1) Although most of these sequencesare assigned to H capitulum by NampK there are somesignificant phylogeographic differences between the 2 cladesIn particular there appears to be an east-west division in thedistribution of the two clades on the Thai-Malay Peninsulawith the Sundaic clade (clade 10) found on the west coast andthe Indochinese clade found on the east coast with sympatricoccurrence in the Muar River Nevertheless mean sequencedivergence between the two clades is 119901 = 29 (05) avalue that is indicative of population-level or subspecies

divergence The two clades are thus best considered ascomprising one species

mtDNA Clade 10 The Sundaic clade includes a number ofclosely related sequences that comprise part of H capitulumand H nemurus Very little genetic structure is apparentand bootstrap support for tree topology within this group isgenerally very weak (Figure 11) A widely spread genotype(NCLCTBA20) characterized 6 of 17 specimens from CiTarum at the Cirata reservoir (West Java) (Table S1) and5 were identified as H nemurus in the analysis of NampK(ZRC 42564) (Table S1) The remaining fish characterized bythis sequence were obtained from Ci Liwong at Sawangan(West Java) and one fish was obtained from the Barito Riverat Banjarmassin south Borneo This latter specimen (ZRC40553) was identified by NampK as H capitulum (Table S1)Another common genotype (NBHKPBR09) was identified aseither H capitulum H hoevenii or H nemurus dependingon the river of origin (Table S1) Many of the remainingsequences of the Sundaic clade appear to characterize fish thatNampK identified asH capitulum A geographically widespreadgenotype (CPEBEMUKA36) characterized 9 fish sampledon the Perak River at Gerik that were identified by NampKas H capitulum (ZRC 41150 Table S1) Another commongenotype within this clade (CABHMS20) included 2 fishfrom the Batang Hari at Jambi and identified by NampK asH capitulum (ZRC40534 Table S1) Another 2 sequences(CABAR0203 CABAR0305) characterized 8 fish sampledon the Barito River at Banjarmasin and were identified as

14 Advances in Evolutionary Biology

100∘

10∘

110∘

0∘

120∘

0

0 250

250

500

500

750(km)

(mi)

53

64

99

85

64

0002

HJOMSKA11

HOBAH0601

HOBHMS05

HMUBHKABM23

HOBAH0701

HOKAP0501

H hoevenii ldquoBrdquo

H hoevenii ldquoArdquo

Shelf minus75mShelf minus120 m

Figure 10 Phylogenetic relationships withinH hoevenii comprised of 2 subclades and 6 sequences The subtree is extracted from the taxonidentification phenogram inferred using theNeighbor-Joiningmethod (see Figure 2)The percentage of replicate trees in which the associatedsequences clustered together in the bootstrap test (500 replicates) is shown next to the branches The distribution of these sequences isillustrated in the right panel

110∘

XXMAH0110

CABAR0305

96 CAKPR0201

74CAKAP0401

5931 CAKAP0601

35 CPEBEMUKA36

646 CAKAP0903

33 CAKAP080114 CAKAP0701

CARAJ0210

4 CABAR0203CABRA0204

20

CBHKPBR0919

CABRA0301

15CABHMS20

24 NCLCTBA20

63 NECIT0201

H capitulumldquoSundaic claderdquo

H nemurus

0002 120∘

0 250 500 750

(km)

0 250 500

(mi)100

10∘

0∘

Shelf minus75mShelf minus120 m

Figure 11 Phylogenetic relationships within theH capitulum ldquoSundaicrdquo clade including 2 sequences associated withH nemurusThe subtreeis extracted from the taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicatetrees in which the associated sequences clustered together in the bootstrap test (500 replicates) is shown next to the branchesThe distributionof these sequences is illustrated in the right panel

Advances in Evolutionary Biology 15

H capitulum

0∘

10∘

100∘

110∘

120∘

66

63

88

59

38

39 68

71

6991

57

9754

88 4944

39

53

89

0002

ldquoIndochineserdquogroup ldquoErdquo

H capitulumldquoIndochineserdquo

group ldquoDrdquoH capitulumldquoIndochineserdquo

group ldquoCrdquo

H capitulumldquoIndochineserdquo

group ldquoBrdquo

H capitulumldquoIndochineserdquo

group ldquoArdquo0

0

250

250

500

500

750

(km)

(mi)

CATER0301CATERR0401

CKETEMU23

CAMEK0601CACHP0301

CAMKPA03

CAMEK0910CAMEK1003

XXMUBM02

CABMTU09

CABRM0601

CAKAP1201CAKAP1001

CAKAP1101CAKAP1301

CABRM0501

CASAD0102

CASAD0220

CAEND0301

CABAH0801

CAPABH05CATEEN03

CATER0701CATERR0801

CAKETE04CATER0501

CAPAH0303

Shelf minus75mShelf minus120 m

Figure 12 Phylogenetic relationships within theH capitulum ldquoIndochineserdquo clade encompassing 5 subclades identified as A B C D and EThe subtree is extracted from the taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2)The percentageof replicate trees in which the associated sequences clustered together in the bootstrap test (500 replicates) is shown next to the branchesThedistribution of these sequences is illustrated in the right panel

H capitulum by NampK (ZRC 40553 Table S1) Although notincluded in the analysis of NampK samples from the Sundaicclade were also found in the Kapuas River (West Borneo)Finally 10 fish sampled on the Mahakam River at Samarindaeast Borneo are all characterized by one relatively divergentgenotype (CAMAH0110) butwere considered asH capitulumin the analysis of NampK (ZRC 40533 Table S1)TheMahakampopulation sample is the most genetically distinct of theSundaic clade (119901 = 17 (04)) but nevertheless indicativeof intraspecific population-level divergence (Figure 12)

The topology of the Sundaic clade does not permit usto draw a clear distinction between H nemurus and theSundaic H capitulum The mean within-group divergenceof the Sundaic clade is only 119901 = 08 (02) well withinpopulation-level divergence values We postulate that Hnemurus may be one of several regional variants of awidespread Sundaic H capitulum clade that has radiatedrelatively recently This may explain the existence of manyshared sequences throughout the Sundaic islands assigned todifferent species in different regions

mtDNA Clade 11 Finally the Indochinese clade of Hcapitulum represents a geographically widespread andweakly diversified clade (within-group mean sequencedivergence 119901 = 11 (02) Figure 12) Although few fishfrom this genetic clade were included in the morphological

analysis of NampK most may be considered as H capitulumgiven their geographic distribution and close geneticrelationship to the Sundaic H capitulum Four sequencesin this clade were identified as H capitulum in the analysisof NampK 3 specimens characterized by one sequence(CABMTU09) found in the Tutong River of westernBorneo (ZRC 40482) 6 characterized by the same sequencefrom the Baram River (ZRC 40498) another 2 sequencesfrom the Baram River (ZRC 40498) and 20 specimenscharacterized by an abundant genotype (CASAD0220)found at 2 sites in the Sadong River also in western Borneo(ZRC 39526 5 specimens 39527 3 specimens and 38753 4specimens) (Table S1) Unfortunately none of the specimenscollected on the Indochinese mainland for genetic analysiswere examined by NampK Nevertheless the distribution ofspecimens analyzed for mtDNA resembles the distribution ofspecimens examined by NampK Although weakly structuredwe may recognize 5 subclades One genotype (CAPAH0303)is basal to the phenogram Subclade A encompasses 8sequences mainly found in eastern Peninsular Malaysia(Table S1 Figure 12) These rivers furnished many of thespecimens NampK examined to define H capitulum Howeversome sequences in this group occur in sympatry withsequences identified within the Sundaic H capitulum groupTwo sequences from subclade A occurred in the Batang HariSumatra (Table S1) Subclade E also encompasses sequences

16 Advances in Evolutionary Biology

found in Peninsular Malaysia as well as in the Mekong ChaoPhraya and Mea Khlong Rivers (Figure 12) In addition3 subclades encompass sequences found principally inwestern Borneo subclade B in the Baram and Sadong RiversSarawak subclade C in the Kapuas River West Kalimantanand subclade D in the Baram and Tutong Rivers of Sarawakand Brunei respectively (Figure 12) This latter subclade alsoincludes one genotype found in the Muar River of westernpeninsular Malaysia Finally subclade E encompasses 5sequences from the Mekong Chao Praya and Mae KhlongRivers all within the distribution area of H spilopterus

EvolutionaryHistoryThe evolutionary history of theHemiba-grus species groups analyzed here dates from at least theMiocene 15ndash20Ma (Figure 13) when considering the widelyused molecular clock of 2 sequence divergence per millionyears Bootstrap support for the sister group relationships ofH wyckioides theH guttatus species group andH peguensisare all weak (clearly due to undersampling) and little can besaid of their evolutionary history other than the fact that theyoccupy basal positions in the rooted trees The remainder ofboth the NJ and ML trees is composed of two major sistergroups the H planiceps + H baramensis group and the Hnemurus species group (Figures 2 3 and 13)TheH planiceps+ H baramensis group is strongly supported in both NJ andML analyses (84 and 90 bootstrap support resp Figures2 and 3) The H nemurus species group is also stronglysupported in both analyses (98 and 99 bootstrap supportFigures 2 and 3) These two major phylogenetic groups areestimated to have diverged at least 8 to 12Ma ago during themid to late Miocene (Figure 13)

Much of the evolutionary history of Hemibagrus hasoccurred during the Quaternary starting approximately26Ma ago This is particularly true of the H planicepsand H nemurus species groups The radiation of the 5species included in the H planiceps species group appearsto coincide with the middle Pleistocene transition period ofapproximately 1Ma ago Their high-elevation distributionhowever appears to have largely isolated them from theeffects of lowered sea levels during glacial maxima and theattendant opportunities for widespread dispersal (Figure 5)The evolutionary histories of theH baramensis species groupand the unspecified species group date back to the latePliocene In contrast the low-elevation distribution of theHnemurus species group in Sundaland appears to have favoredtheir widespread distribution during the Quaternary ice ageIn particular H hoevenii appears to be the most recentlydispersed of the species under study with diversification anddispersal occurring approximately 03Ma ago (Figure 13)

4 Discussion

41 The Congruence of Morphological and Genetic AnalysesCombining genetic and morphological analyses to elucidatetaxonomy allowed us to better understand the diversificationand evolutionary relationships within this speciose group offishes Given the rapid and relatively constant rate of mtDNAevolution the deeper the branches within the phylogenetictree are the greater our ability to distinguish valid taxa

should be Conversely the lack of complete congruencebetween morphological- and genetic-based identificationsis expected to increase among the shallower branches of thephylogenetic tree resulting in the appearance of polyphyleticspecies Although 2 of the 8 species groups defined by Ngand Kottelat [7] were not sampled in this study the validityof the remaining 6 species groups and their componentspecies defined on the basis of morphological similaritywas largely confirmed by the analysis of the mitochondrialCyt 119887 bar code As expected congruence is strongest at thebase of the NJ and ML trees where the H guttatus speciesgroup formed a well-defined monophyletic group The Hmenoda and H wyckii species group were each representedby only one species but they were clearly distinct relativeto sequences characterizing the neighboring species groupsThe H planiceps H baramensis and H nemurus speciesgroups and their component species were also generallycongruent in morphological and genetic analyses Also asexpected most cases of discrepancy between morphological-based species identification and genetic-based speciesidentification occurred on relatively shallow branches of thephylogenetic tree These involve principally H spilopterusand the two mtDNA subclades composing H capitulumHowever a far more unexpected lack of congruence involvesthe definition of a deeply divergent unspecified genetic groupconfounding members of theH baramensis species group inthe morphological analysis of Ng and Kottelat [7]

Interpreting Polyphyly The apparent polyphyletic origin ofa species may be due to incomplete lineage sorting amongrecently diverged species the introgression of mtDNA dueto species hybridization or the erroneous identificationof species due to the lumping of morphologically crypticspecies In the absence of nuclear DNA analyses it isoften difficult to distinguish between introgression andincomplete lineage sorting For example several sequencescharacterizing the Indochinese clade of H capitulum(subclade E) are found in the Mekong and Chao PhrayaRivers well within the distributional area of H spilopterusH spilopterus and H capitulum first diverged at least 3MaOn the one hand this apparent case of polyphyly mayhave resulted from incomplete lineage sorting betweenrecently divergedH spilopterus andH capitulum We wouldhowever have expected similar cases of polyphyly amongpopulations of the Indochinese clade of H capitulum butthis was not detected On the other hand the sympatricoccurrence of H spilopterus subclade A and subcladeB sequences with H capitulum Indochinese subclade Esequences in the Mekong River may represent a migrationevent and local introgression of H capitulum with Hspilopterus A similar event may have occurred in theChao Phraya and Mae Khlong Rivers where H spilopterussequences cooccur withH capitulum sequences Computingthe divergence of H capitulum sequences (Indochinesesubclade E) cooccurring with H spilopterus and Hcapitulum sequences occurring in eastern PeninsularMalaysia (subclade A) reveals a mean sequence divergence119901 = 15 (04) Thus the H capitulum sequences occurringin sympatry with H spilopterus diverged at most 750 ka ago

Advances in Evolutionary Biology 17

H fortis

XXRAJ0604H furcatus

H sabanusH semotusH divaricatus

H planicepsGREND0206

VEBAH0907

H bonganPESAL0102

H wyckioidesH macropterus

H guttatusXXRAJ0301Leiocassis longirostris

Ma

4971

92

47

51

9999

99

9999

99

99

99

9999

99

99

99

99

65

96

96

4946

93

79

78

70

91

93

98

86

87

91

95

97

71

85

9999

05101520

005010015020 000

H capitulum Indochinese clades AndashE

H capitulum Sundaic clade

H hoevenii B

H hoevenii A

H spilopterus C

H spilopterus B

H spilopterus AH spilopterus A

H spp CH spp B

H spp A

Figure 13 Linearized tree with branch lengths estimated with the molecular clock using a mutation rate of 1 per Ma (=divergence rate of2 per Ma) The tree topology is identical to the ML reconstruction illustrated in Figure 3 (see the specificities of the tree reconstruction inthe caption of Figure 3) Boxes represent 95 confidence intervals of divergence time estimates for each node Vertical dashed lines redirectto dates indicated on the timescale (Ma) Numbers below timescale indicate the estimated number of substitution changes using Hasegawa-Kishino-Yanomodel (HKY)with gamma distribution (+119866 = 13290) after linearizing the treeH furcatus is synonymizedwithH baramensis

from eastern Peninsular Malaysia populations suggestinga recent introgression event Similarly H capitulum in theKapuas River is characterized by sequences of both theSundaic and the Indochinese clades As both sequences arebroadly sympatric it is not possible to distinguish betweenincomplete lineage sorting and introgression of these closelyrelated intraspecific lineages

A Possible Case of Morphological Stasis Clade 6 of thepresent analysis defines a well-supported monophyletic cladefound in Sabah Sarawak and the Endau River of easternpeninsula Malaysia that has no equivalent taxonomic statusin the revision of Ng and Kottelat [7] In Borneo they occursympatrically with the H baramensis species group and areconfounded as either H baramensis or H semotus These

two clades are genetically highly divergent having divergedbetween 8 and 12Ma ago It thus seems highly unlikely thatthey represent retained polymorphisms due to incompletelineage sorting Sequences previously identified as character-izing H chrysops [11] are part of clade 6 and extend to thesouth (Rajang and Sadong Rivers of Sarawak West Borneo)with one closely related genotype found in eastern peninsularMalaysia (Endau River) where it may well be confoundedwith H capitulum H chrysops was synonymized with Hcapitulum by Ng and Kottelat [7] based on their analysis ofspecimens ofH chrysops from the Rajang RiverThus despitea long and apparently independent phylogenetic historythe unspecified species group appears largely cryptic andconfounded principally with members of the H baramensisspecies group

18 Advances in Evolutionary Biology

We suggest that this situation is an example of mor-phological stasis involving the coexistence of a number ofgenetically divergent yet morphologically cryptic species thatare lumped together [24] A striking example of morpho-logical and ecological stasis among fishes is provided by thecoastal marine bonefishes (Albula spp) Distinct evolution-ary lineages (separated by sequence divergence between 5 and30 [25]) of bonefish coexist in several sample locations yetthey show little morphological or ecological differentiationin sympatry [25] Similarly a proportion of the grey mulletspecies (Mugilidae) also appears to consist of cryptic species[26] For exampleMugil cephalus in Taiwan is characterizedby three divergent lineages that have been shown to charac-terize 3 reproductively isolated but morphologically crypticspecies [26 27]

Here we have discovered 2 highly distinct and structuredclades (each composed of 3 mtDNA subclades) that aremorphologically confounded within different species Forexample H spp C of the unspecified species group isidentified as H baramensis in the Baram River and as Hsemotus in the Padas River We speculate that North Borneoharbors a morphologically cryptic species complex possiblycomposed of several species pairs inhabiting different riversystems The reconstruction of this phylogenetic history andthe taxonomic status of its components remains a majorchallenge to the revision of the Hemibagrus genus

42 Phylogeographic Hypotheses The phylogeography ofthe genus appears to have been principally influenced bytwo geological histories one encompassing the formationof North Borneo the second the emergence of the SundaIslands and their connectivity with the Indochinesemainlandassociated with recurrent sea-level changes North Borneoappears to have acted as a species sink promoting speciationand locally high biodiversity within the genus but with littleevidence of subsequent dispersal out of North Borneo Incontrast the geological events influencing the Indochinesemainland and the Sunda Islands have permitted bothspeciation and differential dispersal depending on thebiology of the species For example the H planiceps speciesgroup is found throughout the Greater Sunda Islands andtheThai-Malay Peninsula as is theH nemurus species groupWhereas the H nemurus species group generally occupiesthe low altitude lower reaches of large rivers the speciescomposing the H planiceps group are found in the upperreaches of large rivers The H nemurus species group iscomposed of a number of widely distributed closely relatedspecies whereas theH planiceps group is composed of a largenumber of species that are highly localized geographicallyIt thus appears that sea-level changes have had little impacton the dispersal history of the H planiceps species group buthave deeply influenced that of the H nemurus species group

The Case of North Borneo From 15Ma to 5Ma greater areasof Borneo became emergent and the central mountainson the Sarawak-Kalimantan border extending into Sabahbecame wider and higher which may have represented newbarriers to dispersal [28 29] Borneo gradually developedinto the present large island by emergence of land in the

north and east The H baramensis species group is restrictedto North Borneo as is the unspecified species group of theHnemurus species group H fortis is also distributed in NorthBorneo The isolation of the freshwater fish communities ofNorth Borneo (particularly in the Labuk-Segama watershed)has long been recognized based on the number of endemicspecies and the concentration of relict species [30] The latePliocene-early Pleistocene diversification of H fortis theunspecified species group and the H baramensis speciesgroup may thus be associated with their dispersal to andisolation within the watersheds of North Borneo Only onesequence of the unspecified species group is found outsideof North Borneo and H fortis appears to be distributedthroughout central and east Borneo [7]

ThePhylogeography of the H nemurus Species Group Periodicmarine transgressions on the Sundaland platform during theearly and middle Miocene [2 29] may have been sufficientto isolate populations of freshwater organisms and promotecladogenesis [31] This may have been responsible for theinitial mid to late Miocene divergence of the H nemurusspecies group The majority of sequences assigned to thisspecies group belong to 3 species (H spilopterus H capitulum(including H nemurus) and H hoevenii) all of which havebeen influenced by the more recent geological history of theSunda Islands and their connectivity with the Indochinesemainland H spilopterus is known from river drainages inIndochina including the Mekong Dong Nai Ban PakongChao Phraya and Mae Klong where it is found in a widevariety of lotic and lentic habitats [7] H spilopterus divergedin the late Pliocene-early Pleistocene possibly isolated tothe north of the Kra Peninsula during the late Pliocenemarine transgression that is purported to have created amajor biogeographic transition zone between the Sundaicand Indochinese biotas ([32] but see [33] for a critique ofthis biogeographic scenario) The existence of 2 subclades(A and B) in the Mekong River separated by 2 sequencedivergence is consistent with intraspecific divergence Thetwo subclades possibly represent 2 historical mtDNA lineagesthat are retained within the species reflecting a Pleistoceneisolation event followed by secondary contact within thecontemporary Mekong River Subclade C from the ChaoPhraya and Mae Khlong Rivers exhibits a level of geneticdivergence consistent with sibling species-level divergenceshowing a 5 sequence divergence relative to Mekong RiverH spilopterus

H capitulum may be considered as part of the Sundaicbiota located principally to the south of the Kra marinetransgression zone that may have isolated H spilopterus tothe north on the Indochinese mainlandThe biogeographicalevent leading to the subsequent formation of two intraspecificgroups within H capitulum is not at all clear The onlyarea where the two clades are clearly geographically separateis on the Thai-Malay Peninsula with the ldquoSundaicrdquo cladeon the west coast and the ldquoIndochineserdquo clade on theeast coast separated by the central Titiwangsa mountainrange The genetic distinctiveness of east and west coastpopulations of H capitulum has also been documented fornuclear DNA [34] A similar pattern of separation has been

Advances in Evolutionary Biology 19

reported for 5 additional freshwater fishes within this region[35] However as the divergence of the two H capitulumclades postdates the formation of the mountain range byapproximately 200Ma the geographic separation of the twoclades most likely represents differential dispersal rather thanancient vicariance The presence of the Sundaic clade on thewest coast of the Thai-Malay Peninsula as well as on theSundaic Islands reflects the historical connections betweeneast Sumatra and west Peninsular Malaysia across the Straitof Malacca This area encompasses the Northern CentralSumatra-Western Malaysia freshwater ecoregion [36] TheMuar River forms its southern border coincident with thesouthernmost distribution of the Sundaic clade on the Thai-Malay Peninsula It is important to note however that mostof Sumatra and Java emerged to achieve their present size andelevation only 5Ma ago and much of East Java continuedto be the site of marine deposition until late Pliocene-earlyPleistocene [29] We may speculate that the Sundaic clade ofH capitulum resulted from the relatively recent colonizationof these islands and Borneo The radiation within the cladeonly dates back to approximately 1Ma ago H nemurus fromWest Java may thus best be considered an isolated regionalpopulation of very recent origin

The diversification of the Indochinese clade dates fromat least 12Ma ago coincident with the mid-Pleistocenetransition at which time low-frequency high-amplitude andquasiperiodic (100 ka) glacial variability emerged It began125Ma ago and was complete by 07Ma ago [37] The tran-sition marked the beginning of major changes in sea levelsduring which the Sunda Shelf periodically emerged and wastraversed by enormous fluvial systems [3] The contributionof these events to the diversification of this clade cannotbe satisfactorily reconstructed One possible explanation isthat high sea levels produced barriers to gene flow and thepopulations of West Borneo (subclades B C and D) wereisolated from those of east Peninsular Malaysia This couldbe due to one or more dispersal events from Borneo via theGreat Sunda River (Figure 1) to east Peninsular Malaysia orlineage sorting whereby sequences of subclades A and E havebeen lost from West Borneo As in the case of the sympatricoccurrence of H spilopterus and H capitulum subclade E(see above) in the Mekong River we are unable to clearlydefine the relative roles of incomplete lineage sorting andintrogression followingmigration Given the recent origins ofthe Indochinese H capitulum sequences incomplete lineagesorting most probably blurs patterns of recent isolation

The last species of the H nemurus species group is Hhoevenii The species is widespread throughout PeninsularMalaysia and the Sundaic Islands It is found primarily inthe lower reaches of rivers and is apparently capable ofwithstanding low salt concentration [7] H hoevenii thusappears to be the most liable of all members of the Hnemurus species group to be impacted by Pleistocene sea-level changes This is clearly reflected in the widespreaddistribution of the sequences of this species Two lineages areevident having diverged no more than 03Ma ago suggest-ing a late Pleistocene isolation event followed by extensivedispersal and admixture in recent times This represents theclearest example of the dispersal opportunities afforded by

Pleistocene sea level changes on the Sunda Shelf In additionthe relatively low genetic diversity detected (only 6 sequencesin 42 fish) suggests a population bottleneck in the recent pastpossibly related to population declines in the lower reaches ofrivers flooded by increasing sea levels

43 Future Directions We have provided a global picture ofthe evolutionary taxonomy of the genus in Southeast AsiaClearly a more rigorous phylogenetic reconstruction of thegenus Hemibagrus requiring the construction of multiplegene trees based on the analysis of both mitochondrial andnuclear DNA markers from a wider taxonomic array ofhemibagrids will involve an even greater effort of samplingthroughout the region In particular we require samplingat smaller spatial and temporal scales For example smallerscale sampling in the Padas River and the Segama Riverof North Borneo using both mitochondrial and nucleargenetic markers would contribute to clarifying the natureof the cryptic species complex believed to inhabit theserivers The demonstration of reproductive isolation andormicroallopatric distribution among morphologically crypticspecies may result in the description of greater speciesrichness within the genus than presently described Thespecies status of H chrysops merits reconsideration givenits highly divergent evolutionary history Nuclear geneticmarkers in conjunction with mtDNA markers could revealcases of historical introgression particularly in the KapuasRiver and the Mekong River The hemibagrid catfishes con-tinue to provide multiple opportunities for the study of theevolutionary taxonomy and biogeography of a major faunalcomponent of the fluvial ecosystems of Southeast Asia

Disclosure

Ethanol-preserved tissue samples of many of the specimensidentified in Table S1 are freely available upon request fromJJD

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Goh Yan Yih for assistance in collectingspecimens and molecular analyses Mrs Francoise Colom-bani for molecular analyses and Dr Heok Hee Ng forspecies identification This work was funded by the NationalUniversity of Singapore grant to P K L Ng and a CanadianNatural Sciences and Engineering Research Council grant toJ J Dodson

References

[1] D J Lohman M de Bruyn T Page et al ldquoBiogeography ofthe Indo-Australian archipelagordquo Annual Review of EcologyEvolution and Systematics vol 42 pp 205ndash226 2011

20 Advances in Evolutionary Biology

[2] D S Woodruff ldquoNeogene marine transgressions palaeogeog-raphy and biogeographic transitions on the Thai-Malay Penin-sulardquo Journal of Biogeography vol 30 no 4 pp 551ndash567 2003

[3] H K Voris ldquoMaps of Pleistocene sea levels in SoutheastAsia shorelines river systems and time durationsrdquo Journal ofBiogeography vol 27 no 5 pp 1153ndash1167 2000

[4] S Y Yap ldquoOn the distributional patterns of Southeast-EastAsian freshwater fish and their historyrdquo Journal of Biogeographyvol 29 no 9 pp 1187ndash1199 2002

[5] S K J McConnell ldquoMapping aquatic faunal exchanges acrossthe Sunda shelf South-East Asia using distributional andgenetic data sets from the cyprinid fish Barbodes gonionotus(Bleeker 1850)rdquo Journal of Natural History vol 38 no 5 pp651ndash670 2004

[6] T Mo Anatomy Relationships and Systematics of the Bagridae(Teleostei Siluroidei) with a Hypothesis of Siluroid Phylogenyvol 17 ofTheses Zoologicae Koeltz Scientific Books 1991

[7] H H Ng and M Kottelat ldquoRevision of the Asian catfish genusHemibagrus bleeker 1862 (Teleostei Siluriformes Bagridae)rdquoRaffles Bulletin of Zoology vol 61 no 1 pp 205ndash291 2013

[8] P K L Ng and H H Ng ldquoHemibagrus gracilis a new speciesof large riverine catfish (Teleostei Bagridae) from PeninsularMalaysiardquo Raffles Bulletin of Zoology vol 43 pp 133ndash142 1995

[9] H H Tan and M Kottelat ldquoRedescription of Betta picta(Teleostei Osphronemidae) and description of Betta falx sp nfrom central Sumatrardquo Revue Suisse de Zoologie vol 105 no 3pp 557ndash568 1998

[10] J J Dodson F Colombani and P K Ng ldquoPhylogeographicstructure in mitochondrial DNA of a South-east Asian fresh-water fish Hemibagrus nemurus (Siluroidei Bagridae) andPleistocene sea-level changes on the Sunda shelfrdquo MolecularEcology vol 4 no 3 pp 331ndash346 1995

[11] H H Ng and J J Dodson ldquoMorphological and genetic descrip-tions of a new species of catfish Hemibagrus chrysops fromSarawak East Malaysia with an assessment of phylogeneticrelationships (Teleostei Bagridae)rdquo Raffles Bulletin of Zoologyvol 47 no 1 pp 45ndash57 1999

[12] P D N Hebert A Cywinska S L Ball and J R DeWaardldquoBiological identifications through DNA barcodesrdquo Proceedingsof the Royal Society B Biological Sciences vol 270 no 1512 pp313ndash321 2003

[13] J C Carolan T EMurray U Fitzpatrick et al ldquoColour patternsdo not diagnose species quantitative evaluation of a DNAbarcoded cryptic bumblebee complexrdquo PLoS ONE vol 7 no 1Article ID e29251 2012

[14] M Kekkonen and P D N Hebert ldquoDNA barcode-baseddelineation of putative species efficient start for taxonomicworkflowsrdquoMolecular Ecology Resources vol 14 no 4 pp 706ndash715 2014

[15] DM Hillis CMoritz and B KMarbleMolecular SystematicsSinauer Sunderland Mass USA 2nd edition 1996

[16] T D Kocher W K Thomas A Meyer et al ldquoDynamics ofmitochondrial DNA evolution in animals amplification andsequencingwith conserved primersrdquoProceedings of theNationalAcademy of Sciences of the United States of America vol 86 no16 pp 6196ndash6200 1989

[17] T A Hall ldquoBioEdit a user-friendly biological sequence align-ment editor and analysis program for Windows 9598NTrdquoNucleic Acids Symposium Series vol 41 pp 95ndash98 1999

[18] D A Benson I Karsch-Mizrachi K Clark D J Lipman JOstell and EW Sayers ldquoGenBankrdquoNucleic Acids Research vol40 no D1 pp D48ndashD53 2012

[19] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[20] X Ku Z Peng R Diogo and S He ldquoMtDNA phylogenyprovides evidence of generic polyphyleticism for East Asianbagrid catfishesrdquoHydrobiologia vol 579 no 1 pp 147ndash159 2007

[21] L Yang and S He ldquoPhylogeography of the freshwater catfishHemibagrus guttatus (Siluriformes Bagridae) implications forSouth China biogeography and influence of sea-level changesrdquoMolecular Phylogenetics and Evolution vol 49 no 1 pp 393ndash398 2008

[22] B W Bowen A L Bass L A Rocha W S Grant and DR Robertson ldquoPhylogeography of the trumpetfishes (Aulosto-mus) ring species complex on a global scalerdquo Evolution vol 55no 5 pp 1029ndash1039 2001

[23] Y P Kartavtsev ldquoDivergence at Cyt-b and Co-1 mtDNA geneson different taxonomic levels and genetics of speciation inanimalsrdquoMitochondrial DNA vol 22 no 3 pp 55ndash65 2011

[24] SWada Y Kameda and S Chiba ldquoLong-term stasis and short-term divergence in the phenotypes of microsnails on oceanicislandsrdquoMolecular Ecology vol 22 no 18 pp 4801ndash4810 2013

[25] J Colborn R E Crabtree J B Shaklee E Pfeiler and B WBowen ldquoThe evolutionary enigma of bonefishes (Albula spp)cryptic species and ancient separations in a globally distributedshorefishrdquo Evolution vol 55 no 4 pp 807ndash820 2001

[26] J-D Durand K-N Shen W-J Chen et al ldquoSystematics of thegrey mullets (Teleostei Mugiliformes Mugilidae) molecularphylogenetic evidence challenges two centuries ofmorphology-based taxonomyrdquo Molecular Phylogenetics and Evolution vol64 no 1 pp 73ndash92 2012

[27] K-N Shen B W Jamandre C-C Hsu W-N Tzeng and J-D Durand ldquoPlio-Pleistocene sea level and temperature fluc-tuations in the northwestern Pacific promoted speciation inthe globally-distributed flathead mullet Mugil cephalusrdquo BMCEvolutionary Biology vol 11 no 1 article 83 2011

[28] R Hall ldquoThe plate tectonics of Cenozoic SE Asia and thedistribution of land and seardquo in Biogeography and GeologicalEvolution of SE Asia R Hall and J D Holloway Eds pp 99ndash131Backhuys Publishers Leiden The Netherlands 1998

[29] R Hall ldquoSoutheast Asiarsquos changing palaeogeographyrdquo Blumeavol 54 no 1ndash3 pp 148ndash161 2009

[30] R F Inger and P K Chen ldquoThe freshwater fishes of NorthBorneordquo Fieldiana Zoology vol 45 pp 1ndash268 1962

[31] J Bohlen V Slechtova H H Tan and R Britz ldquoPhylogenyof the Southeast Asian freshwater fish genus Pangio (Cyprini-formes Cobitidae)rdquoMolecular Phylogenetics and Evolution vol61 no 3 pp 854ndash865 2011

[32] M de Bruyn E Nugroho M M Hossain J C Wilson andP B Mather ldquoPhylogeographic evidence for the existence ofan ancient biogeographic barrier the Isthmus of Kra SeawayrdquoHeredity vol 94 no 3 pp 370ndash378 2005

[33] J Parnell ldquoThe biogeography of the Isthmus of Kra region areviewrdquoNordic Journal of Botany vol 31 no 1 pp 001ndash015 2013

[34] S Usmani S G Tan S S Siraj and K Yusoff ldquoPopulationstructure of the Southeast Asian river catfishMystus nemurusrdquoAnimal Genetics vol 34 no 6 pp 462ndash464 2003

[35] M P Tan A F J Jamsari and M N Siti Azizah ldquoPhylo-geographic pattern of the striped snakehead Channa striatain Sundaland ancient river connectivity geographical and

Advances in Evolutionary Biology 21

anthropogenic singnaturesrdquo PLoS ONE vol 7 no 12 Article IDe52089 2012

[36] R Abell M L Thieme C Revenga et al ldquoFreshwater ecore-gions of the world a new map of biogeographic units forfreshwater biodiversity conservationrdquo BioScience vol 58 no 5pp 403ndash414 2008

[37] P U Clark D Archer D Pollard et al ldquoThemiddle Pleistocenetransition characteristics mechanisms and implications forlong-term changes in atmospheric pCO

2rdquo Quaternary Science

Reviews vol 25 no 23-24 pp 3150ndash3184 2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

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BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

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Nucleic AcidsJournal of

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Stem CellsInternational

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Enzyme Research

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International Journal of

Microbiology

Page 2: ResearchArticle - Université Laval Julian... · ResearchArticle A DNA Barcode-Based Evaluation of the Southeast Asian Catfish Genus Hemibagrus Bleeker, 1862 (Teleostei: Siluriformes;

2 Advances in Evolutionary Biology

Species of the genus Hemibagrus [6] are large catfishesfound in rivers east from Indiarsquos Godavari River and southfrom the Yangtze in China They occupy a wide range ofhabitats ranging from near estuarine to high altitude fast-flowing headstreams The genus reaches its greatest diversityin Southeast Asia [7] Throughout its geographical rangeand particularly in Southeast Asia species of Hemibagrusare valuable food fishes and species identification is ofparamount importance to sustainably exploit this speciescomplex However the taxonomy ofHemibagrus is confusingand the validity of many nominal species is unclear Morsquos [6]rehabilitation of the genusHemibagrus was not accompaniedby a comprehensive examination of types of all the nomi-nal species The complexity of the regionrsquos biogeographicalhistory coupled with a lack of well-defined morphologicalcharacters and considerable plasticity in phenotypic traitscommonly used for diagnosing species in other catfish groupsrenders the identification and phylogenetic reconstruction ofHemibagrus problematical [7] P K L Ng and H H Ng [8]divided the SundaicHemibagrus into four species groups butsubsequent work by Ng and coworkers revealed that themor-phological characters they used may not always distinguishthe species reliably nor do they allow easy assignment ofmany of the species to any of the groups [7]This situation ledto the publication of a revision of the genus byNg andKottelat[7] The revision is based on morphological criteria andrecognizes eight species groups H baramensis H guttatusH menoda H nemurus H olyroides H planiceps Hpluriradiatus and H wyckii species groups A species groupas defined in Tan and Kottelat [9] is ldquoan assemblage ofspecies sharing a set of diagnostic characters which mayor may not be a monophyletic lineagerdquo Ng and Kottelat [7]employed various morphometric and meristic characters todefine species and species groups including various bodydimensions fin lengths head dimensions eye diameter andvarious fin ray counts (see [7 Figure 1] for a complete listof morphological variables used by Ng and Kottelat) Thetaxonomic revision of Ng and Kottelat [7] was based onthe diagnosis of unique combinations of characters ratherthan discrete autapomorphies Thirty-two valid species arerecognized in the revision three of which are new [7]

Early molecular phylogenetic and phylogeographic stud-ies of the putativeH nemurus species group in Southeast Asiarevealed extensive genetic subdivision of the group [10 11]The occurrence of genetically divergent groups in sympatryin widely separated locations supported the proposition thatlow sea levels promoted the dispersal and mingling of somegenetic groups [10]These studies also revealed very divergentlineageswithinHemibagrus predating the Pleistocene [10 11]However in the absence of a comprehensive examination oftypes of all the nominal species in the genus the taxonomicstatus of these genetic lineages remained unclearThe revisionof the genus proposed by Ng and Kottelat [7] provides abasis by which we may clarify the taxonomic status andthe biogeography of independent genetic lineages withinHemibagrus

The analysis of single-locus data from genomic regionssuch as short sequences of mitochondrial genes used asDNA barcodes [12] represents a useful tool for species

identification and evaluating taxonomic diagnoses based onmorphological characters (eg [13 14]) Given the phe-notypic plasticity inherent in many of the morphologicalvariables used by Ng and Kottelat in their revision [7] and thepossibility that species groups so defined may not representtrue monophyletic lineages we here employ a DNA barcodederived from the mitochondrial cytochrome 119887 gene (Cyt b)to expand our genetic analyses of the genus in Southeast Asiaand to evaluate the revision of the genus Hemibagrus pro-posed by Ng and Kottelat [7] by testing the congruence of themorphologically and genetically based taxonomiesWe chosethis marker to be consistent with earlier work done on thegenus in Southeast Asia [10 11] Our principal objective wasto test the hypothesis that species groups and nominal speciesdefined with morphological criteria by Ng and Kottelat [7]reflect a hierarchical arrangement of monophyletic groupsbased on the similarity of mtDNA sequences Our secondobjectivewas to provide a tentative evaluation of phylogeneticrelationships among taxa Our third objective was to describethe geographical distribution of taxa and to relate majorcladogenetic events with major geological events that mayhave contributed to the radiation ofHemibagrus in SoutheastAsia We pay particular attention to the phylogeography ofthe widespread H nemurus species group

2 Materials and Methods

A total of 452 hemibagrid catfish particularly those of thepresumedH nemurus species group were sampled through-out their distribution range in Southeast Asia and successfullysequenced encompassing 67 sampling sites located on 37river systems (Figure 1 Table 1) Sampling of the other speciesgroups defined byNg andKottelat [7] was sparser thus not allspecies groups were represented in the genetic analysis Fishwere principally obtained from local fishermen Many fishwere placed in the Zoological Reference Collection (ZRC)of the Raffles Museum of Biodiversity Research Universityof Singapore where they received a ZRC accession number(see Table S1 in Supplementary Material available onlineat httpdxdoiorg1011552015490158) Ethanol-preservedtissue samples of many of the specimens identified in TableS1 are freely available upon request from JJD

Genetic Analysis Total genomic DNA was extracted from100 120583g of tissue (fresh or 95 ethanol-preserved) with astandard phenol-chloroform-isoamyl protocol [15] precipi-tated with cold 95 ethanol and suspended in 200120583L TEbuffer (10 nMTris-HCl pH 8 1mMEDTA) A 605 bp portionfrom the 51015840-end of cytochrome 119887 was amplified (including 8-bp located before the start codon) using derivatives of theuniversal primers of Kocher et al [16] P1 51015840-AAAACC-ACCGTTGTTATTCAACTACA-31015840 (light strand) and P251015840-GGGTTGTTTGATCCTGTTTCGTG-31015840 (heavy strand)Polymerase chain reaction (PCR) conditions were carriedout in 50 120583L total volumes containing (final concentrations)200120583M of each of dATP dGTP dCTP and dTTP 400 nM ofeach primer 1ndash25Uof TaqDNApolymerase 25mMMgCl

2

10mMTris-HCl (pH 83) 50mMKCl and between 50 and200 ng (3ndash5 120583L) of template (purified mtDNA) DNA was

Advances in Evolutionary Biology 3

Table 1 Rivers river code (as presented in Figure 1) sampling sites latitudelongitude and distance upstream from riverrsquos mouth of samplingsites and the number of specimens of Hemibagrus spp sequenced

River code Site code River Sample site Latituded∘ mm1015840 ss10158401015840

Longituded∘ mm1015840 ss10158401015840

Distance upstreamfrom river mouth

(km)

Number ofspecimens

1a 1 Mekong Bungkan 18 21 54 N 103 39 04 E 1285 41b 2 Mekong Bannan 17 58 00 N 104 14 00 E 1200 21c 3 Mekong That Phanom 17 24 00 N 104 48 00 E 1100 21d 4 Mekong Warinchamrap 15 12 00 N 104 53 00 E 885 31e 5 Mekong Mun River 15 17 00 N 105 04 00 E 800 131f 6 Mekong Phibum 15 14 00 N 105 14 00 E 845 41g 7 Mekong Nomh Penh 11 33 00 N 104 55 00 E 250 42a 8 Chao Phraya Market 14 35 00 N 100 27 00 E 100 82b 9 Chao Phraya Nakhom Sawan 15 41 00 N 100 07 00 E 275 53a 10 Chanthaburi 12 48 00 N 102 09 00 E 60 64a 11 Mea Khlong Kanchanaburi 14 01 00 N 99 32 00 E 100 55a 12 Thai Pen Phatthalung 7 37 00 N 100 05 00 E 90 16a 13 Kelantan 6 00 00 N 102 10 00 E 50 127a 14 Terengganu Kuala Berang 5 04 00 N 103 01 00 E 40 147b 15 Terengganu Sekayu 4 58 00 N 103 01 00 E 80 3

8a 16 Pahang KampongMengkarak 3 19 00 N 102 27 00 E 100 9

9a 17 Endau Park 2 40 00 N 103 15 00 E 100 69b 18 Endau Kahang 2 18 00 N 103 35 00 E 50 19c 19 Endau Ambat 2 11 00 N 103 51 00 E 70 29d 20 Endau Mersing 2 25 00 N 103 56 00 E 5 1010a 21 Johor Kota Tingi 1 4 00 N 103 54 00 E 45 311a 22 Perak 5 12 00 N 101 02 00 E 180 1311b 23 Perak Chenderoh 4 58 00 N 100 57 00 E 160 611c 24 Perak Gerik 5 25 00 N 101 08 00 E 200 1212a 25 Bernam Sabak 3 46 00 N 100 59 00 E 50 113a 26 Muar Selabu 2 9 00 N 102 36 00 E 20 613b 27 Muar Market 2 14 00 N 102 46 00 E 40 413c 28 Muar Kundang Ulu 2 14 00 N 102 46 00 E 40 213d 29 Muar Panchor 2 10 00 N 102 43 00 E 35 414a 30 Batang Hari Jambi 1 36 00 S 103 37 00 E 100 2714b 31 Batang Hari Kerinci 2 05 00 S 101 23 00 E 800 715a 32 Musi Palembang 2 55 00 S 103 37 00 E 70 1516a 33 Ci Liwong Sawagangan 6 24 00 S 106 46 00 E 45 217a 34 Ci Sadane Bogor 6 35 00 S 106 47 00 E 60 118a 35 Ci Tarum Cirata 6 33 00 S 107 18 00 E 90 1819a 36 Ci Manuk Garut 7 13 00 S 107 54 00 E 150 1420a 37 Kali Progo Mayudan 7 45 00 S 110 11 00 E 30 221a 38 Brantas River Surabaya 7 28 00 S 112 26 00 E 50 622a 39 Mahakam Samarinda 0 24 00 S 116 58 00 E 50 1023a 40 Barito Banjarmassin 3 5 00 S 114 38 00 E 50 924a 41 Kapuas Pontianak 0 17 00 S 109 19 00 E 20 1224b 42 Kapuas Sanggau 0 08 00 N 110 36 00 E 120 224c 43 Kapuas Nanga Pinoh 0 20 00 S 111 44 00 E 320 3

4 Advances in Evolutionary Biology

Table 1 Continued

River code Site code River Sample site Latituded∘ mm1015840 ss10158401015840

Longituded∘ mm1015840 ss10158401015840

Distance upstreamfrom river mouth

(km)

Number ofspecimens

24d 44 Kapuas Sintang 0 04 00 N 111 30 00 E 270 324e 45 Kapuas Danau Sentarum 0 51 00 N 112 6 00 E 550 224f 46 Kapuas Putussibau 0 50 00 N 112 56 00 E 600 224g 47 Kapuas Sibau 0 55 00 N 112 57 00 E 750 224h 48 Kapuas Mendalam 1 00 00 N 113 16 00 E 800 625a 49 Sadong Serian 1 10 00 N 110 34 00 E 50 2525b 50 Sadong Balai Ringin 1 03 00 N 110 45 00 E 70 1126a 51 Rajang Kapit 2 01 00 N 112 56 00 E 210 726b 52 Rajang Sibu 2 18 00 N 111 49 00 E 80 1026c 53 Rajang Katibas 1 43 00 N 112 33 00 E 170 1227a 54 Baram Mulu 3 30 00 N 115 30 00 E 380 327b 55 Baram Bakung 4 17 00 N 114 14 00 E 60 627c 56 Baram Marudi 4 11 00 N 114 19 00 E 80 1528a 57 Tutong Merimbun 4 36 00 N 114 40 00 E 40 329a 58 Belait Ingei 4 10 00 N 114 43 00 E 100 330a 59 Padas Tenom 5 08 00 N 115 57 00 E 70 931a 60 Segaliud Sandakan 5 45 00 N 117 52 00 E 50 132a 61 Segama Danum Valley 5 01 00 N 117 47 00 E 170 1632b 62 Segama Lahad Datu 5 02 00 N 118 19 00 E 90 633a 63 Kinabatangan Batu Putih 5 25 00 N 117 55 00 E 100 634a 64 Umas-Umas Tawau 4 22 00 N 117 44 00 E 15 235 65 Salween Myanmar mdash mdash mdash 236 66 Red Hanoi mdash mdash mdash 237 67 Pearl Guangdong mdash mdash mdash 8

Total 452mdash exact origin unknown

amplified in a programmable thermal cycler using the follow-ing profile one single preliminary denaturation at 95∘C for5min 40 cycles of amplification with denaturation at 94∘Cfor 60 s primer annealing at 45∘C for 60 s primer extensionat 72∘C for 90 s and a final single extension step performed at72∘C for 5min Total 50 120583L PCR products were purified on a2 agarose gel After electrophoresis at 90V for 2 hours theamplified fragments were excised from the gel and extractedand purified using the QIAquick Gel Extraction Kit Protocol(using a microcentrifuge) fromQIAGEN to a final volume of15 to 50120583L (in EB buffer 10mMTris Cl pH 75)

Double stranded purified PCR products were sequencedin both directions (complementary strand was sequenced inall cases) using the ABI PRISM BigDye Terminator CycleSequencing Ready Reaction Kit (Applied Biosystems Inc)Sequencing reactions were carried out in 20120583L total volumecontaining 32 picomoles of the sequencing primer 30 to60 ng of purified PCR product 6 120583L of 25X dilution buffer(CSA seq Buffer Perkin Elmer) and 2 120583L of ABI PRISMBigDye Terminator Ready Reaction Mix Reactions wereperformed in thermal cycler GeneAmp PCR system 9600using the following profile 10 s at 96∘C 5 s at 50∘C and

4min at 60∘C for 25 cycles Sequences were separated byelectrophoresis at 1680V (150W 500mA) for 5 to 7 h on ABIPRISM 377 DNA Sequencer (Applied Biosystems Inc)

Sequences were extracted from the gel image using ABIPRISM Sequencing Analysis 30 program The sequences ofthe complementary strands were then imported in SequenceNavigator program for comparative alignment and correctionof ambiguities (Applied Biosystems Inc) Sequences werealigned using the BioEdit sequence alignment editor [17]Each sequence was identified by a unique code designatingthe origin of the sample (river name) the cumulativenumber of the sequences found in that river and thenumber of individuals characterized by that particularsequence For example the code MEK1502 designates the15th sequence found in the Mekong River and occurring intwo specimens Following the comparison of morphologicaland genetic designations mtDNA codes were preceded bythe putative species designation (eg SPMEK1502 indicatesthat specimens bearing sequence MEK1502 were assignedto Hemibagrus spilopterus) Sequences found in multiplerivers were identified by river name and the total number ofspecimens Sequences were deposited in GenBank [18] with

Advances in Evolutionary Biology 5

(1) Mekong(2) Chao Phraya(3) Chanthaburi(4) Mea Khlong(5) Phatthalung(6) Kelatan(7) Terengannu(8) Pahang(9) Endau(10) Johor(11) Perak(12) Bernam

(13) Muar(14) Batang Hari(15) Musi(16) Ci Liwong(17) Ci Sadane(18) Ci Tarum(19) Ci Manuk(20) Kali Progo(21) Brantas(22) Mahakam(23) Barito(24) Kapuas(25) Sadong(26) Rajang

(27) Baram(28) Tutong(29) Belait(30) Padas(31) Segaliud(32) Segama(33) Kinabatangan(34) Umas-Umas(35) Salween(36) Red(37) Pearl

0 250 500 750

(km)

0 250 500

(mi)100

∘110

∘120

0∘

10∘

Siam RiverNor

th Su

nda R

iver

East Sunda River

35 36

37

1

5

11

12

17

8 9

10

14

16

17

18

19

25

24

23

20

21

22

27

26

28

29 3031

32

33

34

15

6

13

2

3

4

Shelf minus75mShelf minus120 m

Figure 1 Location map of Southeast Asia illustrating the 37 rivers sampled in this study Black dots along rivers represent the sampling sitesas identified in Table 1 and Table S1 Dark grey shading illustrates the extent of the emerging Sunda Shelf at sea levels 75m below present daylevels light grey denotes emerging shelf at sea levels 120m below present day levels (as illustrated by Voris 2000) Rivers illustrated on theSunda Shelf represent drowned Pleistocene river drainage systems according to Voris (2000)

accession numbers corresponding to sequence codes andreported in Supplementary Material Table S1

Data Analysis We first used the Neighbor-Joining (NJ)algorithm in MEGA 52 (Tamura et al 2011) to develop ataxon identification phenogram ofHemibagrusThe Tamura-Nei distance matrix considering invariable site contribution

(+119868) was selected according to the model selection algo-rithm implemented in MEGA 52 [19] The rate variationamong sites was modeled with a gamma distribution (shapeparameter = 1) Recognizing the limitations of single-locusanalyses in phylogeny reconstruction and the objectionsof cladists to distance-based methodology we neverthelesssought to provide a tentative evaluation of phylogenetic

6 Advances in Evolutionary Biology

relationships among taxa To do so the Cyt 119887 sequence of theChinese longsnout catfish (Leiocassis longirostris) obtainedfrom GenBank (accession number DQ3217551) was usedas an outgroup to root the phenogram Chinese speciescurrently assigned to Leiocassismay be considered membersof the genus Pseudobagrus [20] Furthermore we employeda maximum likelihood (ML) phylogenetic reconstructionbased on the comparison of nucleotides within sites to assessthe congruence of the taxon identification phenogram andthe ML phylogenetic reconstruction In the latter case theevolutionary model used was the Hasegawa-Kishino-Yanomodel (HKY)with gammadistribution (+119866 = 13290) Initialtree(s) for the heuristic search were obtained by applyingthe Neighbor-Joining (NJ) method to a matrix of pairwisedistances estimated using the maximum composite likeli-hood (MCL) approach The rate-variation model allowedfor some sites to be evolutionarily invariable (+119868 4644of sites) The ML tree was rooted with the Cyt 119887 sequenceof the Chinese longsnout catfish (Leiocassis longirostris) andconstructed using MEGA52 [19]

TheNJ phenogram classifiedmtDNA sequences based onoverall similarity Ng and Kottelat [7] also defined speciesgroups based on overall similarity but according to mor-phological criteria Of the 8 species groups defined by Ngand Kottelat [7] two groups (H olyroides (1 species) and Hpluriradiatus (5 species)) were not included in our samplesWe thus evaluated the congruence of the two methods indefining the remaining six species groups (H baramensisH guttatus H menoda H nemurus H planiceps and Hwyckii species groups) When possible mtDNA sequenceswere linked to ZRC accession numbers identifying specimensexamined by Ng and Kottelat [7] in their morphologicalrevision of the genus (Table S1)

To provide time estimates for the major phylogeneticevents we present a calibrated linearized tree obtained fromour ML reconstruction (MEGA 52) Time of divergencebetween Hemibagrus lineages has been approximated byapplying a rate of between 10 and 15 genetic divergence permillion years as reported for bagrid fish by Yang and He [21]However the justification for this rate is not clear We thusapplied the more widely used rate of 2 sequence divergenceper million years [22] As there is undoubtedly a wide errorassociated with any such molecular clock calibrations esti-mated divergence times are used to discriminate more recentfrom more ancient cladogenetic events while providing veryapproximate dates for such events

Genetic divergence estimates using 119901-distances (119901) wereused in the present study to identify the different taxonomiclevels observed among the Hemibagrus spp analyzed Sincesuch information is readily available for thousands of animalgroups [23] we used 119901-distance (119901) as a benchmark for taxo-nomic levelsThe survey of 20 731 vertebrate and invertebratespecies reported by Kartavtsev [23] revealed increasing levelsof 119901-distances () of the mtDNA Cyt 119887 gene with taxonomiclevel populations within species 119901 = 138 plusmn 030 (SD)subspecies or sibling species119901 = 510plusmn091 morphologicallydistinct species within genera 119901 = 1031 plusmn 093 generawithin a family 119901 = 1786 plusmn 136 and families within anorder 119901 = 2636 plusmn 388 Although heterogeneity in the

gene evolution rate differs amongdifferent taxonomic groupsthese estimates provide an order of magnitude evaluationof intraspecific versus interspecific divergence and permittedus to discriminate between population- and species-leveldivergences However as 119901-distances underestimate truegenetic distance (eg site saturation and back-mutation)divergence times based on119901-distances greater than about 10are generally underestimated We calculated 119901-distances inMEGA 52 and report them as percentages (standard error)

3 Results

The 452 samples yielded a total of 118 distinct sequences(not including the outgroup taxon) The Neighbor-Joiningphenogram clustered the 119 sequences into a large numberof hierarchically organized monophyletic clades To facilitatepresentation we first present a compressed NJ tree andidentify 11 major mtDNA clades (Figure 2) One unidentifiedfish from the Rajang River was characterized by a genotypethat clustered with the outgroup taxon (XXRAJ0604GenBank accession number KP322872 Figures 2 and3) A second fish from the Rajang was characterized byan isolated genotype (XXRAJ0301 GenBank accessionnumber KP322869) of unknown affiliation (Figures 2and 3) To assess the congruence of the species groupsdefined by Ng and Kottelat ([7] hereafter referred toas NampK) and the mtDNA groupings revealed by thepresent analysis we provide a direct comparison of thetwo methods following the order determined by the taxonidentification phenogram (Figure 2 Table S1) Table S1 alsoprovides additional information about the geographicaldistribution of sequences Gene trees of individual cladesincluding all sequences are presented at each step in theanalysis along with their geographical distributions We alsopresent a compressed maximum likelihood phylogenetictree (Figure 3) The phenogram and the ML tree providedcongruent phylogenetic reconstructions However both treesfailed to resolve the phylogenetic relationships of H wyckiiH guttatus and H menoda species groups most likelydue to undersampling of these lineages The relationshipsamong an unspecified species group H spilopterus and Hfortis are also not well resolved Their position in the NJtree receives weak bootstrap support and they appear as amultifurcation along with a clade composed of H capitulumand H hoevenii in the ML phylogenetic tree (Figures 2 and3) To facilitate comparisons with the taxonomic scheme ofNampK we follow the sequence of taxa as identified in Figure 2

mtDNAClade 1The H wyckii Species GroupThe first clade isclearly consistent with theH wyckii species group (Figure 4)This species group is composed of 4 valid speciesHmaydelliH microphthalmus H wyckii andH wyckioides (NampK)Thelatter species is known from the Mekong and Chao Phrayadrainage Only specimens of H wyckioides obtained fromthe Mekong and the Chao Phraya were available for DNAanalysis Three specimens from the Chao Phraya bearingsequence WYCHP0203 (Table S1) were identified as Hwyckioides (H H Ng personal communication) although

Advances in Evolutionary Biology 7

8811

10

9

8

7

6

5

4

3

1

2

66

85

44

32

98

91

81

8432

57

99

99

99

49

002

34

15

43

86

99

96

H capitulum (Indochinese clade)

H capitulum (Sundaic clade)

H hoeveniiH fortis

H spilopterus

H baramensis species group

H planiceps species groupH menoda species group

H guttatus species group

H wyckii species group

XXRAJ0604

PESAL0102

XXRAJ0301Leiocassis longirostris

mtDNAclade

H nemurusspecies group

Unspecified species group

Figure 2 The taxon identification phenogram of Hemibagrus inferred using the Neighbor-Joining method in MEGA 52 The optimal treewith the sumof branch length = 186668598 is shown Bootstrap values are indicated on each node of the tree (500 replicates) Bar indicates thenumber of substitutions changes estimated using Tamura-Nei distance considering invariable site contribution (+I)The analysis involved 119nucleotide sequences All positions containing gaps and missing data were eliminatedThere were a total of 597 positions in the final dataset

mtDNAclade

H capitulum (Indochinese clade) 11

93

97H capitulum (Sundaic clade)H hoevenii

9999

84H fortis 8

9

10

H spilopterus 773 92

Unspecified species group 678

XXRAJ060454 34

90 H baramensis species group 5

49 H planiceps species group 4

H menoda species groupPESAL0102 3

H wyckii species group 199

99

99H guttatus species group 2

XXRAJ030149

47

Leiocassis longirostris

005

H nemurusspecies group

94

72

Figure 3 Evolutionary history of Hemibagrus inferred by using the maximum likelihood method based on the Hasegawa-Kishino-YanomodelThe tree with the highest log likelihood (minus56774825) is shown Bootstrap values are indicated on each node of the tree (500 replicates)Bar indicates the number of substitution changes estimated using Hasegawa-Kishino-Yano model (HKY) with gamma distribution (+119866 =13290) The analysis involved 119 nucleotide sequences All positions containing gaps and missing data were eliminated There were a totalof 597 positions in the final dataset

their ZRC numbers do not appear in the analysis of NampK Asno specimens of the other 3 species comprising the H wyckiispecies group were available for analysis we cannot confirmif this species group is monophyletic

mtDNA Clade 2 The H guttatus Species Group The secondclade of the Hemibagrus gene tree (Figure 2) is composed oftwo reciprocally monophyletic subclades clearly reflectingthe H guttatus species group as defined by NampK (Figure 4)

8 Advances in Evolutionary Biology

100∘

10∘

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120∘

0

0 250

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750(km)

(mi)

002

98

43

84

34

99

99

79

96

51

59

99

78

49

H peguensis

H guttatus

H macropterus

H wyckioides

PESAL0102

XXRAJ0301Leiocassis longirostris

MAPEA0201MAPEA0302MAPEA0101

GUPEA0501GURED0102

GUPEA0401GUPEA0602

WYCHP0401WYMEK1101

WYCHP0203

See Figures 7ndash12

See Figures 5 and 6

Shelf minus75mShelf minus120 m

Figure 4 Phylogenetic relationships of the individual sequences comprising four species H guttatus H macropterus (together forming theH guttatus species group) H wyckioides and H peguensisThe subtree is extracted from the taxon identification phenogram inferred usingthe Neighbor-Joining method (see Figure 2) The percentage of replicate trees in which the associated sequences clustered together in thebootstrap test (500 replicates) is shown next to the branches The distribution of these sequences is illustrated in the right panel

The group comprises three valid species H guttatus Hmacropterus andH vietnamicus (NampK) Specimens availablefor DNA analysis were obtained from the Pearl River andRed River (11 fish 7 sequences Table 1 and Table S1) Thetwo reciprocally monophyletic subclades comprising theH guttatus species group represent H guttatus and Hmacropterus (Figure 4) Although no ZRC specimens wereexamined by NampK species identification was confirmed(H H Ng personal communication) No H vietnamicusspecimens were available for DNA analysis The twospecies composing the group are closely related (119901 = 51(SE = 08))

mtDNA ldquoCladerdquo 3 The H menoda Species Group Thisisolated genotype (Figure 4) most likely identifies a cladeencompassing the H menoda species group although the

single sequence (Table S1) is shared by only 2 specimensof H peguensis from the Salween River Myanmar alsoincluded in the analysis of NampK (where its provenance wasidentified as the neighboring Irrawaddy River) The otherspecies comprising this group (H menoda H caveatusand H punctatus) were not sampled for DNA analysis Themonophyletic nature of this species group thus remains to bedemonstrated

31 The H planiceps + H baramensis Group The remainderof the gene tree is split into two large sister groups withmarginal bootstrap support (73 in ML and 43 in NJ)the H planiceps + H baramensis group and the H nemurusspecies group (Figures 2 and 3) The H planiceps + Hbaramensis group is strongly supported in both NJ and MLanalyses (84 and 90 bootstrap support respectively

Advances in Evolutionary Biology 9

110∘

47

53

69

81

69

5199

52

95

97

78

001

H bongan

H velox

H gracilis

H planiceps

H divaricatus

BOKAP1801

BOKAP1601BOKAP1701

BOKAP1402BOKAP1503

VEBAH0907GREND0206

PLCIM0114PLCIS0101

DIPER0401DIPER0301

DIPER0201

100∘

10∘

0∘

120∘

0

0 250 500

250 500

750(km)

(mi)

Shelf minus75mShelf minus120 m

Figure 5 Phylogenetic relationships of the individual sequences and species comprising theH planiceps species groupH bongan H veloxH gracilis H planiceps and H divaricatus The subtree is extracted from the taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicate trees in which the associated sequences clustered together in the bootstrap test(500 replicates) is shown next to the branches The distribution of these sequences is illustrated in the right panel

Figures 2 and 3) The two groups are genetically similarwith a genetic divergence estimate of 119901 = 121 (SE = 09)Although theH planiceps species group is strongly supportedin both trees (99) bootstrap support of the H baramensisspecies group is weak (Figure 2 32 Figure 3 34) Thegeographical distribution of the two groups differs Membersof the H planiceps species group are found throughout theGreater Sunda Islands and Peninsular Malaysia but they areisolated in the upper reaches of large rivers (Figure 5) Incontrast members of theH baramensis species group appearto be restricted to Sabah North Borneo (Figure 6)

mtDNA Clade 4 The H planiceps Species Group The Hplaniceps species group (Figure 5) is comprised of 5 closelyrelated but genetically distinct species (mean within-groupdivergence 119901 = 20 (SD = 033) H planiceps (Java)H gracilis (eastern Peninsular Malaysia) H divaricatus(western Peninsular Malaysia) H bongan (Borneo) andH velox (Sumatra)) H lacustrinus the sixth species of thisgroup (NampK) was not sampled in the present study WithintheH planiceps species groupH planiceps formed a distinctspecies (Figure 5) comprising two sequences One sequence(PLCIM0114 Table S1) characterized 14 specimens obtainedfrom Ci Manuk West Java 9 of which were identified as Hplaniceps by NampK (Table S1) A second sequence (PLCIS0101

Table S1) was obtained from one specimen sampled atCi Sadane West Java that we classified as H planicepsH bongan is represented by 5 sequences obtained from 8specimens sampled in the Kapuas River (Figure 5) Two ofthese specimens (ZRC 43013 and 43015) were identified asH bongan by NampK (Table S1) H divaricatus is composedof 3 sequences from three specimens obtained at Gerik onthe Perak River (western Peninsular Malaysia) One of thesespecimens (ZRC 41151) is the holotype of this new species(NampK) H gracilis is represented by only one sequence(GREND0206 Table S1) shared by 6 specimens sampledat Ulu Endau Jasin River eastern Peninsular Malaysiafour of which were identified by NampK (Table S1) ZRC21484 comprises the holotype and ZRC 21482 21486 and21487 comprise the paratypes (NampK) of this species (TableS1) Finally H velox is represented by only one sequence(VEBAH0907) from 7 specimens sampled at Kerinci BatangHari River Sumatra Three of these specimens (ZRC 41503Table S1) represent 3 paratypes of the species (NampK)

mtDNA Clade 5 The H baramensis Species Group The threespecies comprising the H baramensis species group (Figures2 and 3) form three monophyletic subclades (mean within-group divergence 119901 = 106 (08) Figure 6) H semotus iscomposed of two sequences (SEPAD0101 SEPAD0202) that

10 Advances in Evolutionary Biology

110∘

002

H sabanus

H semotus

H baramensis

SAKIN0204SASGM0706

BASGM0402BASGM0301

BASGM0201BASGM0104

SESGM0502SEUMA0101

SEPAD0202SEPAD0101

See Figure 5

100∘

10∘

0∘

120∘

0

0 250 500

250 500

750(km)

(mi)

99

57

32

83

99

72

99

56

66

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Shelf minus75mShelf minus120 m

Figure 6 Phylogenetic relationships of the individual sequences and three species comprising the H baramensis species group H semotusH sabanus and H baramensis (furcatus) The subtree is extracted from the taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicate trees in which the associated sequences clustered together in the bootstrap test(500 replicates) is shown next to the branches The distribution of these sequences is illustrated in the right panel

characterize fish that are the holotype and paratypes of Hsemotus (ZRC 46122 Table S1) along with two somewhatmore divergent sequences (Figure 6) One of these sequences(SGM0502) was found in the Segama River (Danum Valley)and identified as H baramensis by NampK (ZRC 40525)(Table S1) The second sequence (UMA0101) was from afish sampled at Tawau Umas Umas River (ZRC 46123) alsoidentified as H baramensis by NampK These sequences mostlikely reflect distinct lineages within H semotus inhabitingdifferent rivers as the mean intraspecies sequence divergence(119901 = 16) indicates population-level divergence

H sabanus and H furcatus (synonymized with H bara-mensis by NampK) cluster together to form a distinct subcladebut clearly diverge at the species level (Figure 6) H sabanuscomprises 2 sequences that are shared respectively by 6specimens (ZRC 42741) from the Segama River Sabah and4 specimens (part of ZRC 42743) from the KinabatanganRiver Sabah (Table S1) All of these specimenswere identifiedas H sabanus by NampK (Table S1) Finally H baramensisforms a distant subclade (147 mean sequence divergencebetween H baramensis and H sabanus) composed of 4sequences (Figure 6) shared by 8 specimens all sampled inthe Segama River Danum Valley Conservation Area SabahSix of these specimens (ZRC 40523 (2 specimens) ZRC40527 (2 specimens) and ZRC 40526 (2 specimens) Table S1)comprise the paratypes of H baramensis (furcatus) (NampK)

32 The H nemurus Species Group In both NJ and ML trees(Figures 2 and 3) the H nemurus species group is strongly

supported (bootstrap support 98 in NJ and 99 in ML)indicating that it forms a distinct and widespread geneticentity within the genus Hemibagrus However relationshipswithin the group are not so clear In general the H nemurusspecies group is composed of 4 clades The first clade may beconsidered as a species group nested within the H nemurusspecies group (85 and 93 bootstrap support in NJ andML resp) encompassing three clades H hoevenii (clade9) the H capitulum Sundaic clade (clade 10 including Hnemurus) and the H capitulum Indochinese clade (clade 11)(Figures 2 and 3) An additional three clades (Figures 2 and3) are formed by an unidentified species group (clade 6) Hspilopterus (clade 7) andH fortis (clade 8)These three latterclades are distinct at the congeneric species level (unspecifiedversus H spilopterus 119901 = 77 (07) unspecified versus Hfortis 119901 = 85 (07) and H spilopterus versus H fortis119901 = 75 (08)) The distribution of these three clades issomewhat disparate H fortis and the unspecified speciesgroup are principally found in Borneo whereasH spilopterusis widely distributed on the Indochinese mainland

mtDNA Clade 6 Unspecified Species Group This geneticallydiverse group encompassing at least 3 subclades (H spp AB and C Figure 7 within-group mean sequence divergence119901 = 68(06)) is confoundedwith theH baramensis speciesgroup byNampK It is composed of sequences fromfish sampledin Sarawak Sabah and eastern PeninsularMalaysia Subclade119867 spp A (Figure 7) is particularly interesting as it contains2 sequences (CHSARA20 CHRAJ0401) that characterize

Advances in Evolutionary Biology 11

110∘

XXPAD0303XXPAD0601

XXPAD0401XXBEL0202

XXBRM0202XXRAJ0511

XXPAD0501

XXEND0410CHSARA20CHRAJ0401

XXBEL0101XXSEG0101

100∘

10∘

0∘

120∘

0

0 250 500

250 500

750(km)

(mi)

91

86

83

99

98

47

81

001

91

72

99

80

H spp C

H spp B

H spp A

Shelf minus75mShelf minus120 m

Figure 7 Phylogenetic relationships of the individual sequences comprising the unspecified species group containing three subcladesidentified as H spp A B and C The subtree is extracted from the taxon identification phenogram inferred using the Neighbor-Joiningmethod (see Figure 2) The percentage of replicate trees in which the associated sequences clustered together in the bootstrap test (500replicates) is shown next to the branches The distribution of these sequences is illustrated in the right panel

H chrysops a new species identified by Ng and Dodson [11]However specimens of H chrysops from the Rajang Riverwere subsequently synonymized by NampK with H capitulumof the H nemurus species group (ZRC 23008ndash23014 TableS1) However an additional 14 fish from the Sadong Riverat Serian bearing genotype SARA20 (Table S1) and thusgenetically identical to the Rajang H chrysops samples wereidentified by NampK as H spilopterus (ZRC 29583ndash96 TableS1) These specimens however were erroneously identifiedin NampK as originating in the Chao Phraya River (Thailand)Subclade A also contains one sequence obtained from 10 fishsampled on the Endau River (Mersing) in eastern PeninsularMalaysia These fish were not morphologically examinedFinally subclade A also contains one sequence from oneunidentified fish sampled in the Segaliud River Sabah andanother unidentified fish sampled in the Belait River BruneiSubclade H spp B encompasses 2 sequences one of whichcomes from a fish sampled in the Padas River (XXPAD0501)and part of the ZRC 46122 fish lot identified as H semotusby NampK (Table S1) The second sequence (XXRAJ0511) wasshared by 11 unidentified fish sampled at Katibas on theRajang River Sarawak (Table S1) Finally subclade 119867 sppC encompasses 5 sequences Three sequences (XXPAD0303XXPAD0401 and XXPAD0601) were obtained from thePadas River and identified byNampKasH semotus (ZRC46122Table S1) The fourth sequence (XXBRM0202) was obtainedfrom 2 fish sampled in the Baram River and was identified

as H baramensis by NampK (ZRC 42742) The 5th sequencecomposing this group was obtained from 2 unidentified fishsampled in the Belait River Brunei (Table S1)

mtDNA clade 6 is thus problematical within the contextof the revision of the genus Hemibagrus Two highlydivergent mtDNA clades (clade 5 and clade 6 Figures 2and 3) occur in sympatry principally in the Padas River ofSabah North Borneo (Figures 6 and 7) Specimens of theunspecified species group are morphologically similar tothe H baramensis species group where they are assigned inthe analysis of NampK However the mean genetic distancebetween the unspecified species group and theH baramensisspecies group is 119901 = 193 (28) close to genus leveldivergence Furthermore subclade 119867 spp A (Figure 7)contains not only H chrysops but also an unidentifiedpopulation from eastern Peninsular Malaysia (XXEND0410Table S1) and two additional Sabah sequences On theother hand the unspecified species group clusters with allother members of the H nemurus species group (meangenetic divergence of unspecified group versus H capitulum(Sundaic clade) 119901 = 76 (07) (Figure 2)) with strongbootstrap support Such genetic clustering may justify theinclusion of H chrysops and associated sequences in theH nemurus species group but its synonymization with Hcapitulum (NampK) is difficult to defend on genetic groundsWe tentatively conclude that H chrysops is a valid speciesbased on genetic identity [11] forming part of an unknown

12 Advances in Evolutionary Biology

110∘

SPCPCHMK12

SPMEK0202SPMEK0401

SPMEK0301SPMEK0103

SPMEK1401SPMEK0501

SPMEK1201SPMEK1301

SPMEK1601SPMEK1502

SPMEK0701SPMEK0801SPMEK1701

SPCHP0501SPCHA0201

SPCHA0304SPPAT0101

100∘

10∘

0∘

120∘

0

0 250 500

250 500

750(km)

(mi)

000499

99 98

60

47

48

54

96

7091

38

86

87

99

80

58

H spilopterus ldquoCrdquo

H spilopterus ldquoBrdquo

H spilopterus ldquoArdquo

Shelf minus75mShelf minus120 m

Figure 8 Phylogenetic relationships within H spilopterus comprised of 3 subclades identified as A B and C The subtree is extracted fromthe taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicate trees in whichthe associated sequences clustered together in the bootstrap test (500 replicates) is shown next to the branches The distribution of thesesequences is illustrated in the right panel

species group found throughout North Borneo (and toa lesser degree in eastern Peninsular Malaysia) that maybe considered as part of the H nemurus species groupHowever the morphological confusion of the unspecifiedspecies group with the sympatric highly divergent Hbaramensis species group suggests the existence of a crypticspecies assemblage (see Section 4)

mtDNA Clade 7 Clade 7 (Figures 2 and 3) is composed of atleast 3 distinct subclades that appear to reflect 3 geneticallydivergent populations ofH spilopterus Subclade A (Figure 8)encompasses 7 sequences obtained from 8 fish sampled inthe Mekong River Four fish bearing 3 of these sequenceswere identified as paratypes (ZRC 43324) of H spilopterusby NampK (Table S1) One additional fish was identified asH spilopterus by NampK (ZRC 46157 Table S1) Subclade Bencompasses 6 sequences obtained from 9 fish all sampledfrom the Mekong River (Table S1) The fish sampled at ThatPhanom (ZRC 46012) was identified as H spilopterus inthe analysis of HampK (Table S1) Subclade C encompasses 5sequences obtained from 19 fish sampled in the Chao PhrayaMae Khlong and Chanthaburi Rivers Thailand Onesequence (CPCHMK12) was found in all of these drainages(Table S1) A closely allied sequence was found in one fishsampled at Phatthalung just south of the Isthmus of Kra onthe Thai-Malay Peninsula None of these fish were examined

by NampK and thus we are unable to definitively identifythe species However given the widespread occurrence ofH spilopterus in the Chao Phraya and Mae Klong Riversreported by NampK we conclude that these fish are part of awidespread H spilopterus population complex The meanwithin-group (clade 7) genetic divergence (119901 = 29 (04))suggests intraspecific population-level divergence

mtDNA Clade 8 Clade 8 encompasses 3 sequencesobtained from 9 fish sampled in east Sabah specifically theKinabatangan Segama and Umas Umas Rivers (Figure 9)The Kinabatangan sequence is somewhat divergent withinthe clade and the specimen was not identified by NampK Onesequence (SGM0606) characterized 6 fish from the SegamaRiver 2 of which were identified as H fortis (ZEC 4052140522 Table S1) by NampK (Table S1) Finally one fish bearinga closely related genotype from the Umas Umas River wasidentified as H baramensis by NampK (Table S1)

mtDNA Clade 9 Clade 9 is a sister group to the remaining2 clades composing the H nemurus species group Thiswell-defined clade (Figure 10) clusters 6 sequences from42 fish that are clearly identified as H hoevenii (NampK)Some of these haplotypes are geographically widespread onesequence (JOMSKA11 Table S1) was found in the Johor River(East Peninsular Malaysia) the Musi River (Sumatra) and

Advances in Evolutionary Biology 13

100∘ 110∘

0∘

120∘

00 250

250500

500750

(km)

(mi)

FOKIN0102FOSGM0606

FOUMA0201H fortis

10∘

99

86

001

Shelf minus75mShelf minus120 m

Figure 9 Phylogenetic relationships within H fortis comprised of 3 sequences The subtree is extracted from the taxon identificationphenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicate trees in which the associated sequencesclustered together in the bootstrap test (500 replicates) is shown next to the branches The distribution of these sequences is illustrated in theright panel

the Kapuas River (West Borneo) and a second sequence(HMUBHKABM23 Table S1) was found inMuarRiver (WestPeninsular Malaysia) and the Batang Hari (Sumatra) theKapuas (Sarawak) and the Baram Rivers (Sarawak) Many ofthese fish were identified as H hoevenii by NampK (Table S1)The existence of twodistinct but very closely related subclades(Figure 10 119901 = 06 (03)) that are not geographicallysegregated suggests some recent isolation event followed bywidespread dispersal and secondary contact

33 The H capitulum Group The final two monophyleticgroups of the Hemibagrus gene tree are approximatelyequivalent to the remaining species composing the Hnemurus species group as defined by NampK Dodson etal [10] and Ng and Dodson [11] have previously referredto clade 10 as the Sundaic clade as most sequences wereobtained from fish sampled on the Greater Sunda Islandsand clade 11 as the Indochinese clade asmany sequences wereobtained from fish sampled on the Indochinese mainland(Figure 2 Table S1) Although most of these sequencesare assigned to H capitulum by NampK there are somesignificant phylogeographic differences between the 2 cladesIn particular there appears to be an east-west division in thedistribution of the two clades on the Thai-Malay Peninsulawith the Sundaic clade (clade 10) found on the west coast andthe Indochinese clade found on the east coast with sympatricoccurrence in the Muar River Nevertheless mean sequencedivergence between the two clades is 119901 = 29 (05) avalue that is indicative of population-level or subspecies

divergence The two clades are thus best considered ascomprising one species

mtDNA Clade 10 The Sundaic clade includes a number ofclosely related sequences that comprise part of H capitulumand H nemurus Very little genetic structure is apparentand bootstrap support for tree topology within this group isgenerally very weak (Figure 11) A widely spread genotype(NCLCTBA20) characterized 6 of 17 specimens from CiTarum at the Cirata reservoir (West Java) (Table S1) and5 were identified as H nemurus in the analysis of NampK(ZRC 42564) (Table S1) The remaining fish characterized bythis sequence were obtained from Ci Liwong at Sawangan(West Java) and one fish was obtained from the Barito Riverat Banjarmassin south Borneo This latter specimen (ZRC40553) was identified by NampK as H capitulum (Table S1)Another common genotype (NBHKPBR09) was identified aseither H capitulum H hoevenii or H nemurus dependingon the river of origin (Table S1) Many of the remainingsequences of the Sundaic clade appear to characterize fish thatNampK identified asH capitulum A geographically widespreadgenotype (CPEBEMUKA36) characterized 9 fish sampledon the Perak River at Gerik that were identified by NampKas H capitulum (ZRC 41150 Table S1) Another commongenotype within this clade (CABHMS20) included 2 fishfrom the Batang Hari at Jambi and identified by NampK asH capitulum (ZRC40534 Table S1) Another 2 sequences(CABAR0203 CABAR0305) characterized 8 fish sampledon the Barito River at Banjarmasin and were identified as

14 Advances in Evolutionary Biology

100∘

10∘

110∘

0∘

120∘

0

0 250

250

500

500

750(km)

(mi)

53

64

99

85

64

0002

HJOMSKA11

HOBAH0601

HOBHMS05

HMUBHKABM23

HOBAH0701

HOKAP0501

H hoevenii ldquoBrdquo

H hoevenii ldquoArdquo

Shelf minus75mShelf minus120 m

Figure 10 Phylogenetic relationships withinH hoevenii comprised of 2 subclades and 6 sequences The subtree is extracted from the taxonidentification phenogram inferred using theNeighbor-Joiningmethod (see Figure 2)The percentage of replicate trees in which the associatedsequences clustered together in the bootstrap test (500 replicates) is shown next to the branches The distribution of these sequences isillustrated in the right panel

110∘

XXMAH0110

CABAR0305

96 CAKPR0201

74CAKAP0401

5931 CAKAP0601

35 CPEBEMUKA36

646 CAKAP0903

33 CAKAP080114 CAKAP0701

CARAJ0210

4 CABAR0203CABRA0204

20

CBHKPBR0919

CABRA0301

15CABHMS20

24 NCLCTBA20

63 NECIT0201

H capitulumldquoSundaic claderdquo

H nemurus

0002 120∘

0 250 500 750

(km)

0 250 500

(mi)100

10∘

0∘

Shelf minus75mShelf minus120 m

Figure 11 Phylogenetic relationships within theH capitulum ldquoSundaicrdquo clade including 2 sequences associated withH nemurusThe subtreeis extracted from the taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicatetrees in which the associated sequences clustered together in the bootstrap test (500 replicates) is shown next to the branchesThe distributionof these sequences is illustrated in the right panel

Advances in Evolutionary Biology 15

H capitulum

0∘

10∘

100∘

110∘

120∘

66

63

88

59

38

39 68

71

6991

57

9754

88 4944

39

53

89

0002

ldquoIndochineserdquogroup ldquoErdquo

H capitulumldquoIndochineserdquo

group ldquoDrdquoH capitulumldquoIndochineserdquo

group ldquoCrdquo

H capitulumldquoIndochineserdquo

group ldquoBrdquo

H capitulumldquoIndochineserdquo

group ldquoArdquo0

0

250

250

500

500

750

(km)

(mi)

CATER0301CATERR0401

CKETEMU23

CAMEK0601CACHP0301

CAMKPA03

CAMEK0910CAMEK1003

XXMUBM02

CABMTU09

CABRM0601

CAKAP1201CAKAP1001

CAKAP1101CAKAP1301

CABRM0501

CASAD0102

CASAD0220

CAEND0301

CABAH0801

CAPABH05CATEEN03

CATER0701CATERR0801

CAKETE04CATER0501

CAPAH0303

Shelf minus75mShelf minus120 m

Figure 12 Phylogenetic relationships within theH capitulum ldquoIndochineserdquo clade encompassing 5 subclades identified as A B C D and EThe subtree is extracted from the taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2)The percentageof replicate trees in which the associated sequences clustered together in the bootstrap test (500 replicates) is shown next to the branchesThedistribution of these sequences is illustrated in the right panel

H capitulum by NampK (ZRC 40553 Table S1) Although notincluded in the analysis of NampK samples from the Sundaicclade were also found in the Kapuas River (West Borneo)Finally 10 fish sampled on the Mahakam River at Samarindaeast Borneo are all characterized by one relatively divergentgenotype (CAMAH0110) butwere considered asH capitulumin the analysis of NampK (ZRC 40533 Table S1)TheMahakampopulation sample is the most genetically distinct of theSundaic clade (119901 = 17 (04)) but nevertheless indicativeof intraspecific population-level divergence (Figure 12)

The topology of the Sundaic clade does not permit usto draw a clear distinction between H nemurus and theSundaic H capitulum The mean within-group divergenceof the Sundaic clade is only 119901 = 08 (02) well withinpopulation-level divergence values We postulate that Hnemurus may be one of several regional variants of awidespread Sundaic H capitulum clade that has radiatedrelatively recently This may explain the existence of manyshared sequences throughout the Sundaic islands assigned todifferent species in different regions

mtDNA Clade 11 Finally the Indochinese clade of Hcapitulum represents a geographically widespread andweakly diversified clade (within-group mean sequencedivergence 119901 = 11 (02) Figure 12) Although few fishfrom this genetic clade were included in the morphological

analysis of NampK most may be considered as H capitulumgiven their geographic distribution and close geneticrelationship to the Sundaic H capitulum Four sequencesin this clade were identified as H capitulum in the analysisof NampK 3 specimens characterized by one sequence(CABMTU09) found in the Tutong River of westernBorneo (ZRC 40482) 6 characterized by the same sequencefrom the Baram River (ZRC 40498) another 2 sequencesfrom the Baram River (ZRC 40498) and 20 specimenscharacterized by an abundant genotype (CASAD0220)found at 2 sites in the Sadong River also in western Borneo(ZRC 39526 5 specimens 39527 3 specimens and 38753 4specimens) (Table S1) Unfortunately none of the specimenscollected on the Indochinese mainland for genetic analysiswere examined by NampK Nevertheless the distribution ofspecimens analyzed for mtDNA resembles the distribution ofspecimens examined by NampK Although weakly structuredwe may recognize 5 subclades One genotype (CAPAH0303)is basal to the phenogram Subclade A encompasses 8sequences mainly found in eastern Peninsular Malaysia(Table S1 Figure 12) These rivers furnished many of thespecimens NampK examined to define H capitulum Howeversome sequences in this group occur in sympatry withsequences identified within the Sundaic H capitulum groupTwo sequences from subclade A occurred in the Batang HariSumatra (Table S1) Subclade E also encompasses sequences

16 Advances in Evolutionary Biology

found in Peninsular Malaysia as well as in the Mekong ChaoPhraya and Mea Khlong Rivers (Figure 12) In addition3 subclades encompass sequences found principally inwestern Borneo subclade B in the Baram and Sadong RiversSarawak subclade C in the Kapuas River West Kalimantanand subclade D in the Baram and Tutong Rivers of Sarawakand Brunei respectively (Figure 12) This latter subclade alsoincludes one genotype found in the Muar River of westernpeninsular Malaysia Finally subclade E encompasses 5sequences from the Mekong Chao Praya and Mae KhlongRivers all within the distribution area of H spilopterus

EvolutionaryHistoryThe evolutionary history of theHemiba-grus species groups analyzed here dates from at least theMiocene 15ndash20Ma (Figure 13) when considering the widelyused molecular clock of 2 sequence divergence per millionyears Bootstrap support for the sister group relationships ofH wyckioides theH guttatus species group andH peguensisare all weak (clearly due to undersampling) and little can besaid of their evolutionary history other than the fact that theyoccupy basal positions in the rooted trees The remainder ofboth the NJ and ML trees is composed of two major sistergroups the H planiceps + H baramensis group and the Hnemurus species group (Figures 2 3 and 13)TheH planiceps+ H baramensis group is strongly supported in both NJ andML analyses (84 and 90 bootstrap support resp Figures2 and 3) The H nemurus species group is also stronglysupported in both analyses (98 and 99 bootstrap supportFigures 2 and 3) These two major phylogenetic groups areestimated to have diverged at least 8 to 12Ma ago during themid to late Miocene (Figure 13)

Much of the evolutionary history of Hemibagrus hasoccurred during the Quaternary starting approximately26Ma ago This is particularly true of the H planicepsand H nemurus species groups The radiation of the 5species included in the H planiceps species group appearsto coincide with the middle Pleistocene transition period ofapproximately 1Ma ago Their high-elevation distributionhowever appears to have largely isolated them from theeffects of lowered sea levels during glacial maxima and theattendant opportunities for widespread dispersal (Figure 5)The evolutionary histories of theH baramensis species groupand the unspecified species group date back to the latePliocene In contrast the low-elevation distribution of theHnemurus species group in Sundaland appears to have favoredtheir widespread distribution during the Quaternary ice ageIn particular H hoevenii appears to be the most recentlydispersed of the species under study with diversification anddispersal occurring approximately 03Ma ago (Figure 13)

4 Discussion

41 The Congruence of Morphological and Genetic AnalysesCombining genetic and morphological analyses to elucidatetaxonomy allowed us to better understand the diversificationand evolutionary relationships within this speciose group offishes Given the rapid and relatively constant rate of mtDNAevolution the deeper the branches within the phylogenetictree are the greater our ability to distinguish valid taxa

should be Conversely the lack of complete congruencebetween morphological- and genetic-based identificationsis expected to increase among the shallower branches of thephylogenetic tree resulting in the appearance of polyphyleticspecies Although 2 of the 8 species groups defined by Ngand Kottelat [7] were not sampled in this study the validityof the remaining 6 species groups and their componentspecies defined on the basis of morphological similaritywas largely confirmed by the analysis of the mitochondrialCyt 119887 bar code As expected congruence is strongest at thebase of the NJ and ML trees where the H guttatus speciesgroup formed a well-defined monophyletic group The Hmenoda and H wyckii species group were each representedby only one species but they were clearly distinct relativeto sequences characterizing the neighboring species groupsThe H planiceps H baramensis and H nemurus speciesgroups and their component species were also generallycongruent in morphological and genetic analyses Also asexpected most cases of discrepancy between morphological-based species identification and genetic-based speciesidentification occurred on relatively shallow branches of thephylogenetic tree These involve principally H spilopterusand the two mtDNA subclades composing H capitulumHowever a far more unexpected lack of congruence involvesthe definition of a deeply divergent unspecified genetic groupconfounding members of theH baramensis species group inthe morphological analysis of Ng and Kottelat [7]

Interpreting Polyphyly The apparent polyphyletic origin ofa species may be due to incomplete lineage sorting amongrecently diverged species the introgression of mtDNA dueto species hybridization or the erroneous identificationof species due to the lumping of morphologically crypticspecies In the absence of nuclear DNA analyses it isoften difficult to distinguish between introgression andincomplete lineage sorting For example several sequencescharacterizing the Indochinese clade of H capitulum(subclade E) are found in the Mekong and Chao PhrayaRivers well within the distributional area of H spilopterusH spilopterus and H capitulum first diverged at least 3MaOn the one hand this apparent case of polyphyly mayhave resulted from incomplete lineage sorting betweenrecently divergedH spilopterus andH capitulum We wouldhowever have expected similar cases of polyphyly amongpopulations of the Indochinese clade of H capitulum butthis was not detected On the other hand the sympatricoccurrence of H spilopterus subclade A and subcladeB sequences with H capitulum Indochinese subclade Esequences in the Mekong River may represent a migrationevent and local introgression of H capitulum with Hspilopterus A similar event may have occurred in theChao Phraya and Mae Khlong Rivers where H spilopterussequences cooccur withH capitulum sequences Computingthe divergence of H capitulum sequences (Indochinesesubclade E) cooccurring with H spilopterus and Hcapitulum sequences occurring in eastern PeninsularMalaysia (subclade A) reveals a mean sequence divergence119901 = 15 (04) Thus the H capitulum sequences occurringin sympatry with H spilopterus diverged at most 750 ka ago

Advances in Evolutionary Biology 17

H fortis

XXRAJ0604H furcatus

H sabanusH semotusH divaricatus

H planicepsGREND0206

VEBAH0907

H bonganPESAL0102

H wyckioidesH macropterus

H guttatusXXRAJ0301Leiocassis longirostris

Ma

4971

92

47

51

9999

99

9999

99

99

99

9999

99

99

99

99

65

96

96

4946

93

79

78

70

91

93

98

86

87

91

95

97

71

85

9999

05101520

005010015020 000

H capitulum Indochinese clades AndashE

H capitulum Sundaic clade

H hoevenii B

H hoevenii A

H spilopterus C

H spilopterus B

H spilopterus AH spilopterus A

H spp CH spp B

H spp A

Figure 13 Linearized tree with branch lengths estimated with the molecular clock using a mutation rate of 1 per Ma (=divergence rate of2 per Ma) The tree topology is identical to the ML reconstruction illustrated in Figure 3 (see the specificities of the tree reconstruction inthe caption of Figure 3) Boxes represent 95 confidence intervals of divergence time estimates for each node Vertical dashed lines redirectto dates indicated on the timescale (Ma) Numbers below timescale indicate the estimated number of substitution changes using Hasegawa-Kishino-Yanomodel (HKY)with gamma distribution (+119866 = 13290) after linearizing the treeH furcatus is synonymizedwithH baramensis

from eastern Peninsular Malaysia populations suggestinga recent introgression event Similarly H capitulum in theKapuas River is characterized by sequences of both theSundaic and the Indochinese clades As both sequences arebroadly sympatric it is not possible to distinguish betweenincomplete lineage sorting and introgression of these closelyrelated intraspecific lineages

A Possible Case of Morphological Stasis Clade 6 of thepresent analysis defines a well-supported monophyletic cladefound in Sabah Sarawak and the Endau River of easternpeninsula Malaysia that has no equivalent taxonomic statusin the revision of Ng and Kottelat [7] In Borneo they occursympatrically with the H baramensis species group and areconfounded as either H baramensis or H semotus These

two clades are genetically highly divergent having divergedbetween 8 and 12Ma ago It thus seems highly unlikely thatthey represent retained polymorphisms due to incompletelineage sorting Sequences previously identified as character-izing H chrysops [11] are part of clade 6 and extend to thesouth (Rajang and Sadong Rivers of Sarawak West Borneo)with one closely related genotype found in eastern peninsularMalaysia (Endau River) where it may well be confoundedwith H capitulum H chrysops was synonymized with Hcapitulum by Ng and Kottelat [7] based on their analysis ofspecimens ofH chrysops from the Rajang RiverThus despitea long and apparently independent phylogenetic historythe unspecified species group appears largely cryptic andconfounded principally with members of the H baramensisspecies group

18 Advances in Evolutionary Biology

We suggest that this situation is an example of mor-phological stasis involving the coexistence of a number ofgenetically divergent yet morphologically cryptic species thatare lumped together [24] A striking example of morpho-logical and ecological stasis among fishes is provided by thecoastal marine bonefishes (Albula spp) Distinct evolution-ary lineages (separated by sequence divergence between 5 and30 [25]) of bonefish coexist in several sample locations yetthey show little morphological or ecological differentiationin sympatry [25] Similarly a proportion of the grey mulletspecies (Mugilidae) also appears to consist of cryptic species[26] For exampleMugil cephalus in Taiwan is characterizedby three divergent lineages that have been shown to charac-terize 3 reproductively isolated but morphologically crypticspecies [26 27]

Here we have discovered 2 highly distinct and structuredclades (each composed of 3 mtDNA subclades) that aremorphologically confounded within different species Forexample H spp C of the unspecified species group isidentified as H baramensis in the Baram River and as Hsemotus in the Padas River We speculate that North Borneoharbors a morphologically cryptic species complex possiblycomposed of several species pairs inhabiting different riversystems The reconstruction of this phylogenetic history andthe taxonomic status of its components remains a majorchallenge to the revision of the Hemibagrus genus

42 Phylogeographic Hypotheses The phylogeography ofthe genus appears to have been principally influenced bytwo geological histories one encompassing the formationof North Borneo the second the emergence of the SundaIslands and their connectivity with the Indochinesemainlandassociated with recurrent sea-level changes North Borneoappears to have acted as a species sink promoting speciationand locally high biodiversity within the genus but with littleevidence of subsequent dispersal out of North Borneo Incontrast the geological events influencing the Indochinesemainland and the Sunda Islands have permitted bothspeciation and differential dispersal depending on thebiology of the species For example the H planiceps speciesgroup is found throughout the Greater Sunda Islands andtheThai-Malay Peninsula as is theH nemurus species groupWhereas the H nemurus species group generally occupiesthe low altitude lower reaches of large rivers the speciescomposing the H planiceps group are found in the upperreaches of large rivers The H nemurus species group iscomposed of a number of widely distributed closely relatedspecies whereas theH planiceps group is composed of a largenumber of species that are highly localized geographicallyIt thus appears that sea-level changes have had little impacton the dispersal history of the H planiceps species group buthave deeply influenced that of the H nemurus species group

The Case of North Borneo From 15Ma to 5Ma greater areasof Borneo became emergent and the central mountainson the Sarawak-Kalimantan border extending into Sabahbecame wider and higher which may have represented newbarriers to dispersal [28 29] Borneo gradually developedinto the present large island by emergence of land in the

north and east The H baramensis species group is restrictedto North Borneo as is the unspecified species group of theHnemurus species group H fortis is also distributed in NorthBorneo The isolation of the freshwater fish communities ofNorth Borneo (particularly in the Labuk-Segama watershed)has long been recognized based on the number of endemicspecies and the concentration of relict species [30] The latePliocene-early Pleistocene diversification of H fortis theunspecified species group and the H baramensis speciesgroup may thus be associated with their dispersal to andisolation within the watersheds of North Borneo Only onesequence of the unspecified species group is found outsideof North Borneo and H fortis appears to be distributedthroughout central and east Borneo [7]

ThePhylogeography of the H nemurus Species Group Periodicmarine transgressions on the Sundaland platform during theearly and middle Miocene [2 29] may have been sufficientto isolate populations of freshwater organisms and promotecladogenesis [31] This may have been responsible for theinitial mid to late Miocene divergence of the H nemurusspecies group The majority of sequences assigned to thisspecies group belong to 3 species (H spilopterus H capitulum(including H nemurus) and H hoevenii) all of which havebeen influenced by the more recent geological history of theSunda Islands and their connectivity with the Indochinesemainland H spilopterus is known from river drainages inIndochina including the Mekong Dong Nai Ban PakongChao Phraya and Mae Klong where it is found in a widevariety of lotic and lentic habitats [7] H spilopterus divergedin the late Pliocene-early Pleistocene possibly isolated tothe north of the Kra Peninsula during the late Pliocenemarine transgression that is purported to have created amajor biogeographic transition zone between the Sundaicand Indochinese biotas ([32] but see [33] for a critique ofthis biogeographic scenario) The existence of 2 subclades(A and B) in the Mekong River separated by 2 sequencedivergence is consistent with intraspecific divergence Thetwo subclades possibly represent 2 historical mtDNA lineagesthat are retained within the species reflecting a Pleistoceneisolation event followed by secondary contact within thecontemporary Mekong River Subclade C from the ChaoPhraya and Mae Khlong Rivers exhibits a level of geneticdivergence consistent with sibling species-level divergenceshowing a 5 sequence divergence relative to Mekong RiverH spilopterus

H capitulum may be considered as part of the Sundaicbiota located principally to the south of the Kra marinetransgression zone that may have isolated H spilopterus tothe north on the Indochinese mainlandThe biogeographicalevent leading to the subsequent formation of two intraspecificgroups within H capitulum is not at all clear The onlyarea where the two clades are clearly geographically separateis on the Thai-Malay Peninsula with the ldquoSundaicrdquo cladeon the west coast and the ldquoIndochineserdquo clade on theeast coast separated by the central Titiwangsa mountainrange The genetic distinctiveness of east and west coastpopulations of H capitulum has also been documented fornuclear DNA [34] A similar pattern of separation has been

Advances in Evolutionary Biology 19

reported for 5 additional freshwater fishes within this region[35] However as the divergence of the two H capitulumclades postdates the formation of the mountain range byapproximately 200Ma the geographic separation of the twoclades most likely represents differential dispersal rather thanancient vicariance The presence of the Sundaic clade on thewest coast of the Thai-Malay Peninsula as well as on theSundaic Islands reflects the historical connections betweeneast Sumatra and west Peninsular Malaysia across the Straitof Malacca This area encompasses the Northern CentralSumatra-Western Malaysia freshwater ecoregion [36] TheMuar River forms its southern border coincident with thesouthernmost distribution of the Sundaic clade on the Thai-Malay Peninsula It is important to note however that mostof Sumatra and Java emerged to achieve their present size andelevation only 5Ma ago and much of East Java continuedto be the site of marine deposition until late Pliocene-earlyPleistocene [29] We may speculate that the Sundaic clade ofH capitulum resulted from the relatively recent colonizationof these islands and Borneo The radiation within the cladeonly dates back to approximately 1Ma ago H nemurus fromWest Java may thus best be considered an isolated regionalpopulation of very recent origin

The diversification of the Indochinese clade dates fromat least 12Ma ago coincident with the mid-Pleistocenetransition at which time low-frequency high-amplitude andquasiperiodic (100 ka) glacial variability emerged It began125Ma ago and was complete by 07Ma ago [37] The tran-sition marked the beginning of major changes in sea levelsduring which the Sunda Shelf periodically emerged and wastraversed by enormous fluvial systems [3] The contributionof these events to the diversification of this clade cannotbe satisfactorily reconstructed One possible explanation isthat high sea levels produced barriers to gene flow and thepopulations of West Borneo (subclades B C and D) wereisolated from those of east Peninsular Malaysia This couldbe due to one or more dispersal events from Borneo via theGreat Sunda River (Figure 1) to east Peninsular Malaysia orlineage sorting whereby sequences of subclades A and E havebeen lost from West Borneo As in the case of the sympatricoccurrence of H spilopterus and H capitulum subclade E(see above) in the Mekong River we are unable to clearlydefine the relative roles of incomplete lineage sorting andintrogression followingmigration Given the recent origins ofthe Indochinese H capitulum sequences incomplete lineagesorting most probably blurs patterns of recent isolation

The last species of the H nemurus species group is Hhoevenii The species is widespread throughout PeninsularMalaysia and the Sundaic Islands It is found primarily inthe lower reaches of rivers and is apparently capable ofwithstanding low salt concentration [7] H hoevenii thusappears to be the most liable of all members of the Hnemurus species group to be impacted by Pleistocene sea-level changes This is clearly reflected in the widespreaddistribution of the sequences of this species Two lineages areevident having diverged no more than 03Ma ago suggest-ing a late Pleistocene isolation event followed by extensivedispersal and admixture in recent times This represents theclearest example of the dispersal opportunities afforded by

Pleistocene sea level changes on the Sunda Shelf In additionthe relatively low genetic diversity detected (only 6 sequencesin 42 fish) suggests a population bottleneck in the recent pastpossibly related to population declines in the lower reaches ofrivers flooded by increasing sea levels

43 Future Directions We have provided a global picture ofthe evolutionary taxonomy of the genus in Southeast AsiaClearly a more rigorous phylogenetic reconstruction of thegenus Hemibagrus requiring the construction of multiplegene trees based on the analysis of both mitochondrial andnuclear DNA markers from a wider taxonomic array ofhemibagrids will involve an even greater effort of samplingthroughout the region In particular we require samplingat smaller spatial and temporal scales For example smallerscale sampling in the Padas River and the Segama Riverof North Borneo using both mitochondrial and nucleargenetic markers would contribute to clarifying the natureof the cryptic species complex believed to inhabit theserivers The demonstration of reproductive isolation andormicroallopatric distribution among morphologically crypticspecies may result in the description of greater speciesrichness within the genus than presently described Thespecies status of H chrysops merits reconsideration givenits highly divergent evolutionary history Nuclear geneticmarkers in conjunction with mtDNA markers could revealcases of historical introgression particularly in the KapuasRiver and the Mekong River The hemibagrid catfishes con-tinue to provide multiple opportunities for the study of theevolutionary taxonomy and biogeography of a major faunalcomponent of the fluvial ecosystems of Southeast Asia

Disclosure

Ethanol-preserved tissue samples of many of the specimensidentified in Table S1 are freely available upon request fromJJD

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Goh Yan Yih for assistance in collectingspecimens and molecular analyses Mrs Francoise Colom-bani for molecular analyses and Dr Heok Hee Ng forspecies identification This work was funded by the NationalUniversity of Singapore grant to P K L Ng and a CanadianNatural Sciences and Engineering Research Council grant toJ J Dodson

References

[1] D J Lohman M de Bruyn T Page et al ldquoBiogeography ofthe Indo-Australian archipelagordquo Annual Review of EcologyEvolution and Systematics vol 42 pp 205ndash226 2011

20 Advances in Evolutionary Biology

[2] D S Woodruff ldquoNeogene marine transgressions palaeogeog-raphy and biogeographic transitions on the Thai-Malay Penin-sulardquo Journal of Biogeography vol 30 no 4 pp 551ndash567 2003

[3] H K Voris ldquoMaps of Pleistocene sea levels in SoutheastAsia shorelines river systems and time durationsrdquo Journal ofBiogeography vol 27 no 5 pp 1153ndash1167 2000

[4] S Y Yap ldquoOn the distributional patterns of Southeast-EastAsian freshwater fish and their historyrdquo Journal of Biogeographyvol 29 no 9 pp 1187ndash1199 2002

[5] S K J McConnell ldquoMapping aquatic faunal exchanges acrossthe Sunda shelf South-East Asia using distributional andgenetic data sets from the cyprinid fish Barbodes gonionotus(Bleeker 1850)rdquo Journal of Natural History vol 38 no 5 pp651ndash670 2004

[6] T Mo Anatomy Relationships and Systematics of the Bagridae(Teleostei Siluroidei) with a Hypothesis of Siluroid Phylogenyvol 17 ofTheses Zoologicae Koeltz Scientific Books 1991

[7] H H Ng and M Kottelat ldquoRevision of the Asian catfish genusHemibagrus bleeker 1862 (Teleostei Siluriformes Bagridae)rdquoRaffles Bulletin of Zoology vol 61 no 1 pp 205ndash291 2013

[8] P K L Ng and H H Ng ldquoHemibagrus gracilis a new speciesof large riverine catfish (Teleostei Bagridae) from PeninsularMalaysiardquo Raffles Bulletin of Zoology vol 43 pp 133ndash142 1995

[9] H H Tan and M Kottelat ldquoRedescription of Betta picta(Teleostei Osphronemidae) and description of Betta falx sp nfrom central Sumatrardquo Revue Suisse de Zoologie vol 105 no 3pp 557ndash568 1998

[10] J J Dodson F Colombani and P K Ng ldquoPhylogeographicstructure in mitochondrial DNA of a South-east Asian fresh-water fish Hemibagrus nemurus (Siluroidei Bagridae) andPleistocene sea-level changes on the Sunda shelfrdquo MolecularEcology vol 4 no 3 pp 331ndash346 1995

[11] H H Ng and J J Dodson ldquoMorphological and genetic descrip-tions of a new species of catfish Hemibagrus chrysops fromSarawak East Malaysia with an assessment of phylogeneticrelationships (Teleostei Bagridae)rdquo Raffles Bulletin of Zoologyvol 47 no 1 pp 45ndash57 1999

[12] P D N Hebert A Cywinska S L Ball and J R DeWaardldquoBiological identifications through DNA barcodesrdquo Proceedingsof the Royal Society B Biological Sciences vol 270 no 1512 pp313ndash321 2003

[13] J C Carolan T EMurray U Fitzpatrick et al ldquoColour patternsdo not diagnose species quantitative evaluation of a DNAbarcoded cryptic bumblebee complexrdquo PLoS ONE vol 7 no 1Article ID e29251 2012

[14] M Kekkonen and P D N Hebert ldquoDNA barcode-baseddelineation of putative species efficient start for taxonomicworkflowsrdquoMolecular Ecology Resources vol 14 no 4 pp 706ndash715 2014

[15] DM Hillis CMoritz and B KMarbleMolecular SystematicsSinauer Sunderland Mass USA 2nd edition 1996

[16] T D Kocher W K Thomas A Meyer et al ldquoDynamics ofmitochondrial DNA evolution in animals amplification andsequencingwith conserved primersrdquoProceedings of theNationalAcademy of Sciences of the United States of America vol 86 no16 pp 6196ndash6200 1989

[17] T A Hall ldquoBioEdit a user-friendly biological sequence align-ment editor and analysis program for Windows 9598NTrdquoNucleic Acids Symposium Series vol 41 pp 95ndash98 1999

[18] D A Benson I Karsch-Mizrachi K Clark D J Lipman JOstell and EW Sayers ldquoGenBankrdquoNucleic Acids Research vol40 no D1 pp D48ndashD53 2012

[19] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[20] X Ku Z Peng R Diogo and S He ldquoMtDNA phylogenyprovides evidence of generic polyphyleticism for East Asianbagrid catfishesrdquoHydrobiologia vol 579 no 1 pp 147ndash159 2007

[21] L Yang and S He ldquoPhylogeography of the freshwater catfishHemibagrus guttatus (Siluriformes Bagridae) implications forSouth China biogeography and influence of sea-level changesrdquoMolecular Phylogenetics and Evolution vol 49 no 1 pp 393ndash398 2008

[22] B W Bowen A L Bass L A Rocha W S Grant and DR Robertson ldquoPhylogeography of the trumpetfishes (Aulosto-mus) ring species complex on a global scalerdquo Evolution vol 55no 5 pp 1029ndash1039 2001

[23] Y P Kartavtsev ldquoDivergence at Cyt-b and Co-1 mtDNA geneson different taxonomic levels and genetics of speciation inanimalsrdquoMitochondrial DNA vol 22 no 3 pp 55ndash65 2011

[24] SWada Y Kameda and S Chiba ldquoLong-term stasis and short-term divergence in the phenotypes of microsnails on oceanicislandsrdquoMolecular Ecology vol 22 no 18 pp 4801ndash4810 2013

[25] J Colborn R E Crabtree J B Shaklee E Pfeiler and B WBowen ldquoThe evolutionary enigma of bonefishes (Albula spp)cryptic species and ancient separations in a globally distributedshorefishrdquo Evolution vol 55 no 4 pp 807ndash820 2001

[26] J-D Durand K-N Shen W-J Chen et al ldquoSystematics of thegrey mullets (Teleostei Mugiliformes Mugilidae) molecularphylogenetic evidence challenges two centuries ofmorphology-based taxonomyrdquo Molecular Phylogenetics and Evolution vol64 no 1 pp 73ndash92 2012

[27] K-N Shen B W Jamandre C-C Hsu W-N Tzeng and J-D Durand ldquoPlio-Pleistocene sea level and temperature fluc-tuations in the northwestern Pacific promoted speciation inthe globally-distributed flathead mullet Mugil cephalusrdquo BMCEvolutionary Biology vol 11 no 1 article 83 2011

[28] R Hall ldquoThe plate tectonics of Cenozoic SE Asia and thedistribution of land and seardquo in Biogeography and GeologicalEvolution of SE Asia R Hall and J D Holloway Eds pp 99ndash131Backhuys Publishers Leiden The Netherlands 1998

[29] R Hall ldquoSoutheast Asiarsquos changing palaeogeographyrdquo Blumeavol 54 no 1ndash3 pp 148ndash161 2009

[30] R F Inger and P K Chen ldquoThe freshwater fishes of NorthBorneordquo Fieldiana Zoology vol 45 pp 1ndash268 1962

[31] J Bohlen V Slechtova H H Tan and R Britz ldquoPhylogenyof the Southeast Asian freshwater fish genus Pangio (Cyprini-formes Cobitidae)rdquoMolecular Phylogenetics and Evolution vol61 no 3 pp 854ndash865 2011

[32] M de Bruyn E Nugroho M M Hossain J C Wilson andP B Mather ldquoPhylogeographic evidence for the existence ofan ancient biogeographic barrier the Isthmus of Kra SeawayrdquoHeredity vol 94 no 3 pp 370ndash378 2005

[33] J Parnell ldquoThe biogeography of the Isthmus of Kra region areviewrdquoNordic Journal of Botany vol 31 no 1 pp 001ndash015 2013

[34] S Usmani S G Tan S S Siraj and K Yusoff ldquoPopulationstructure of the Southeast Asian river catfishMystus nemurusrdquoAnimal Genetics vol 34 no 6 pp 462ndash464 2003

[35] M P Tan A F J Jamsari and M N Siti Azizah ldquoPhylo-geographic pattern of the striped snakehead Channa striatain Sundaland ancient river connectivity geographical and

Advances in Evolutionary Biology 21

anthropogenic singnaturesrdquo PLoS ONE vol 7 no 12 Article IDe52089 2012

[36] R Abell M L Thieme C Revenga et al ldquoFreshwater ecore-gions of the world a new map of biogeographic units forfreshwater biodiversity conservationrdquo BioScience vol 58 no 5pp 403ndash414 2008

[37] P U Clark D Archer D Pollard et al ldquoThemiddle Pleistocenetransition characteristics mechanisms and implications forlong-term changes in atmospheric pCO

2rdquo Quaternary Science

Reviews vol 25 no 23-24 pp 3150ndash3184 2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 3: ResearchArticle - Université Laval Julian... · ResearchArticle A DNA Barcode-Based Evaluation of the Southeast Asian Catfish Genus Hemibagrus Bleeker, 1862 (Teleostei: Siluriformes;

Advances in Evolutionary Biology 3

Table 1 Rivers river code (as presented in Figure 1) sampling sites latitudelongitude and distance upstream from riverrsquos mouth of samplingsites and the number of specimens of Hemibagrus spp sequenced

River code Site code River Sample site Latituded∘ mm1015840 ss10158401015840

Longituded∘ mm1015840 ss10158401015840

Distance upstreamfrom river mouth

(km)

Number ofspecimens

1a 1 Mekong Bungkan 18 21 54 N 103 39 04 E 1285 41b 2 Mekong Bannan 17 58 00 N 104 14 00 E 1200 21c 3 Mekong That Phanom 17 24 00 N 104 48 00 E 1100 21d 4 Mekong Warinchamrap 15 12 00 N 104 53 00 E 885 31e 5 Mekong Mun River 15 17 00 N 105 04 00 E 800 131f 6 Mekong Phibum 15 14 00 N 105 14 00 E 845 41g 7 Mekong Nomh Penh 11 33 00 N 104 55 00 E 250 42a 8 Chao Phraya Market 14 35 00 N 100 27 00 E 100 82b 9 Chao Phraya Nakhom Sawan 15 41 00 N 100 07 00 E 275 53a 10 Chanthaburi 12 48 00 N 102 09 00 E 60 64a 11 Mea Khlong Kanchanaburi 14 01 00 N 99 32 00 E 100 55a 12 Thai Pen Phatthalung 7 37 00 N 100 05 00 E 90 16a 13 Kelantan 6 00 00 N 102 10 00 E 50 127a 14 Terengganu Kuala Berang 5 04 00 N 103 01 00 E 40 147b 15 Terengganu Sekayu 4 58 00 N 103 01 00 E 80 3

8a 16 Pahang KampongMengkarak 3 19 00 N 102 27 00 E 100 9

9a 17 Endau Park 2 40 00 N 103 15 00 E 100 69b 18 Endau Kahang 2 18 00 N 103 35 00 E 50 19c 19 Endau Ambat 2 11 00 N 103 51 00 E 70 29d 20 Endau Mersing 2 25 00 N 103 56 00 E 5 1010a 21 Johor Kota Tingi 1 4 00 N 103 54 00 E 45 311a 22 Perak 5 12 00 N 101 02 00 E 180 1311b 23 Perak Chenderoh 4 58 00 N 100 57 00 E 160 611c 24 Perak Gerik 5 25 00 N 101 08 00 E 200 1212a 25 Bernam Sabak 3 46 00 N 100 59 00 E 50 113a 26 Muar Selabu 2 9 00 N 102 36 00 E 20 613b 27 Muar Market 2 14 00 N 102 46 00 E 40 413c 28 Muar Kundang Ulu 2 14 00 N 102 46 00 E 40 213d 29 Muar Panchor 2 10 00 N 102 43 00 E 35 414a 30 Batang Hari Jambi 1 36 00 S 103 37 00 E 100 2714b 31 Batang Hari Kerinci 2 05 00 S 101 23 00 E 800 715a 32 Musi Palembang 2 55 00 S 103 37 00 E 70 1516a 33 Ci Liwong Sawagangan 6 24 00 S 106 46 00 E 45 217a 34 Ci Sadane Bogor 6 35 00 S 106 47 00 E 60 118a 35 Ci Tarum Cirata 6 33 00 S 107 18 00 E 90 1819a 36 Ci Manuk Garut 7 13 00 S 107 54 00 E 150 1420a 37 Kali Progo Mayudan 7 45 00 S 110 11 00 E 30 221a 38 Brantas River Surabaya 7 28 00 S 112 26 00 E 50 622a 39 Mahakam Samarinda 0 24 00 S 116 58 00 E 50 1023a 40 Barito Banjarmassin 3 5 00 S 114 38 00 E 50 924a 41 Kapuas Pontianak 0 17 00 S 109 19 00 E 20 1224b 42 Kapuas Sanggau 0 08 00 N 110 36 00 E 120 224c 43 Kapuas Nanga Pinoh 0 20 00 S 111 44 00 E 320 3

4 Advances in Evolutionary Biology

Table 1 Continued

River code Site code River Sample site Latituded∘ mm1015840 ss10158401015840

Longituded∘ mm1015840 ss10158401015840

Distance upstreamfrom river mouth

(km)

Number ofspecimens

24d 44 Kapuas Sintang 0 04 00 N 111 30 00 E 270 324e 45 Kapuas Danau Sentarum 0 51 00 N 112 6 00 E 550 224f 46 Kapuas Putussibau 0 50 00 N 112 56 00 E 600 224g 47 Kapuas Sibau 0 55 00 N 112 57 00 E 750 224h 48 Kapuas Mendalam 1 00 00 N 113 16 00 E 800 625a 49 Sadong Serian 1 10 00 N 110 34 00 E 50 2525b 50 Sadong Balai Ringin 1 03 00 N 110 45 00 E 70 1126a 51 Rajang Kapit 2 01 00 N 112 56 00 E 210 726b 52 Rajang Sibu 2 18 00 N 111 49 00 E 80 1026c 53 Rajang Katibas 1 43 00 N 112 33 00 E 170 1227a 54 Baram Mulu 3 30 00 N 115 30 00 E 380 327b 55 Baram Bakung 4 17 00 N 114 14 00 E 60 627c 56 Baram Marudi 4 11 00 N 114 19 00 E 80 1528a 57 Tutong Merimbun 4 36 00 N 114 40 00 E 40 329a 58 Belait Ingei 4 10 00 N 114 43 00 E 100 330a 59 Padas Tenom 5 08 00 N 115 57 00 E 70 931a 60 Segaliud Sandakan 5 45 00 N 117 52 00 E 50 132a 61 Segama Danum Valley 5 01 00 N 117 47 00 E 170 1632b 62 Segama Lahad Datu 5 02 00 N 118 19 00 E 90 633a 63 Kinabatangan Batu Putih 5 25 00 N 117 55 00 E 100 634a 64 Umas-Umas Tawau 4 22 00 N 117 44 00 E 15 235 65 Salween Myanmar mdash mdash mdash 236 66 Red Hanoi mdash mdash mdash 237 67 Pearl Guangdong mdash mdash mdash 8

Total 452mdash exact origin unknown

amplified in a programmable thermal cycler using the follow-ing profile one single preliminary denaturation at 95∘C for5min 40 cycles of amplification with denaturation at 94∘Cfor 60 s primer annealing at 45∘C for 60 s primer extensionat 72∘C for 90 s and a final single extension step performed at72∘C for 5min Total 50 120583L PCR products were purified on a2 agarose gel After electrophoresis at 90V for 2 hours theamplified fragments were excised from the gel and extractedand purified using the QIAquick Gel Extraction Kit Protocol(using a microcentrifuge) fromQIAGEN to a final volume of15 to 50120583L (in EB buffer 10mMTris Cl pH 75)

Double stranded purified PCR products were sequencedin both directions (complementary strand was sequenced inall cases) using the ABI PRISM BigDye Terminator CycleSequencing Ready Reaction Kit (Applied Biosystems Inc)Sequencing reactions were carried out in 20120583L total volumecontaining 32 picomoles of the sequencing primer 30 to60 ng of purified PCR product 6 120583L of 25X dilution buffer(CSA seq Buffer Perkin Elmer) and 2 120583L of ABI PRISMBigDye Terminator Ready Reaction Mix Reactions wereperformed in thermal cycler GeneAmp PCR system 9600using the following profile 10 s at 96∘C 5 s at 50∘C and

4min at 60∘C for 25 cycles Sequences were separated byelectrophoresis at 1680V (150W 500mA) for 5 to 7 h on ABIPRISM 377 DNA Sequencer (Applied Biosystems Inc)

Sequences were extracted from the gel image using ABIPRISM Sequencing Analysis 30 program The sequences ofthe complementary strands were then imported in SequenceNavigator program for comparative alignment and correctionof ambiguities (Applied Biosystems Inc) Sequences werealigned using the BioEdit sequence alignment editor [17]Each sequence was identified by a unique code designatingthe origin of the sample (river name) the cumulativenumber of the sequences found in that river and thenumber of individuals characterized by that particularsequence For example the code MEK1502 designates the15th sequence found in the Mekong River and occurring intwo specimens Following the comparison of morphologicaland genetic designations mtDNA codes were preceded bythe putative species designation (eg SPMEK1502 indicatesthat specimens bearing sequence MEK1502 were assignedto Hemibagrus spilopterus) Sequences found in multiplerivers were identified by river name and the total number ofspecimens Sequences were deposited in GenBank [18] with

Advances in Evolutionary Biology 5

(1) Mekong(2) Chao Phraya(3) Chanthaburi(4) Mea Khlong(5) Phatthalung(6) Kelatan(7) Terengannu(8) Pahang(9) Endau(10) Johor(11) Perak(12) Bernam

(13) Muar(14) Batang Hari(15) Musi(16) Ci Liwong(17) Ci Sadane(18) Ci Tarum(19) Ci Manuk(20) Kali Progo(21) Brantas(22) Mahakam(23) Barito(24) Kapuas(25) Sadong(26) Rajang

(27) Baram(28) Tutong(29) Belait(30) Padas(31) Segaliud(32) Segama(33) Kinabatangan(34) Umas-Umas(35) Salween(36) Red(37) Pearl

0 250 500 750

(km)

0 250 500

(mi)100

∘110

∘120

0∘

10∘

Siam RiverNor

th Su

nda R

iver

East Sunda River

35 36

37

1

5

11

12

17

8 9

10

14

16

17

18

19

25

24

23

20

21

22

27

26

28

29 3031

32

33

34

15

6

13

2

3

4

Shelf minus75mShelf minus120 m

Figure 1 Location map of Southeast Asia illustrating the 37 rivers sampled in this study Black dots along rivers represent the sampling sitesas identified in Table 1 and Table S1 Dark grey shading illustrates the extent of the emerging Sunda Shelf at sea levels 75m below present daylevels light grey denotes emerging shelf at sea levels 120m below present day levels (as illustrated by Voris 2000) Rivers illustrated on theSunda Shelf represent drowned Pleistocene river drainage systems according to Voris (2000)

accession numbers corresponding to sequence codes andreported in Supplementary Material Table S1

Data Analysis We first used the Neighbor-Joining (NJ)algorithm in MEGA 52 (Tamura et al 2011) to develop ataxon identification phenogram ofHemibagrusThe Tamura-Nei distance matrix considering invariable site contribution

(+119868) was selected according to the model selection algo-rithm implemented in MEGA 52 [19] The rate variationamong sites was modeled with a gamma distribution (shapeparameter = 1) Recognizing the limitations of single-locusanalyses in phylogeny reconstruction and the objectionsof cladists to distance-based methodology we neverthelesssought to provide a tentative evaluation of phylogenetic

6 Advances in Evolutionary Biology

relationships among taxa To do so the Cyt 119887 sequence of theChinese longsnout catfish (Leiocassis longirostris) obtainedfrom GenBank (accession number DQ3217551) was usedas an outgroup to root the phenogram Chinese speciescurrently assigned to Leiocassismay be considered membersof the genus Pseudobagrus [20] Furthermore we employeda maximum likelihood (ML) phylogenetic reconstructionbased on the comparison of nucleotides within sites to assessthe congruence of the taxon identification phenogram andthe ML phylogenetic reconstruction In the latter case theevolutionary model used was the Hasegawa-Kishino-Yanomodel (HKY)with gammadistribution (+119866 = 13290) Initialtree(s) for the heuristic search were obtained by applyingthe Neighbor-Joining (NJ) method to a matrix of pairwisedistances estimated using the maximum composite likeli-hood (MCL) approach The rate-variation model allowedfor some sites to be evolutionarily invariable (+119868 4644of sites) The ML tree was rooted with the Cyt 119887 sequenceof the Chinese longsnout catfish (Leiocassis longirostris) andconstructed using MEGA52 [19]

TheNJ phenogram classifiedmtDNA sequences based onoverall similarity Ng and Kottelat [7] also defined speciesgroups based on overall similarity but according to mor-phological criteria Of the 8 species groups defined by Ngand Kottelat [7] two groups (H olyroides (1 species) and Hpluriradiatus (5 species)) were not included in our samplesWe thus evaluated the congruence of the two methods indefining the remaining six species groups (H baramensisH guttatus H menoda H nemurus H planiceps and Hwyckii species groups) When possible mtDNA sequenceswere linked to ZRC accession numbers identifying specimensexamined by Ng and Kottelat [7] in their morphologicalrevision of the genus (Table S1)

To provide time estimates for the major phylogeneticevents we present a calibrated linearized tree obtained fromour ML reconstruction (MEGA 52) Time of divergencebetween Hemibagrus lineages has been approximated byapplying a rate of between 10 and 15 genetic divergence permillion years as reported for bagrid fish by Yang and He [21]However the justification for this rate is not clear We thusapplied the more widely used rate of 2 sequence divergenceper million years [22] As there is undoubtedly a wide errorassociated with any such molecular clock calibrations esti-mated divergence times are used to discriminate more recentfrom more ancient cladogenetic events while providing veryapproximate dates for such events

Genetic divergence estimates using 119901-distances (119901) wereused in the present study to identify the different taxonomiclevels observed among the Hemibagrus spp analyzed Sincesuch information is readily available for thousands of animalgroups [23] we used 119901-distance (119901) as a benchmark for taxo-nomic levelsThe survey of 20 731 vertebrate and invertebratespecies reported by Kartavtsev [23] revealed increasing levelsof 119901-distances () of the mtDNA Cyt 119887 gene with taxonomiclevel populations within species 119901 = 138 plusmn 030 (SD)subspecies or sibling species119901 = 510plusmn091 morphologicallydistinct species within genera 119901 = 1031 plusmn 093 generawithin a family 119901 = 1786 plusmn 136 and families within anorder 119901 = 2636 plusmn 388 Although heterogeneity in the

gene evolution rate differs amongdifferent taxonomic groupsthese estimates provide an order of magnitude evaluationof intraspecific versus interspecific divergence and permittedus to discriminate between population- and species-leveldivergences However as 119901-distances underestimate truegenetic distance (eg site saturation and back-mutation)divergence times based on119901-distances greater than about 10are generally underestimated We calculated 119901-distances inMEGA 52 and report them as percentages (standard error)

3 Results

The 452 samples yielded a total of 118 distinct sequences(not including the outgroup taxon) The Neighbor-Joiningphenogram clustered the 119 sequences into a large numberof hierarchically organized monophyletic clades To facilitatepresentation we first present a compressed NJ tree andidentify 11 major mtDNA clades (Figure 2) One unidentifiedfish from the Rajang River was characterized by a genotypethat clustered with the outgroup taxon (XXRAJ0604GenBank accession number KP322872 Figures 2 and3) A second fish from the Rajang was characterized byan isolated genotype (XXRAJ0301 GenBank accessionnumber KP322869) of unknown affiliation (Figures 2and 3) To assess the congruence of the species groupsdefined by Ng and Kottelat ([7] hereafter referred toas NampK) and the mtDNA groupings revealed by thepresent analysis we provide a direct comparison of thetwo methods following the order determined by the taxonidentification phenogram (Figure 2 Table S1) Table S1 alsoprovides additional information about the geographicaldistribution of sequences Gene trees of individual cladesincluding all sequences are presented at each step in theanalysis along with their geographical distributions We alsopresent a compressed maximum likelihood phylogenetictree (Figure 3) The phenogram and the ML tree providedcongruent phylogenetic reconstructions However both treesfailed to resolve the phylogenetic relationships of H wyckiiH guttatus and H menoda species groups most likelydue to undersampling of these lineages The relationshipsamong an unspecified species group H spilopterus and Hfortis are also not well resolved Their position in the NJtree receives weak bootstrap support and they appear as amultifurcation along with a clade composed of H capitulumand H hoevenii in the ML phylogenetic tree (Figures 2 and3) To facilitate comparisons with the taxonomic scheme ofNampK we follow the sequence of taxa as identified in Figure 2

mtDNAClade 1The H wyckii Species GroupThe first clade isclearly consistent with theH wyckii species group (Figure 4)This species group is composed of 4 valid speciesHmaydelliH microphthalmus H wyckii andH wyckioides (NampK)Thelatter species is known from the Mekong and Chao Phrayadrainage Only specimens of H wyckioides obtained fromthe Mekong and the Chao Phraya were available for DNAanalysis Three specimens from the Chao Phraya bearingsequence WYCHP0203 (Table S1) were identified as Hwyckioides (H H Ng personal communication) although

Advances in Evolutionary Biology 7

8811

10

9

8

7

6

5

4

3

1

2

66

85

44

32

98

91

81

8432

57

99

99

99

49

002

34

15

43

86

99

96

H capitulum (Indochinese clade)

H capitulum (Sundaic clade)

H hoeveniiH fortis

H spilopterus

H baramensis species group

H planiceps species groupH menoda species group

H guttatus species group

H wyckii species group

XXRAJ0604

PESAL0102

XXRAJ0301Leiocassis longirostris

mtDNAclade

H nemurusspecies group

Unspecified species group

Figure 2 The taxon identification phenogram of Hemibagrus inferred using the Neighbor-Joining method in MEGA 52 The optimal treewith the sumof branch length = 186668598 is shown Bootstrap values are indicated on each node of the tree (500 replicates) Bar indicates thenumber of substitutions changes estimated using Tamura-Nei distance considering invariable site contribution (+I)The analysis involved 119nucleotide sequences All positions containing gaps and missing data were eliminatedThere were a total of 597 positions in the final dataset

mtDNAclade

H capitulum (Indochinese clade) 11

93

97H capitulum (Sundaic clade)H hoevenii

9999

84H fortis 8

9

10

H spilopterus 773 92

Unspecified species group 678

XXRAJ060454 34

90 H baramensis species group 5

49 H planiceps species group 4

H menoda species groupPESAL0102 3

H wyckii species group 199

99

99H guttatus species group 2

XXRAJ030149

47

Leiocassis longirostris

005

H nemurusspecies group

94

72

Figure 3 Evolutionary history of Hemibagrus inferred by using the maximum likelihood method based on the Hasegawa-Kishino-YanomodelThe tree with the highest log likelihood (minus56774825) is shown Bootstrap values are indicated on each node of the tree (500 replicates)Bar indicates the number of substitution changes estimated using Hasegawa-Kishino-Yano model (HKY) with gamma distribution (+119866 =13290) The analysis involved 119 nucleotide sequences All positions containing gaps and missing data were eliminated There were a totalof 597 positions in the final dataset

their ZRC numbers do not appear in the analysis of NampK Asno specimens of the other 3 species comprising the H wyckiispecies group were available for analysis we cannot confirmif this species group is monophyletic

mtDNA Clade 2 The H guttatus Species Group The secondclade of the Hemibagrus gene tree (Figure 2) is composed oftwo reciprocally monophyletic subclades clearly reflectingthe H guttatus species group as defined by NampK (Figure 4)

8 Advances in Evolutionary Biology

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500

750(km)

(mi)

002

98

43

84

34

99

99

79

96

51

59

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78

49

H peguensis

H guttatus

H macropterus

H wyckioides

PESAL0102

XXRAJ0301Leiocassis longirostris

MAPEA0201MAPEA0302MAPEA0101

GUPEA0501GURED0102

GUPEA0401GUPEA0602

WYCHP0401WYMEK1101

WYCHP0203

See Figures 7ndash12

See Figures 5 and 6

Shelf minus75mShelf minus120 m

Figure 4 Phylogenetic relationships of the individual sequences comprising four species H guttatus H macropterus (together forming theH guttatus species group) H wyckioides and H peguensisThe subtree is extracted from the taxon identification phenogram inferred usingthe Neighbor-Joining method (see Figure 2) The percentage of replicate trees in which the associated sequences clustered together in thebootstrap test (500 replicates) is shown next to the branches The distribution of these sequences is illustrated in the right panel

The group comprises three valid species H guttatus Hmacropterus andH vietnamicus (NampK) Specimens availablefor DNA analysis were obtained from the Pearl River andRed River (11 fish 7 sequences Table 1 and Table S1) Thetwo reciprocally monophyletic subclades comprising theH guttatus species group represent H guttatus and Hmacropterus (Figure 4) Although no ZRC specimens wereexamined by NampK species identification was confirmed(H H Ng personal communication) No H vietnamicusspecimens were available for DNA analysis The twospecies composing the group are closely related (119901 = 51(SE = 08))

mtDNA ldquoCladerdquo 3 The H menoda Species Group Thisisolated genotype (Figure 4) most likely identifies a cladeencompassing the H menoda species group although the

single sequence (Table S1) is shared by only 2 specimensof H peguensis from the Salween River Myanmar alsoincluded in the analysis of NampK (where its provenance wasidentified as the neighboring Irrawaddy River) The otherspecies comprising this group (H menoda H caveatusand H punctatus) were not sampled for DNA analysis Themonophyletic nature of this species group thus remains to bedemonstrated

31 The H planiceps + H baramensis Group The remainderof the gene tree is split into two large sister groups withmarginal bootstrap support (73 in ML and 43 in NJ)the H planiceps + H baramensis group and the H nemurusspecies group (Figures 2 and 3) The H planiceps + Hbaramensis group is strongly supported in both NJ and MLanalyses (84 and 90 bootstrap support respectively

Advances in Evolutionary Biology 9

110∘

47

53

69

81

69

5199

52

95

97

78

001

H bongan

H velox

H gracilis

H planiceps

H divaricatus

BOKAP1801

BOKAP1601BOKAP1701

BOKAP1402BOKAP1503

VEBAH0907GREND0206

PLCIM0114PLCIS0101

DIPER0401DIPER0301

DIPER0201

100∘

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0

0 250 500

250 500

750(km)

(mi)

Shelf minus75mShelf minus120 m

Figure 5 Phylogenetic relationships of the individual sequences and species comprising theH planiceps species groupH bongan H veloxH gracilis H planiceps and H divaricatus The subtree is extracted from the taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicate trees in which the associated sequences clustered together in the bootstrap test(500 replicates) is shown next to the branches The distribution of these sequences is illustrated in the right panel

Figures 2 and 3) The two groups are genetically similarwith a genetic divergence estimate of 119901 = 121 (SE = 09)Although theH planiceps species group is strongly supportedin both trees (99) bootstrap support of the H baramensisspecies group is weak (Figure 2 32 Figure 3 34) Thegeographical distribution of the two groups differs Membersof the H planiceps species group are found throughout theGreater Sunda Islands and Peninsular Malaysia but they areisolated in the upper reaches of large rivers (Figure 5) Incontrast members of theH baramensis species group appearto be restricted to Sabah North Borneo (Figure 6)

mtDNA Clade 4 The H planiceps Species Group The Hplaniceps species group (Figure 5) is comprised of 5 closelyrelated but genetically distinct species (mean within-groupdivergence 119901 = 20 (SD = 033) H planiceps (Java)H gracilis (eastern Peninsular Malaysia) H divaricatus(western Peninsular Malaysia) H bongan (Borneo) andH velox (Sumatra)) H lacustrinus the sixth species of thisgroup (NampK) was not sampled in the present study WithintheH planiceps species groupH planiceps formed a distinctspecies (Figure 5) comprising two sequences One sequence(PLCIM0114 Table S1) characterized 14 specimens obtainedfrom Ci Manuk West Java 9 of which were identified as Hplaniceps by NampK (Table S1) A second sequence (PLCIS0101

Table S1) was obtained from one specimen sampled atCi Sadane West Java that we classified as H planicepsH bongan is represented by 5 sequences obtained from 8specimens sampled in the Kapuas River (Figure 5) Two ofthese specimens (ZRC 43013 and 43015) were identified asH bongan by NampK (Table S1) H divaricatus is composedof 3 sequences from three specimens obtained at Gerik onthe Perak River (western Peninsular Malaysia) One of thesespecimens (ZRC 41151) is the holotype of this new species(NampK) H gracilis is represented by only one sequence(GREND0206 Table S1) shared by 6 specimens sampledat Ulu Endau Jasin River eastern Peninsular Malaysiafour of which were identified by NampK (Table S1) ZRC21484 comprises the holotype and ZRC 21482 21486 and21487 comprise the paratypes (NampK) of this species (TableS1) Finally H velox is represented by only one sequence(VEBAH0907) from 7 specimens sampled at Kerinci BatangHari River Sumatra Three of these specimens (ZRC 41503Table S1) represent 3 paratypes of the species (NampK)

mtDNA Clade 5 The H baramensis Species Group The threespecies comprising the H baramensis species group (Figures2 and 3) form three monophyletic subclades (mean within-group divergence 119901 = 106 (08) Figure 6) H semotus iscomposed of two sequences (SEPAD0101 SEPAD0202) that

10 Advances in Evolutionary Biology

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002

H sabanus

H semotus

H baramensis

SAKIN0204SASGM0706

BASGM0402BASGM0301

BASGM0201BASGM0104

SESGM0502SEUMA0101

SEPAD0202SEPAD0101

See Figure 5

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0 250 500

250 500

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(mi)

99

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83

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72

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56

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Shelf minus75mShelf minus120 m

Figure 6 Phylogenetic relationships of the individual sequences and three species comprising the H baramensis species group H semotusH sabanus and H baramensis (furcatus) The subtree is extracted from the taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicate trees in which the associated sequences clustered together in the bootstrap test(500 replicates) is shown next to the branches The distribution of these sequences is illustrated in the right panel

characterize fish that are the holotype and paratypes of Hsemotus (ZRC 46122 Table S1) along with two somewhatmore divergent sequences (Figure 6) One of these sequences(SGM0502) was found in the Segama River (Danum Valley)and identified as H baramensis by NampK (ZRC 40525)(Table S1) The second sequence (UMA0101) was from afish sampled at Tawau Umas Umas River (ZRC 46123) alsoidentified as H baramensis by NampK These sequences mostlikely reflect distinct lineages within H semotus inhabitingdifferent rivers as the mean intraspecies sequence divergence(119901 = 16) indicates population-level divergence

H sabanus and H furcatus (synonymized with H bara-mensis by NampK) cluster together to form a distinct subcladebut clearly diverge at the species level (Figure 6) H sabanuscomprises 2 sequences that are shared respectively by 6specimens (ZRC 42741) from the Segama River Sabah and4 specimens (part of ZRC 42743) from the KinabatanganRiver Sabah (Table S1) All of these specimenswere identifiedas H sabanus by NampK (Table S1) Finally H baramensisforms a distant subclade (147 mean sequence divergencebetween H baramensis and H sabanus) composed of 4sequences (Figure 6) shared by 8 specimens all sampled inthe Segama River Danum Valley Conservation Area SabahSix of these specimens (ZRC 40523 (2 specimens) ZRC40527 (2 specimens) and ZRC 40526 (2 specimens) Table S1)comprise the paratypes of H baramensis (furcatus) (NampK)

32 The H nemurus Species Group In both NJ and ML trees(Figures 2 and 3) the H nemurus species group is strongly

supported (bootstrap support 98 in NJ and 99 in ML)indicating that it forms a distinct and widespread geneticentity within the genus Hemibagrus However relationshipswithin the group are not so clear In general the H nemurusspecies group is composed of 4 clades The first clade may beconsidered as a species group nested within the H nemurusspecies group (85 and 93 bootstrap support in NJ andML resp) encompassing three clades H hoevenii (clade9) the H capitulum Sundaic clade (clade 10 including Hnemurus) and the H capitulum Indochinese clade (clade 11)(Figures 2 and 3) An additional three clades (Figures 2 and3) are formed by an unidentified species group (clade 6) Hspilopterus (clade 7) andH fortis (clade 8)These three latterclades are distinct at the congeneric species level (unspecifiedversus H spilopterus 119901 = 77 (07) unspecified versus Hfortis 119901 = 85 (07) and H spilopterus versus H fortis119901 = 75 (08)) The distribution of these three clades issomewhat disparate H fortis and the unspecified speciesgroup are principally found in Borneo whereasH spilopterusis widely distributed on the Indochinese mainland

mtDNA Clade 6 Unspecified Species Group This geneticallydiverse group encompassing at least 3 subclades (H spp AB and C Figure 7 within-group mean sequence divergence119901 = 68(06)) is confoundedwith theH baramensis speciesgroup byNampK It is composed of sequences fromfish sampledin Sarawak Sabah and eastern PeninsularMalaysia Subclade119867 spp A (Figure 7) is particularly interesting as it contains2 sequences (CHSARA20 CHRAJ0401) that characterize

Advances in Evolutionary Biology 11

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XXPAD0303XXPAD0601

XXPAD0401XXBEL0202

XXBRM0202XXRAJ0511

XXPAD0501

XXEND0410CHSARA20CHRAJ0401

XXBEL0101XXSEG0101

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91

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H spp C

H spp B

H spp A

Shelf minus75mShelf minus120 m

Figure 7 Phylogenetic relationships of the individual sequences comprising the unspecified species group containing three subcladesidentified as H spp A B and C The subtree is extracted from the taxon identification phenogram inferred using the Neighbor-Joiningmethod (see Figure 2) The percentage of replicate trees in which the associated sequences clustered together in the bootstrap test (500replicates) is shown next to the branches The distribution of these sequences is illustrated in the right panel

H chrysops a new species identified by Ng and Dodson [11]However specimens of H chrysops from the Rajang Riverwere subsequently synonymized by NampK with H capitulumof the H nemurus species group (ZRC 23008ndash23014 TableS1) However an additional 14 fish from the Sadong Riverat Serian bearing genotype SARA20 (Table S1) and thusgenetically identical to the Rajang H chrysops samples wereidentified by NampK as H spilopterus (ZRC 29583ndash96 TableS1) These specimens however were erroneously identifiedin NampK as originating in the Chao Phraya River (Thailand)Subclade A also contains one sequence obtained from 10 fishsampled on the Endau River (Mersing) in eastern PeninsularMalaysia These fish were not morphologically examinedFinally subclade A also contains one sequence from oneunidentified fish sampled in the Segaliud River Sabah andanother unidentified fish sampled in the Belait River BruneiSubclade H spp B encompasses 2 sequences one of whichcomes from a fish sampled in the Padas River (XXPAD0501)and part of the ZRC 46122 fish lot identified as H semotusby NampK (Table S1) The second sequence (XXRAJ0511) wasshared by 11 unidentified fish sampled at Katibas on theRajang River Sarawak (Table S1) Finally subclade 119867 sppC encompasses 5 sequences Three sequences (XXPAD0303XXPAD0401 and XXPAD0601) were obtained from thePadas River and identified byNampKasH semotus (ZRC46122Table S1) The fourth sequence (XXBRM0202) was obtainedfrom 2 fish sampled in the Baram River and was identified

as H baramensis by NampK (ZRC 42742) The 5th sequencecomposing this group was obtained from 2 unidentified fishsampled in the Belait River Brunei (Table S1)

mtDNA clade 6 is thus problematical within the contextof the revision of the genus Hemibagrus Two highlydivergent mtDNA clades (clade 5 and clade 6 Figures 2and 3) occur in sympatry principally in the Padas River ofSabah North Borneo (Figures 6 and 7) Specimens of theunspecified species group are morphologically similar tothe H baramensis species group where they are assigned inthe analysis of NampK However the mean genetic distancebetween the unspecified species group and theH baramensisspecies group is 119901 = 193 (28) close to genus leveldivergence Furthermore subclade 119867 spp A (Figure 7)contains not only H chrysops but also an unidentifiedpopulation from eastern Peninsular Malaysia (XXEND0410Table S1) and two additional Sabah sequences On theother hand the unspecified species group clusters with allother members of the H nemurus species group (meangenetic divergence of unspecified group versus H capitulum(Sundaic clade) 119901 = 76 (07) (Figure 2)) with strongbootstrap support Such genetic clustering may justify theinclusion of H chrysops and associated sequences in theH nemurus species group but its synonymization with Hcapitulum (NampK) is difficult to defend on genetic groundsWe tentatively conclude that H chrysops is a valid speciesbased on genetic identity [11] forming part of an unknown

12 Advances in Evolutionary Biology

110∘

SPCPCHMK12

SPMEK0202SPMEK0401

SPMEK0301SPMEK0103

SPMEK1401SPMEK0501

SPMEK1201SPMEK1301

SPMEK1601SPMEK1502

SPMEK0701SPMEK0801SPMEK1701

SPCHP0501SPCHA0201

SPCHA0304SPPAT0101

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(mi)

000499

99 98

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47

48

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7091

38

86

87

99

80

58

H spilopterus ldquoCrdquo

H spilopterus ldquoBrdquo

H spilopterus ldquoArdquo

Shelf minus75mShelf minus120 m

Figure 8 Phylogenetic relationships within H spilopterus comprised of 3 subclades identified as A B and C The subtree is extracted fromthe taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicate trees in whichthe associated sequences clustered together in the bootstrap test (500 replicates) is shown next to the branches The distribution of thesesequences is illustrated in the right panel

species group found throughout North Borneo (and toa lesser degree in eastern Peninsular Malaysia) that maybe considered as part of the H nemurus species groupHowever the morphological confusion of the unspecifiedspecies group with the sympatric highly divergent Hbaramensis species group suggests the existence of a crypticspecies assemblage (see Section 4)

mtDNA Clade 7 Clade 7 (Figures 2 and 3) is composed of atleast 3 distinct subclades that appear to reflect 3 geneticallydivergent populations ofH spilopterus Subclade A (Figure 8)encompasses 7 sequences obtained from 8 fish sampled inthe Mekong River Four fish bearing 3 of these sequenceswere identified as paratypes (ZRC 43324) of H spilopterusby NampK (Table S1) One additional fish was identified asH spilopterus by NampK (ZRC 46157 Table S1) Subclade Bencompasses 6 sequences obtained from 9 fish all sampledfrom the Mekong River (Table S1) The fish sampled at ThatPhanom (ZRC 46012) was identified as H spilopterus inthe analysis of HampK (Table S1) Subclade C encompasses 5sequences obtained from 19 fish sampled in the Chao PhrayaMae Khlong and Chanthaburi Rivers Thailand Onesequence (CPCHMK12) was found in all of these drainages(Table S1) A closely allied sequence was found in one fishsampled at Phatthalung just south of the Isthmus of Kra onthe Thai-Malay Peninsula None of these fish were examined

by NampK and thus we are unable to definitively identifythe species However given the widespread occurrence ofH spilopterus in the Chao Phraya and Mae Klong Riversreported by NampK we conclude that these fish are part of awidespread H spilopterus population complex The meanwithin-group (clade 7) genetic divergence (119901 = 29 (04))suggests intraspecific population-level divergence

mtDNA Clade 8 Clade 8 encompasses 3 sequencesobtained from 9 fish sampled in east Sabah specifically theKinabatangan Segama and Umas Umas Rivers (Figure 9)The Kinabatangan sequence is somewhat divergent withinthe clade and the specimen was not identified by NampK Onesequence (SGM0606) characterized 6 fish from the SegamaRiver 2 of which were identified as H fortis (ZEC 4052140522 Table S1) by NampK (Table S1) Finally one fish bearinga closely related genotype from the Umas Umas River wasidentified as H baramensis by NampK (Table S1)

mtDNA Clade 9 Clade 9 is a sister group to the remaining2 clades composing the H nemurus species group Thiswell-defined clade (Figure 10) clusters 6 sequences from42 fish that are clearly identified as H hoevenii (NampK)Some of these haplotypes are geographically widespread onesequence (JOMSKA11 Table S1) was found in the Johor River(East Peninsular Malaysia) the Musi River (Sumatra) and

Advances in Evolutionary Biology 13

100∘ 110∘

0∘

120∘

00 250

250500

500750

(km)

(mi)

FOKIN0102FOSGM0606

FOUMA0201H fortis

10∘

99

86

001

Shelf minus75mShelf minus120 m

Figure 9 Phylogenetic relationships within H fortis comprised of 3 sequences The subtree is extracted from the taxon identificationphenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicate trees in which the associated sequencesclustered together in the bootstrap test (500 replicates) is shown next to the branches The distribution of these sequences is illustrated in theright panel

the Kapuas River (West Borneo) and a second sequence(HMUBHKABM23 Table S1) was found inMuarRiver (WestPeninsular Malaysia) and the Batang Hari (Sumatra) theKapuas (Sarawak) and the Baram Rivers (Sarawak) Many ofthese fish were identified as H hoevenii by NampK (Table S1)The existence of twodistinct but very closely related subclades(Figure 10 119901 = 06 (03)) that are not geographicallysegregated suggests some recent isolation event followed bywidespread dispersal and secondary contact

33 The H capitulum Group The final two monophyleticgroups of the Hemibagrus gene tree are approximatelyequivalent to the remaining species composing the Hnemurus species group as defined by NampK Dodson etal [10] and Ng and Dodson [11] have previously referredto clade 10 as the Sundaic clade as most sequences wereobtained from fish sampled on the Greater Sunda Islandsand clade 11 as the Indochinese clade asmany sequences wereobtained from fish sampled on the Indochinese mainland(Figure 2 Table S1) Although most of these sequencesare assigned to H capitulum by NampK there are somesignificant phylogeographic differences between the 2 cladesIn particular there appears to be an east-west division in thedistribution of the two clades on the Thai-Malay Peninsulawith the Sundaic clade (clade 10) found on the west coast andthe Indochinese clade found on the east coast with sympatricoccurrence in the Muar River Nevertheless mean sequencedivergence between the two clades is 119901 = 29 (05) avalue that is indicative of population-level or subspecies

divergence The two clades are thus best considered ascomprising one species

mtDNA Clade 10 The Sundaic clade includes a number ofclosely related sequences that comprise part of H capitulumand H nemurus Very little genetic structure is apparentand bootstrap support for tree topology within this group isgenerally very weak (Figure 11) A widely spread genotype(NCLCTBA20) characterized 6 of 17 specimens from CiTarum at the Cirata reservoir (West Java) (Table S1) and5 were identified as H nemurus in the analysis of NampK(ZRC 42564) (Table S1) The remaining fish characterized bythis sequence were obtained from Ci Liwong at Sawangan(West Java) and one fish was obtained from the Barito Riverat Banjarmassin south Borneo This latter specimen (ZRC40553) was identified by NampK as H capitulum (Table S1)Another common genotype (NBHKPBR09) was identified aseither H capitulum H hoevenii or H nemurus dependingon the river of origin (Table S1) Many of the remainingsequences of the Sundaic clade appear to characterize fish thatNampK identified asH capitulum A geographically widespreadgenotype (CPEBEMUKA36) characterized 9 fish sampledon the Perak River at Gerik that were identified by NampKas H capitulum (ZRC 41150 Table S1) Another commongenotype within this clade (CABHMS20) included 2 fishfrom the Batang Hari at Jambi and identified by NampK asH capitulum (ZRC40534 Table S1) Another 2 sequences(CABAR0203 CABAR0305) characterized 8 fish sampledon the Barito River at Banjarmasin and were identified as

14 Advances in Evolutionary Biology

100∘

10∘

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0

0 250

250

500

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750(km)

(mi)

53

64

99

85

64

0002

HJOMSKA11

HOBAH0601

HOBHMS05

HMUBHKABM23

HOBAH0701

HOKAP0501

H hoevenii ldquoBrdquo

H hoevenii ldquoArdquo

Shelf minus75mShelf minus120 m

Figure 10 Phylogenetic relationships withinH hoevenii comprised of 2 subclades and 6 sequences The subtree is extracted from the taxonidentification phenogram inferred using theNeighbor-Joiningmethod (see Figure 2)The percentage of replicate trees in which the associatedsequences clustered together in the bootstrap test (500 replicates) is shown next to the branches The distribution of these sequences isillustrated in the right panel

110∘

XXMAH0110

CABAR0305

96 CAKPR0201

74CAKAP0401

5931 CAKAP0601

35 CPEBEMUKA36

646 CAKAP0903

33 CAKAP080114 CAKAP0701

CARAJ0210

4 CABAR0203CABRA0204

20

CBHKPBR0919

CABRA0301

15CABHMS20

24 NCLCTBA20

63 NECIT0201

H capitulumldquoSundaic claderdquo

H nemurus

0002 120∘

0 250 500 750

(km)

0 250 500

(mi)100

10∘

0∘

Shelf minus75mShelf minus120 m

Figure 11 Phylogenetic relationships within theH capitulum ldquoSundaicrdquo clade including 2 sequences associated withH nemurusThe subtreeis extracted from the taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicatetrees in which the associated sequences clustered together in the bootstrap test (500 replicates) is shown next to the branchesThe distributionof these sequences is illustrated in the right panel

Advances in Evolutionary Biology 15

H capitulum

0∘

10∘

100∘

110∘

120∘

66

63

88

59

38

39 68

71

6991

57

9754

88 4944

39

53

89

0002

ldquoIndochineserdquogroup ldquoErdquo

H capitulumldquoIndochineserdquo

group ldquoDrdquoH capitulumldquoIndochineserdquo

group ldquoCrdquo

H capitulumldquoIndochineserdquo

group ldquoBrdquo

H capitulumldquoIndochineserdquo

group ldquoArdquo0

0

250

250

500

500

750

(km)

(mi)

CATER0301CATERR0401

CKETEMU23

CAMEK0601CACHP0301

CAMKPA03

CAMEK0910CAMEK1003

XXMUBM02

CABMTU09

CABRM0601

CAKAP1201CAKAP1001

CAKAP1101CAKAP1301

CABRM0501

CASAD0102

CASAD0220

CAEND0301

CABAH0801

CAPABH05CATEEN03

CATER0701CATERR0801

CAKETE04CATER0501

CAPAH0303

Shelf minus75mShelf minus120 m

Figure 12 Phylogenetic relationships within theH capitulum ldquoIndochineserdquo clade encompassing 5 subclades identified as A B C D and EThe subtree is extracted from the taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2)The percentageof replicate trees in which the associated sequences clustered together in the bootstrap test (500 replicates) is shown next to the branchesThedistribution of these sequences is illustrated in the right panel

H capitulum by NampK (ZRC 40553 Table S1) Although notincluded in the analysis of NampK samples from the Sundaicclade were also found in the Kapuas River (West Borneo)Finally 10 fish sampled on the Mahakam River at Samarindaeast Borneo are all characterized by one relatively divergentgenotype (CAMAH0110) butwere considered asH capitulumin the analysis of NampK (ZRC 40533 Table S1)TheMahakampopulation sample is the most genetically distinct of theSundaic clade (119901 = 17 (04)) but nevertheless indicativeof intraspecific population-level divergence (Figure 12)

The topology of the Sundaic clade does not permit usto draw a clear distinction between H nemurus and theSundaic H capitulum The mean within-group divergenceof the Sundaic clade is only 119901 = 08 (02) well withinpopulation-level divergence values We postulate that Hnemurus may be one of several regional variants of awidespread Sundaic H capitulum clade that has radiatedrelatively recently This may explain the existence of manyshared sequences throughout the Sundaic islands assigned todifferent species in different regions

mtDNA Clade 11 Finally the Indochinese clade of Hcapitulum represents a geographically widespread andweakly diversified clade (within-group mean sequencedivergence 119901 = 11 (02) Figure 12) Although few fishfrom this genetic clade were included in the morphological

analysis of NampK most may be considered as H capitulumgiven their geographic distribution and close geneticrelationship to the Sundaic H capitulum Four sequencesin this clade were identified as H capitulum in the analysisof NampK 3 specimens characterized by one sequence(CABMTU09) found in the Tutong River of westernBorneo (ZRC 40482) 6 characterized by the same sequencefrom the Baram River (ZRC 40498) another 2 sequencesfrom the Baram River (ZRC 40498) and 20 specimenscharacterized by an abundant genotype (CASAD0220)found at 2 sites in the Sadong River also in western Borneo(ZRC 39526 5 specimens 39527 3 specimens and 38753 4specimens) (Table S1) Unfortunately none of the specimenscollected on the Indochinese mainland for genetic analysiswere examined by NampK Nevertheless the distribution ofspecimens analyzed for mtDNA resembles the distribution ofspecimens examined by NampK Although weakly structuredwe may recognize 5 subclades One genotype (CAPAH0303)is basal to the phenogram Subclade A encompasses 8sequences mainly found in eastern Peninsular Malaysia(Table S1 Figure 12) These rivers furnished many of thespecimens NampK examined to define H capitulum Howeversome sequences in this group occur in sympatry withsequences identified within the Sundaic H capitulum groupTwo sequences from subclade A occurred in the Batang HariSumatra (Table S1) Subclade E also encompasses sequences

16 Advances in Evolutionary Biology

found in Peninsular Malaysia as well as in the Mekong ChaoPhraya and Mea Khlong Rivers (Figure 12) In addition3 subclades encompass sequences found principally inwestern Borneo subclade B in the Baram and Sadong RiversSarawak subclade C in the Kapuas River West Kalimantanand subclade D in the Baram and Tutong Rivers of Sarawakand Brunei respectively (Figure 12) This latter subclade alsoincludes one genotype found in the Muar River of westernpeninsular Malaysia Finally subclade E encompasses 5sequences from the Mekong Chao Praya and Mae KhlongRivers all within the distribution area of H spilopterus

EvolutionaryHistoryThe evolutionary history of theHemiba-grus species groups analyzed here dates from at least theMiocene 15ndash20Ma (Figure 13) when considering the widelyused molecular clock of 2 sequence divergence per millionyears Bootstrap support for the sister group relationships ofH wyckioides theH guttatus species group andH peguensisare all weak (clearly due to undersampling) and little can besaid of their evolutionary history other than the fact that theyoccupy basal positions in the rooted trees The remainder ofboth the NJ and ML trees is composed of two major sistergroups the H planiceps + H baramensis group and the Hnemurus species group (Figures 2 3 and 13)TheH planiceps+ H baramensis group is strongly supported in both NJ andML analyses (84 and 90 bootstrap support resp Figures2 and 3) The H nemurus species group is also stronglysupported in both analyses (98 and 99 bootstrap supportFigures 2 and 3) These two major phylogenetic groups areestimated to have diverged at least 8 to 12Ma ago during themid to late Miocene (Figure 13)

Much of the evolutionary history of Hemibagrus hasoccurred during the Quaternary starting approximately26Ma ago This is particularly true of the H planicepsand H nemurus species groups The radiation of the 5species included in the H planiceps species group appearsto coincide with the middle Pleistocene transition period ofapproximately 1Ma ago Their high-elevation distributionhowever appears to have largely isolated them from theeffects of lowered sea levels during glacial maxima and theattendant opportunities for widespread dispersal (Figure 5)The evolutionary histories of theH baramensis species groupand the unspecified species group date back to the latePliocene In contrast the low-elevation distribution of theHnemurus species group in Sundaland appears to have favoredtheir widespread distribution during the Quaternary ice ageIn particular H hoevenii appears to be the most recentlydispersed of the species under study with diversification anddispersal occurring approximately 03Ma ago (Figure 13)

4 Discussion

41 The Congruence of Morphological and Genetic AnalysesCombining genetic and morphological analyses to elucidatetaxonomy allowed us to better understand the diversificationand evolutionary relationships within this speciose group offishes Given the rapid and relatively constant rate of mtDNAevolution the deeper the branches within the phylogenetictree are the greater our ability to distinguish valid taxa

should be Conversely the lack of complete congruencebetween morphological- and genetic-based identificationsis expected to increase among the shallower branches of thephylogenetic tree resulting in the appearance of polyphyleticspecies Although 2 of the 8 species groups defined by Ngand Kottelat [7] were not sampled in this study the validityof the remaining 6 species groups and their componentspecies defined on the basis of morphological similaritywas largely confirmed by the analysis of the mitochondrialCyt 119887 bar code As expected congruence is strongest at thebase of the NJ and ML trees where the H guttatus speciesgroup formed a well-defined monophyletic group The Hmenoda and H wyckii species group were each representedby only one species but they were clearly distinct relativeto sequences characterizing the neighboring species groupsThe H planiceps H baramensis and H nemurus speciesgroups and their component species were also generallycongruent in morphological and genetic analyses Also asexpected most cases of discrepancy between morphological-based species identification and genetic-based speciesidentification occurred on relatively shallow branches of thephylogenetic tree These involve principally H spilopterusand the two mtDNA subclades composing H capitulumHowever a far more unexpected lack of congruence involvesthe definition of a deeply divergent unspecified genetic groupconfounding members of theH baramensis species group inthe morphological analysis of Ng and Kottelat [7]

Interpreting Polyphyly The apparent polyphyletic origin ofa species may be due to incomplete lineage sorting amongrecently diverged species the introgression of mtDNA dueto species hybridization or the erroneous identificationof species due to the lumping of morphologically crypticspecies In the absence of nuclear DNA analyses it isoften difficult to distinguish between introgression andincomplete lineage sorting For example several sequencescharacterizing the Indochinese clade of H capitulum(subclade E) are found in the Mekong and Chao PhrayaRivers well within the distributional area of H spilopterusH spilopterus and H capitulum first diverged at least 3MaOn the one hand this apparent case of polyphyly mayhave resulted from incomplete lineage sorting betweenrecently divergedH spilopterus andH capitulum We wouldhowever have expected similar cases of polyphyly amongpopulations of the Indochinese clade of H capitulum butthis was not detected On the other hand the sympatricoccurrence of H spilopterus subclade A and subcladeB sequences with H capitulum Indochinese subclade Esequences in the Mekong River may represent a migrationevent and local introgression of H capitulum with Hspilopterus A similar event may have occurred in theChao Phraya and Mae Khlong Rivers where H spilopterussequences cooccur withH capitulum sequences Computingthe divergence of H capitulum sequences (Indochinesesubclade E) cooccurring with H spilopterus and Hcapitulum sequences occurring in eastern PeninsularMalaysia (subclade A) reveals a mean sequence divergence119901 = 15 (04) Thus the H capitulum sequences occurringin sympatry with H spilopterus diverged at most 750 ka ago

Advances in Evolutionary Biology 17

H fortis

XXRAJ0604H furcatus

H sabanusH semotusH divaricatus

H planicepsGREND0206

VEBAH0907

H bonganPESAL0102

H wyckioidesH macropterus

H guttatusXXRAJ0301Leiocassis longirostris

Ma

4971

92

47

51

9999

99

9999

99

99

99

9999

99

99

99

99

65

96

96

4946

93

79

78

70

91

93

98

86

87

91

95

97

71

85

9999

05101520

005010015020 000

H capitulum Indochinese clades AndashE

H capitulum Sundaic clade

H hoevenii B

H hoevenii A

H spilopterus C

H spilopterus B

H spilopterus AH spilopterus A

H spp CH spp B

H spp A

Figure 13 Linearized tree with branch lengths estimated with the molecular clock using a mutation rate of 1 per Ma (=divergence rate of2 per Ma) The tree topology is identical to the ML reconstruction illustrated in Figure 3 (see the specificities of the tree reconstruction inthe caption of Figure 3) Boxes represent 95 confidence intervals of divergence time estimates for each node Vertical dashed lines redirectto dates indicated on the timescale (Ma) Numbers below timescale indicate the estimated number of substitution changes using Hasegawa-Kishino-Yanomodel (HKY)with gamma distribution (+119866 = 13290) after linearizing the treeH furcatus is synonymizedwithH baramensis

from eastern Peninsular Malaysia populations suggestinga recent introgression event Similarly H capitulum in theKapuas River is characterized by sequences of both theSundaic and the Indochinese clades As both sequences arebroadly sympatric it is not possible to distinguish betweenincomplete lineage sorting and introgression of these closelyrelated intraspecific lineages

A Possible Case of Morphological Stasis Clade 6 of thepresent analysis defines a well-supported monophyletic cladefound in Sabah Sarawak and the Endau River of easternpeninsula Malaysia that has no equivalent taxonomic statusin the revision of Ng and Kottelat [7] In Borneo they occursympatrically with the H baramensis species group and areconfounded as either H baramensis or H semotus These

two clades are genetically highly divergent having divergedbetween 8 and 12Ma ago It thus seems highly unlikely thatthey represent retained polymorphisms due to incompletelineage sorting Sequences previously identified as character-izing H chrysops [11] are part of clade 6 and extend to thesouth (Rajang and Sadong Rivers of Sarawak West Borneo)with one closely related genotype found in eastern peninsularMalaysia (Endau River) where it may well be confoundedwith H capitulum H chrysops was synonymized with Hcapitulum by Ng and Kottelat [7] based on their analysis ofspecimens ofH chrysops from the Rajang RiverThus despitea long and apparently independent phylogenetic historythe unspecified species group appears largely cryptic andconfounded principally with members of the H baramensisspecies group

18 Advances in Evolutionary Biology

We suggest that this situation is an example of mor-phological stasis involving the coexistence of a number ofgenetically divergent yet morphologically cryptic species thatare lumped together [24] A striking example of morpho-logical and ecological stasis among fishes is provided by thecoastal marine bonefishes (Albula spp) Distinct evolution-ary lineages (separated by sequence divergence between 5 and30 [25]) of bonefish coexist in several sample locations yetthey show little morphological or ecological differentiationin sympatry [25] Similarly a proportion of the grey mulletspecies (Mugilidae) also appears to consist of cryptic species[26] For exampleMugil cephalus in Taiwan is characterizedby three divergent lineages that have been shown to charac-terize 3 reproductively isolated but morphologically crypticspecies [26 27]

Here we have discovered 2 highly distinct and structuredclades (each composed of 3 mtDNA subclades) that aremorphologically confounded within different species Forexample H spp C of the unspecified species group isidentified as H baramensis in the Baram River and as Hsemotus in the Padas River We speculate that North Borneoharbors a morphologically cryptic species complex possiblycomposed of several species pairs inhabiting different riversystems The reconstruction of this phylogenetic history andthe taxonomic status of its components remains a majorchallenge to the revision of the Hemibagrus genus

42 Phylogeographic Hypotheses The phylogeography ofthe genus appears to have been principally influenced bytwo geological histories one encompassing the formationof North Borneo the second the emergence of the SundaIslands and their connectivity with the Indochinesemainlandassociated with recurrent sea-level changes North Borneoappears to have acted as a species sink promoting speciationand locally high biodiversity within the genus but with littleevidence of subsequent dispersal out of North Borneo Incontrast the geological events influencing the Indochinesemainland and the Sunda Islands have permitted bothspeciation and differential dispersal depending on thebiology of the species For example the H planiceps speciesgroup is found throughout the Greater Sunda Islands andtheThai-Malay Peninsula as is theH nemurus species groupWhereas the H nemurus species group generally occupiesthe low altitude lower reaches of large rivers the speciescomposing the H planiceps group are found in the upperreaches of large rivers The H nemurus species group iscomposed of a number of widely distributed closely relatedspecies whereas theH planiceps group is composed of a largenumber of species that are highly localized geographicallyIt thus appears that sea-level changes have had little impacton the dispersal history of the H planiceps species group buthave deeply influenced that of the H nemurus species group

The Case of North Borneo From 15Ma to 5Ma greater areasof Borneo became emergent and the central mountainson the Sarawak-Kalimantan border extending into Sabahbecame wider and higher which may have represented newbarriers to dispersal [28 29] Borneo gradually developedinto the present large island by emergence of land in the

north and east The H baramensis species group is restrictedto North Borneo as is the unspecified species group of theHnemurus species group H fortis is also distributed in NorthBorneo The isolation of the freshwater fish communities ofNorth Borneo (particularly in the Labuk-Segama watershed)has long been recognized based on the number of endemicspecies and the concentration of relict species [30] The latePliocene-early Pleistocene diversification of H fortis theunspecified species group and the H baramensis speciesgroup may thus be associated with their dispersal to andisolation within the watersheds of North Borneo Only onesequence of the unspecified species group is found outsideof North Borneo and H fortis appears to be distributedthroughout central and east Borneo [7]

ThePhylogeography of the H nemurus Species Group Periodicmarine transgressions on the Sundaland platform during theearly and middle Miocene [2 29] may have been sufficientto isolate populations of freshwater organisms and promotecladogenesis [31] This may have been responsible for theinitial mid to late Miocene divergence of the H nemurusspecies group The majority of sequences assigned to thisspecies group belong to 3 species (H spilopterus H capitulum(including H nemurus) and H hoevenii) all of which havebeen influenced by the more recent geological history of theSunda Islands and their connectivity with the Indochinesemainland H spilopterus is known from river drainages inIndochina including the Mekong Dong Nai Ban PakongChao Phraya and Mae Klong where it is found in a widevariety of lotic and lentic habitats [7] H spilopterus divergedin the late Pliocene-early Pleistocene possibly isolated tothe north of the Kra Peninsula during the late Pliocenemarine transgression that is purported to have created amajor biogeographic transition zone between the Sundaicand Indochinese biotas ([32] but see [33] for a critique ofthis biogeographic scenario) The existence of 2 subclades(A and B) in the Mekong River separated by 2 sequencedivergence is consistent with intraspecific divergence Thetwo subclades possibly represent 2 historical mtDNA lineagesthat are retained within the species reflecting a Pleistoceneisolation event followed by secondary contact within thecontemporary Mekong River Subclade C from the ChaoPhraya and Mae Khlong Rivers exhibits a level of geneticdivergence consistent with sibling species-level divergenceshowing a 5 sequence divergence relative to Mekong RiverH spilopterus

H capitulum may be considered as part of the Sundaicbiota located principally to the south of the Kra marinetransgression zone that may have isolated H spilopterus tothe north on the Indochinese mainlandThe biogeographicalevent leading to the subsequent formation of two intraspecificgroups within H capitulum is not at all clear The onlyarea where the two clades are clearly geographically separateis on the Thai-Malay Peninsula with the ldquoSundaicrdquo cladeon the west coast and the ldquoIndochineserdquo clade on theeast coast separated by the central Titiwangsa mountainrange The genetic distinctiveness of east and west coastpopulations of H capitulum has also been documented fornuclear DNA [34] A similar pattern of separation has been

Advances in Evolutionary Biology 19

reported for 5 additional freshwater fishes within this region[35] However as the divergence of the two H capitulumclades postdates the formation of the mountain range byapproximately 200Ma the geographic separation of the twoclades most likely represents differential dispersal rather thanancient vicariance The presence of the Sundaic clade on thewest coast of the Thai-Malay Peninsula as well as on theSundaic Islands reflects the historical connections betweeneast Sumatra and west Peninsular Malaysia across the Straitof Malacca This area encompasses the Northern CentralSumatra-Western Malaysia freshwater ecoregion [36] TheMuar River forms its southern border coincident with thesouthernmost distribution of the Sundaic clade on the Thai-Malay Peninsula It is important to note however that mostof Sumatra and Java emerged to achieve their present size andelevation only 5Ma ago and much of East Java continuedto be the site of marine deposition until late Pliocene-earlyPleistocene [29] We may speculate that the Sundaic clade ofH capitulum resulted from the relatively recent colonizationof these islands and Borneo The radiation within the cladeonly dates back to approximately 1Ma ago H nemurus fromWest Java may thus best be considered an isolated regionalpopulation of very recent origin

The diversification of the Indochinese clade dates fromat least 12Ma ago coincident with the mid-Pleistocenetransition at which time low-frequency high-amplitude andquasiperiodic (100 ka) glacial variability emerged It began125Ma ago and was complete by 07Ma ago [37] The tran-sition marked the beginning of major changes in sea levelsduring which the Sunda Shelf periodically emerged and wastraversed by enormous fluvial systems [3] The contributionof these events to the diversification of this clade cannotbe satisfactorily reconstructed One possible explanation isthat high sea levels produced barriers to gene flow and thepopulations of West Borneo (subclades B C and D) wereisolated from those of east Peninsular Malaysia This couldbe due to one or more dispersal events from Borneo via theGreat Sunda River (Figure 1) to east Peninsular Malaysia orlineage sorting whereby sequences of subclades A and E havebeen lost from West Borneo As in the case of the sympatricoccurrence of H spilopterus and H capitulum subclade E(see above) in the Mekong River we are unable to clearlydefine the relative roles of incomplete lineage sorting andintrogression followingmigration Given the recent origins ofthe Indochinese H capitulum sequences incomplete lineagesorting most probably blurs patterns of recent isolation

The last species of the H nemurus species group is Hhoevenii The species is widespread throughout PeninsularMalaysia and the Sundaic Islands It is found primarily inthe lower reaches of rivers and is apparently capable ofwithstanding low salt concentration [7] H hoevenii thusappears to be the most liable of all members of the Hnemurus species group to be impacted by Pleistocene sea-level changes This is clearly reflected in the widespreaddistribution of the sequences of this species Two lineages areevident having diverged no more than 03Ma ago suggest-ing a late Pleistocene isolation event followed by extensivedispersal and admixture in recent times This represents theclearest example of the dispersal opportunities afforded by

Pleistocene sea level changes on the Sunda Shelf In additionthe relatively low genetic diversity detected (only 6 sequencesin 42 fish) suggests a population bottleneck in the recent pastpossibly related to population declines in the lower reaches ofrivers flooded by increasing sea levels

43 Future Directions We have provided a global picture ofthe evolutionary taxonomy of the genus in Southeast AsiaClearly a more rigorous phylogenetic reconstruction of thegenus Hemibagrus requiring the construction of multiplegene trees based on the analysis of both mitochondrial andnuclear DNA markers from a wider taxonomic array ofhemibagrids will involve an even greater effort of samplingthroughout the region In particular we require samplingat smaller spatial and temporal scales For example smallerscale sampling in the Padas River and the Segama Riverof North Borneo using both mitochondrial and nucleargenetic markers would contribute to clarifying the natureof the cryptic species complex believed to inhabit theserivers The demonstration of reproductive isolation andormicroallopatric distribution among morphologically crypticspecies may result in the description of greater speciesrichness within the genus than presently described Thespecies status of H chrysops merits reconsideration givenits highly divergent evolutionary history Nuclear geneticmarkers in conjunction with mtDNA markers could revealcases of historical introgression particularly in the KapuasRiver and the Mekong River The hemibagrid catfishes con-tinue to provide multiple opportunities for the study of theevolutionary taxonomy and biogeography of a major faunalcomponent of the fluvial ecosystems of Southeast Asia

Disclosure

Ethanol-preserved tissue samples of many of the specimensidentified in Table S1 are freely available upon request fromJJD

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Goh Yan Yih for assistance in collectingspecimens and molecular analyses Mrs Francoise Colom-bani for molecular analyses and Dr Heok Hee Ng forspecies identification This work was funded by the NationalUniversity of Singapore grant to P K L Ng and a CanadianNatural Sciences and Engineering Research Council grant toJ J Dodson

References

[1] D J Lohman M de Bruyn T Page et al ldquoBiogeography ofthe Indo-Australian archipelagordquo Annual Review of EcologyEvolution and Systematics vol 42 pp 205ndash226 2011

20 Advances in Evolutionary Biology

[2] D S Woodruff ldquoNeogene marine transgressions palaeogeog-raphy and biogeographic transitions on the Thai-Malay Penin-sulardquo Journal of Biogeography vol 30 no 4 pp 551ndash567 2003

[3] H K Voris ldquoMaps of Pleistocene sea levels in SoutheastAsia shorelines river systems and time durationsrdquo Journal ofBiogeography vol 27 no 5 pp 1153ndash1167 2000

[4] S Y Yap ldquoOn the distributional patterns of Southeast-EastAsian freshwater fish and their historyrdquo Journal of Biogeographyvol 29 no 9 pp 1187ndash1199 2002

[5] S K J McConnell ldquoMapping aquatic faunal exchanges acrossthe Sunda shelf South-East Asia using distributional andgenetic data sets from the cyprinid fish Barbodes gonionotus(Bleeker 1850)rdquo Journal of Natural History vol 38 no 5 pp651ndash670 2004

[6] T Mo Anatomy Relationships and Systematics of the Bagridae(Teleostei Siluroidei) with a Hypothesis of Siluroid Phylogenyvol 17 ofTheses Zoologicae Koeltz Scientific Books 1991

[7] H H Ng and M Kottelat ldquoRevision of the Asian catfish genusHemibagrus bleeker 1862 (Teleostei Siluriformes Bagridae)rdquoRaffles Bulletin of Zoology vol 61 no 1 pp 205ndash291 2013

[8] P K L Ng and H H Ng ldquoHemibagrus gracilis a new speciesof large riverine catfish (Teleostei Bagridae) from PeninsularMalaysiardquo Raffles Bulletin of Zoology vol 43 pp 133ndash142 1995

[9] H H Tan and M Kottelat ldquoRedescription of Betta picta(Teleostei Osphronemidae) and description of Betta falx sp nfrom central Sumatrardquo Revue Suisse de Zoologie vol 105 no 3pp 557ndash568 1998

[10] J J Dodson F Colombani and P K Ng ldquoPhylogeographicstructure in mitochondrial DNA of a South-east Asian fresh-water fish Hemibagrus nemurus (Siluroidei Bagridae) andPleistocene sea-level changes on the Sunda shelfrdquo MolecularEcology vol 4 no 3 pp 331ndash346 1995

[11] H H Ng and J J Dodson ldquoMorphological and genetic descrip-tions of a new species of catfish Hemibagrus chrysops fromSarawak East Malaysia with an assessment of phylogeneticrelationships (Teleostei Bagridae)rdquo Raffles Bulletin of Zoologyvol 47 no 1 pp 45ndash57 1999

[12] P D N Hebert A Cywinska S L Ball and J R DeWaardldquoBiological identifications through DNA barcodesrdquo Proceedingsof the Royal Society B Biological Sciences vol 270 no 1512 pp313ndash321 2003

[13] J C Carolan T EMurray U Fitzpatrick et al ldquoColour patternsdo not diagnose species quantitative evaluation of a DNAbarcoded cryptic bumblebee complexrdquo PLoS ONE vol 7 no 1Article ID e29251 2012

[14] M Kekkonen and P D N Hebert ldquoDNA barcode-baseddelineation of putative species efficient start for taxonomicworkflowsrdquoMolecular Ecology Resources vol 14 no 4 pp 706ndash715 2014

[15] DM Hillis CMoritz and B KMarbleMolecular SystematicsSinauer Sunderland Mass USA 2nd edition 1996

[16] T D Kocher W K Thomas A Meyer et al ldquoDynamics ofmitochondrial DNA evolution in animals amplification andsequencingwith conserved primersrdquoProceedings of theNationalAcademy of Sciences of the United States of America vol 86 no16 pp 6196ndash6200 1989

[17] T A Hall ldquoBioEdit a user-friendly biological sequence align-ment editor and analysis program for Windows 9598NTrdquoNucleic Acids Symposium Series vol 41 pp 95ndash98 1999

[18] D A Benson I Karsch-Mizrachi K Clark D J Lipman JOstell and EW Sayers ldquoGenBankrdquoNucleic Acids Research vol40 no D1 pp D48ndashD53 2012

[19] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[20] X Ku Z Peng R Diogo and S He ldquoMtDNA phylogenyprovides evidence of generic polyphyleticism for East Asianbagrid catfishesrdquoHydrobiologia vol 579 no 1 pp 147ndash159 2007

[21] L Yang and S He ldquoPhylogeography of the freshwater catfishHemibagrus guttatus (Siluriformes Bagridae) implications forSouth China biogeography and influence of sea-level changesrdquoMolecular Phylogenetics and Evolution vol 49 no 1 pp 393ndash398 2008

[22] B W Bowen A L Bass L A Rocha W S Grant and DR Robertson ldquoPhylogeography of the trumpetfishes (Aulosto-mus) ring species complex on a global scalerdquo Evolution vol 55no 5 pp 1029ndash1039 2001

[23] Y P Kartavtsev ldquoDivergence at Cyt-b and Co-1 mtDNA geneson different taxonomic levels and genetics of speciation inanimalsrdquoMitochondrial DNA vol 22 no 3 pp 55ndash65 2011

[24] SWada Y Kameda and S Chiba ldquoLong-term stasis and short-term divergence in the phenotypes of microsnails on oceanicislandsrdquoMolecular Ecology vol 22 no 18 pp 4801ndash4810 2013

[25] J Colborn R E Crabtree J B Shaklee E Pfeiler and B WBowen ldquoThe evolutionary enigma of bonefishes (Albula spp)cryptic species and ancient separations in a globally distributedshorefishrdquo Evolution vol 55 no 4 pp 807ndash820 2001

[26] J-D Durand K-N Shen W-J Chen et al ldquoSystematics of thegrey mullets (Teleostei Mugiliformes Mugilidae) molecularphylogenetic evidence challenges two centuries ofmorphology-based taxonomyrdquo Molecular Phylogenetics and Evolution vol64 no 1 pp 73ndash92 2012

[27] K-N Shen B W Jamandre C-C Hsu W-N Tzeng and J-D Durand ldquoPlio-Pleistocene sea level and temperature fluc-tuations in the northwestern Pacific promoted speciation inthe globally-distributed flathead mullet Mugil cephalusrdquo BMCEvolutionary Biology vol 11 no 1 article 83 2011

[28] R Hall ldquoThe plate tectonics of Cenozoic SE Asia and thedistribution of land and seardquo in Biogeography and GeologicalEvolution of SE Asia R Hall and J D Holloway Eds pp 99ndash131Backhuys Publishers Leiden The Netherlands 1998

[29] R Hall ldquoSoutheast Asiarsquos changing palaeogeographyrdquo Blumeavol 54 no 1ndash3 pp 148ndash161 2009

[30] R F Inger and P K Chen ldquoThe freshwater fishes of NorthBorneordquo Fieldiana Zoology vol 45 pp 1ndash268 1962

[31] J Bohlen V Slechtova H H Tan and R Britz ldquoPhylogenyof the Southeast Asian freshwater fish genus Pangio (Cyprini-formes Cobitidae)rdquoMolecular Phylogenetics and Evolution vol61 no 3 pp 854ndash865 2011

[32] M de Bruyn E Nugroho M M Hossain J C Wilson andP B Mather ldquoPhylogeographic evidence for the existence ofan ancient biogeographic barrier the Isthmus of Kra SeawayrdquoHeredity vol 94 no 3 pp 370ndash378 2005

[33] J Parnell ldquoThe biogeography of the Isthmus of Kra region areviewrdquoNordic Journal of Botany vol 31 no 1 pp 001ndash015 2013

[34] S Usmani S G Tan S S Siraj and K Yusoff ldquoPopulationstructure of the Southeast Asian river catfishMystus nemurusrdquoAnimal Genetics vol 34 no 6 pp 462ndash464 2003

[35] M P Tan A F J Jamsari and M N Siti Azizah ldquoPhylo-geographic pattern of the striped snakehead Channa striatain Sundaland ancient river connectivity geographical and

Advances in Evolutionary Biology 21

anthropogenic singnaturesrdquo PLoS ONE vol 7 no 12 Article IDe52089 2012

[36] R Abell M L Thieme C Revenga et al ldquoFreshwater ecore-gions of the world a new map of biogeographic units forfreshwater biodiversity conservationrdquo BioScience vol 58 no 5pp 403ndash414 2008

[37] P U Clark D Archer D Pollard et al ldquoThemiddle Pleistocenetransition characteristics mechanisms and implications forlong-term changes in atmospheric pCO

2rdquo Quaternary Science

Reviews vol 25 no 23-24 pp 3150ndash3184 2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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International Journal of

Volume 2014

Zoology

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Microbiology

Page 4: ResearchArticle - Université Laval Julian... · ResearchArticle A DNA Barcode-Based Evaluation of the Southeast Asian Catfish Genus Hemibagrus Bleeker, 1862 (Teleostei: Siluriformes;

4 Advances in Evolutionary Biology

Table 1 Continued

River code Site code River Sample site Latituded∘ mm1015840 ss10158401015840

Longituded∘ mm1015840 ss10158401015840

Distance upstreamfrom river mouth

(km)

Number ofspecimens

24d 44 Kapuas Sintang 0 04 00 N 111 30 00 E 270 324e 45 Kapuas Danau Sentarum 0 51 00 N 112 6 00 E 550 224f 46 Kapuas Putussibau 0 50 00 N 112 56 00 E 600 224g 47 Kapuas Sibau 0 55 00 N 112 57 00 E 750 224h 48 Kapuas Mendalam 1 00 00 N 113 16 00 E 800 625a 49 Sadong Serian 1 10 00 N 110 34 00 E 50 2525b 50 Sadong Balai Ringin 1 03 00 N 110 45 00 E 70 1126a 51 Rajang Kapit 2 01 00 N 112 56 00 E 210 726b 52 Rajang Sibu 2 18 00 N 111 49 00 E 80 1026c 53 Rajang Katibas 1 43 00 N 112 33 00 E 170 1227a 54 Baram Mulu 3 30 00 N 115 30 00 E 380 327b 55 Baram Bakung 4 17 00 N 114 14 00 E 60 627c 56 Baram Marudi 4 11 00 N 114 19 00 E 80 1528a 57 Tutong Merimbun 4 36 00 N 114 40 00 E 40 329a 58 Belait Ingei 4 10 00 N 114 43 00 E 100 330a 59 Padas Tenom 5 08 00 N 115 57 00 E 70 931a 60 Segaliud Sandakan 5 45 00 N 117 52 00 E 50 132a 61 Segama Danum Valley 5 01 00 N 117 47 00 E 170 1632b 62 Segama Lahad Datu 5 02 00 N 118 19 00 E 90 633a 63 Kinabatangan Batu Putih 5 25 00 N 117 55 00 E 100 634a 64 Umas-Umas Tawau 4 22 00 N 117 44 00 E 15 235 65 Salween Myanmar mdash mdash mdash 236 66 Red Hanoi mdash mdash mdash 237 67 Pearl Guangdong mdash mdash mdash 8

Total 452mdash exact origin unknown

amplified in a programmable thermal cycler using the follow-ing profile one single preliminary denaturation at 95∘C for5min 40 cycles of amplification with denaturation at 94∘Cfor 60 s primer annealing at 45∘C for 60 s primer extensionat 72∘C for 90 s and a final single extension step performed at72∘C for 5min Total 50 120583L PCR products were purified on a2 agarose gel After electrophoresis at 90V for 2 hours theamplified fragments were excised from the gel and extractedand purified using the QIAquick Gel Extraction Kit Protocol(using a microcentrifuge) fromQIAGEN to a final volume of15 to 50120583L (in EB buffer 10mMTris Cl pH 75)

Double stranded purified PCR products were sequencedin both directions (complementary strand was sequenced inall cases) using the ABI PRISM BigDye Terminator CycleSequencing Ready Reaction Kit (Applied Biosystems Inc)Sequencing reactions were carried out in 20120583L total volumecontaining 32 picomoles of the sequencing primer 30 to60 ng of purified PCR product 6 120583L of 25X dilution buffer(CSA seq Buffer Perkin Elmer) and 2 120583L of ABI PRISMBigDye Terminator Ready Reaction Mix Reactions wereperformed in thermal cycler GeneAmp PCR system 9600using the following profile 10 s at 96∘C 5 s at 50∘C and

4min at 60∘C for 25 cycles Sequences were separated byelectrophoresis at 1680V (150W 500mA) for 5 to 7 h on ABIPRISM 377 DNA Sequencer (Applied Biosystems Inc)

Sequences were extracted from the gel image using ABIPRISM Sequencing Analysis 30 program The sequences ofthe complementary strands were then imported in SequenceNavigator program for comparative alignment and correctionof ambiguities (Applied Biosystems Inc) Sequences werealigned using the BioEdit sequence alignment editor [17]Each sequence was identified by a unique code designatingthe origin of the sample (river name) the cumulativenumber of the sequences found in that river and thenumber of individuals characterized by that particularsequence For example the code MEK1502 designates the15th sequence found in the Mekong River and occurring intwo specimens Following the comparison of morphologicaland genetic designations mtDNA codes were preceded bythe putative species designation (eg SPMEK1502 indicatesthat specimens bearing sequence MEK1502 were assignedto Hemibagrus spilopterus) Sequences found in multiplerivers were identified by river name and the total number ofspecimens Sequences were deposited in GenBank [18] with

Advances in Evolutionary Biology 5

(1) Mekong(2) Chao Phraya(3) Chanthaburi(4) Mea Khlong(5) Phatthalung(6) Kelatan(7) Terengannu(8) Pahang(9) Endau(10) Johor(11) Perak(12) Bernam

(13) Muar(14) Batang Hari(15) Musi(16) Ci Liwong(17) Ci Sadane(18) Ci Tarum(19) Ci Manuk(20) Kali Progo(21) Brantas(22) Mahakam(23) Barito(24) Kapuas(25) Sadong(26) Rajang

(27) Baram(28) Tutong(29) Belait(30) Padas(31) Segaliud(32) Segama(33) Kinabatangan(34) Umas-Umas(35) Salween(36) Red(37) Pearl

0 250 500 750

(km)

0 250 500

(mi)100

∘110

∘120

0∘

10∘

Siam RiverNor

th Su

nda R

iver

East Sunda River

35 36

37

1

5

11

12

17

8 9

10

14

16

17

18

19

25

24

23

20

21

22

27

26

28

29 3031

32

33

34

15

6

13

2

3

4

Shelf minus75mShelf minus120 m

Figure 1 Location map of Southeast Asia illustrating the 37 rivers sampled in this study Black dots along rivers represent the sampling sitesas identified in Table 1 and Table S1 Dark grey shading illustrates the extent of the emerging Sunda Shelf at sea levels 75m below present daylevels light grey denotes emerging shelf at sea levels 120m below present day levels (as illustrated by Voris 2000) Rivers illustrated on theSunda Shelf represent drowned Pleistocene river drainage systems according to Voris (2000)

accession numbers corresponding to sequence codes andreported in Supplementary Material Table S1

Data Analysis We first used the Neighbor-Joining (NJ)algorithm in MEGA 52 (Tamura et al 2011) to develop ataxon identification phenogram ofHemibagrusThe Tamura-Nei distance matrix considering invariable site contribution

(+119868) was selected according to the model selection algo-rithm implemented in MEGA 52 [19] The rate variationamong sites was modeled with a gamma distribution (shapeparameter = 1) Recognizing the limitations of single-locusanalyses in phylogeny reconstruction and the objectionsof cladists to distance-based methodology we neverthelesssought to provide a tentative evaluation of phylogenetic

6 Advances in Evolutionary Biology

relationships among taxa To do so the Cyt 119887 sequence of theChinese longsnout catfish (Leiocassis longirostris) obtainedfrom GenBank (accession number DQ3217551) was usedas an outgroup to root the phenogram Chinese speciescurrently assigned to Leiocassismay be considered membersof the genus Pseudobagrus [20] Furthermore we employeda maximum likelihood (ML) phylogenetic reconstructionbased on the comparison of nucleotides within sites to assessthe congruence of the taxon identification phenogram andthe ML phylogenetic reconstruction In the latter case theevolutionary model used was the Hasegawa-Kishino-Yanomodel (HKY)with gammadistribution (+119866 = 13290) Initialtree(s) for the heuristic search were obtained by applyingthe Neighbor-Joining (NJ) method to a matrix of pairwisedistances estimated using the maximum composite likeli-hood (MCL) approach The rate-variation model allowedfor some sites to be evolutionarily invariable (+119868 4644of sites) The ML tree was rooted with the Cyt 119887 sequenceof the Chinese longsnout catfish (Leiocassis longirostris) andconstructed using MEGA52 [19]

TheNJ phenogram classifiedmtDNA sequences based onoverall similarity Ng and Kottelat [7] also defined speciesgroups based on overall similarity but according to mor-phological criteria Of the 8 species groups defined by Ngand Kottelat [7] two groups (H olyroides (1 species) and Hpluriradiatus (5 species)) were not included in our samplesWe thus evaluated the congruence of the two methods indefining the remaining six species groups (H baramensisH guttatus H menoda H nemurus H planiceps and Hwyckii species groups) When possible mtDNA sequenceswere linked to ZRC accession numbers identifying specimensexamined by Ng and Kottelat [7] in their morphologicalrevision of the genus (Table S1)

To provide time estimates for the major phylogeneticevents we present a calibrated linearized tree obtained fromour ML reconstruction (MEGA 52) Time of divergencebetween Hemibagrus lineages has been approximated byapplying a rate of between 10 and 15 genetic divergence permillion years as reported for bagrid fish by Yang and He [21]However the justification for this rate is not clear We thusapplied the more widely used rate of 2 sequence divergenceper million years [22] As there is undoubtedly a wide errorassociated with any such molecular clock calibrations esti-mated divergence times are used to discriminate more recentfrom more ancient cladogenetic events while providing veryapproximate dates for such events

Genetic divergence estimates using 119901-distances (119901) wereused in the present study to identify the different taxonomiclevels observed among the Hemibagrus spp analyzed Sincesuch information is readily available for thousands of animalgroups [23] we used 119901-distance (119901) as a benchmark for taxo-nomic levelsThe survey of 20 731 vertebrate and invertebratespecies reported by Kartavtsev [23] revealed increasing levelsof 119901-distances () of the mtDNA Cyt 119887 gene with taxonomiclevel populations within species 119901 = 138 plusmn 030 (SD)subspecies or sibling species119901 = 510plusmn091 morphologicallydistinct species within genera 119901 = 1031 plusmn 093 generawithin a family 119901 = 1786 plusmn 136 and families within anorder 119901 = 2636 plusmn 388 Although heterogeneity in the

gene evolution rate differs amongdifferent taxonomic groupsthese estimates provide an order of magnitude evaluationof intraspecific versus interspecific divergence and permittedus to discriminate between population- and species-leveldivergences However as 119901-distances underestimate truegenetic distance (eg site saturation and back-mutation)divergence times based on119901-distances greater than about 10are generally underestimated We calculated 119901-distances inMEGA 52 and report them as percentages (standard error)

3 Results

The 452 samples yielded a total of 118 distinct sequences(not including the outgroup taxon) The Neighbor-Joiningphenogram clustered the 119 sequences into a large numberof hierarchically organized monophyletic clades To facilitatepresentation we first present a compressed NJ tree andidentify 11 major mtDNA clades (Figure 2) One unidentifiedfish from the Rajang River was characterized by a genotypethat clustered with the outgroup taxon (XXRAJ0604GenBank accession number KP322872 Figures 2 and3) A second fish from the Rajang was characterized byan isolated genotype (XXRAJ0301 GenBank accessionnumber KP322869) of unknown affiliation (Figures 2and 3) To assess the congruence of the species groupsdefined by Ng and Kottelat ([7] hereafter referred toas NampK) and the mtDNA groupings revealed by thepresent analysis we provide a direct comparison of thetwo methods following the order determined by the taxonidentification phenogram (Figure 2 Table S1) Table S1 alsoprovides additional information about the geographicaldistribution of sequences Gene trees of individual cladesincluding all sequences are presented at each step in theanalysis along with their geographical distributions We alsopresent a compressed maximum likelihood phylogenetictree (Figure 3) The phenogram and the ML tree providedcongruent phylogenetic reconstructions However both treesfailed to resolve the phylogenetic relationships of H wyckiiH guttatus and H menoda species groups most likelydue to undersampling of these lineages The relationshipsamong an unspecified species group H spilopterus and Hfortis are also not well resolved Their position in the NJtree receives weak bootstrap support and they appear as amultifurcation along with a clade composed of H capitulumand H hoevenii in the ML phylogenetic tree (Figures 2 and3) To facilitate comparisons with the taxonomic scheme ofNampK we follow the sequence of taxa as identified in Figure 2

mtDNAClade 1The H wyckii Species GroupThe first clade isclearly consistent with theH wyckii species group (Figure 4)This species group is composed of 4 valid speciesHmaydelliH microphthalmus H wyckii andH wyckioides (NampK)Thelatter species is known from the Mekong and Chao Phrayadrainage Only specimens of H wyckioides obtained fromthe Mekong and the Chao Phraya were available for DNAanalysis Three specimens from the Chao Phraya bearingsequence WYCHP0203 (Table S1) were identified as Hwyckioides (H H Ng personal communication) although

Advances in Evolutionary Biology 7

8811

10

9

8

7

6

5

4

3

1

2

66

85

44

32

98

91

81

8432

57

99

99

99

49

002

34

15

43

86

99

96

H capitulum (Indochinese clade)

H capitulum (Sundaic clade)

H hoeveniiH fortis

H spilopterus

H baramensis species group

H planiceps species groupH menoda species group

H guttatus species group

H wyckii species group

XXRAJ0604

PESAL0102

XXRAJ0301Leiocassis longirostris

mtDNAclade

H nemurusspecies group

Unspecified species group

Figure 2 The taxon identification phenogram of Hemibagrus inferred using the Neighbor-Joining method in MEGA 52 The optimal treewith the sumof branch length = 186668598 is shown Bootstrap values are indicated on each node of the tree (500 replicates) Bar indicates thenumber of substitutions changes estimated using Tamura-Nei distance considering invariable site contribution (+I)The analysis involved 119nucleotide sequences All positions containing gaps and missing data were eliminatedThere were a total of 597 positions in the final dataset

mtDNAclade

H capitulum (Indochinese clade) 11

93

97H capitulum (Sundaic clade)H hoevenii

9999

84H fortis 8

9

10

H spilopterus 773 92

Unspecified species group 678

XXRAJ060454 34

90 H baramensis species group 5

49 H planiceps species group 4

H menoda species groupPESAL0102 3

H wyckii species group 199

99

99H guttatus species group 2

XXRAJ030149

47

Leiocassis longirostris

005

H nemurusspecies group

94

72

Figure 3 Evolutionary history of Hemibagrus inferred by using the maximum likelihood method based on the Hasegawa-Kishino-YanomodelThe tree with the highest log likelihood (minus56774825) is shown Bootstrap values are indicated on each node of the tree (500 replicates)Bar indicates the number of substitution changes estimated using Hasegawa-Kishino-Yano model (HKY) with gamma distribution (+119866 =13290) The analysis involved 119 nucleotide sequences All positions containing gaps and missing data were eliminated There were a totalof 597 positions in the final dataset

their ZRC numbers do not appear in the analysis of NampK Asno specimens of the other 3 species comprising the H wyckiispecies group were available for analysis we cannot confirmif this species group is monophyletic

mtDNA Clade 2 The H guttatus Species Group The secondclade of the Hemibagrus gene tree (Figure 2) is composed oftwo reciprocally monophyletic subclades clearly reflectingthe H guttatus species group as defined by NampK (Figure 4)

8 Advances in Evolutionary Biology

100∘

10∘

110∘

0∘

120∘

0

0 250

250

500

500

750(km)

(mi)

002

98

43

84

34

99

99

79

96

51

59

99

78

49

H peguensis

H guttatus

H macropterus

H wyckioides

PESAL0102

XXRAJ0301Leiocassis longirostris

MAPEA0201MAPEA0302MAPEA0101

GUPEA0501GURED0102

GUPEA0401GUPEA0602

WYCHP0401WYMEK1101

WYCHP0203

See Figures 7ndash12

See Figures 5 and 6

Shelf minus75mShelf minus120 m

Figure 4 Phylogenetic relationships of the individual sequences comprising four species H guttatus H macropterus (together forming theH guttatus species group) H wyckioides and H peguensisThe subtree is extracted from the taxon identification phenogram inferred usingthe Neighbor-Joining method (see Figure 2) The percentage of replicate trees in which the associated sequences clustered together in thebootstrap test (500 replicates) is shown next to the branches The distribution of these sequences is illustrated in the right panel

The group comprises three valid species H guttatus Hmacropterus andH vietnamicus (NampK) Specimens availablefor DNA analysis were obtained from the Pearl River andRed River (11 fish 7 sequences Table 1 and Table S1) Thetwo reciprocally monophyletic subclades comprising theH guttatus species group represent H guttatus and Hmacropterus (Figure 4) Although no ZRC specimens wereexamined by NampK species identification was confirmed(H H Ng personal communication) No H vietnamicusspecimens were available for DNA analysis The twospecies composing the group are closely related (119901 = 51(SE = 08))

mtDNA ldquoCladerdquo 3 The H menoda Species Group Thisisolated genotype (Figure 4) most likely identifies a cladeencompassing the H menoda species group although the

single sequence (Table S1) is shared by only 2 specimensof H peguensis from the Salween River Myanmar alsoincluded in the analysis of NampK (where its provenance wasidentified as the neighboring Irrawaddy River) The otherspecies comprising this group (H menoda H caveatusand H punctatus) were not sampled for DNA analysis Themonophyletic nature of this species group thus remains to bedemonstrated

31 The H planiceps + H baramensis Group The remainderof the gene tree is split into two large sister groups withmarginal bootstrap support (73 in ML and 43 in NJ)the H planiceps + H baramensis group and the H nemurusspecies group (Figures 2 and 3) The H planiceps + Hbaramensis group is strongly supported in both NJ and MLanalyses (84 and 90 bootstrap support respectively

Advances in Evolutionary Biology 9

110∘

47

53

69

81

69

5199

52

95

97

78

001

H bongan

H velox

H gracilis

H planiceps

H divaricatus

BOKAP1801

BOKAP1601BOKAP1701

BOKAP1402BOKAP1503

VEBAH0907GREND0206

PLCIM0114PLCIS0101

DIPER0401DIPER0301

DIPER0201

100∘

10∘

0∘

120∘

0

0 250 500

250 500

750(km)

(mi)

Shelf minus75mShelf minus120 m

Figure 5 Phylogenetic relationships of the individual sequences and species comprising theH planiceps species groupH bongan H veloxH gracilis H planiceps and H divaricatus The subtree is extracted from the taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicate trees in which the associated sequences clustered together in the bootstrap test(500 replicates) is shown next to the branches The distribution of these sequences is illustrated in the right panel

Figures 2 and 3) The two groups are genetically similarwith a genetic divergence estimate of 119901 = 121 (SE = 09)Although theH planiceps species group is strongly supportedin both trees (99) bootstrap support of the H baramensisspecies group is weak (Figure 2 32 Figure 3 34) Thegeographical distribution of the two groups differs Membersof the H planiceps species group are found throughout theGreater Sunda Islands and Peninsular Malaysia but they areisolated in the upper reaches of large rivers (Figure 5) Incontrast members of theH baramensis species group appearto be restricted to Sabah North Borneo (Figure 6)

mtDNA Clade 4 The H planiceps Species Group The Hplaniceps species group (Figure 5) is comprised of 5 closelyrelated but genetically distinct species (mean within-groupdivergence 119901 = 20 (SD = 033) H planiceps (Java)H gracilis (eastern Peninsular Malaysia) H divaricatus(western Peninsular Malaysia) H bongan (Borneo) andH velox (Sumatra)) H lacustrinus the sixth species of thisgroup (NampK) was not sampled in the present study WithintheH planiceps species groupH planiceps formed a distinctspecies (Figure 5) comprising two sequences One sequence(PLCIM0114 Table S1) characterized 14 specimens obtainedfrom Ci Manuk West Java 9 of which were identified as Hplaniceps by NampK (Table S1) A second sequence (PLCIS0101

Table S1) was obtained from one specimen sampled atCi Sadane West Java that we classified as H planicepsH bongan is represented by 5 sequences obtained from 8specimens sampled in the Kapuas River (Figure 5) Two ofthese specimens (ZRC 43013 and 43015) were identified asH bongan by NampK (Table S1) H divaricatus is composedof 3 sequences from three specimens obtained at Gerik onthe Perak River (western Peninsular Malaysia) One of thesespecimens (ZRC 41151) is the holotype of this new species(NampK) H gracilis is represented by only one sequence(GREND0206 Table S1) shared by 6 specimens sampledat Ulu Endau Jasin River eastern Peninsular Malaysiafour of which were identified by NampK (Table S1) ZRC21484 comprises the holotype and ZRC 21482 21486 and21487 comprise the paratypes (NampK) of this species (TableS1) Finally H velox is represented by only one sequence(VEBAH0907) from 7 specimens sampled at Kerinci BatangHari River Sumatra Three of these specimens (ZRC 41503Table S1) represent 3 paratypes of the species (NampK)

mtDNA Clade 5 The H baramensis Species Group The threespecies comprising the H baramensis species group (Figures2 and 3) form three monophyletic subclades (mean within-group divergence 119901 = 106 (08) Figure 6) H semotus iscomposed of two sequences (SEPAD0101 SEPAD0202) that

10 Advances in Evolutionary Biology

110∘

002

H sabanus

H semotus

H baramensis

SAKIN0204SASGM0706

BASGM0402BASGM0301

BASGM0201BASGM0104

SESGM0502SEUMA0101

SEPAD0202SEPAD0101

See Figure 5

100∘

10∘

0∘

120∘

0

0 250 500

250 500

750(km)

(mi)

99

57

32

83

99

72

99

56

66

54

99

Shelf minus75mShelf minus120 m

Figure 6 Phylogenetic relationships of the individual sequences and three species comprising the H baramensis species group H semotusH sabanus and H baramensis (furcatus) The subtree is extracted from the taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicate trees in which the associated sequences clustered together in the bootstrap test(500 replicates) is shown next to the branches The distribution of these sequences is illustrated in the right panel

characterize fish that are the holotype and paratypes of Hsemotus (ZRC 46122 Table S1) along with two somewhatmore divergent sequences (Figure 6) One of these sequences(SGM0502) was found in the Segama River (Danum Valley)and identified as H baramensis by NampK (ZRC 40525)(Table S1) The second sequence (UMA0101) was from afish sampled at Tawau Umas Umas River (ZRC 46123) alsoidentified as H baramensis by NampK These sequences mostlikely reflect distinct lineages within H semotus inhabitingdifferent rivers as the mean intraspecies sequence divergence(119901 = 16) indicates population-level divergence

H sabanus and H furcatus (synonymized with H bara-mensis by NampK) cluster together to form a distinct subcladebut clearly diverge at the species level (Figure 6) H sabanuscomprises 2 sequences that are shared respectively by 6specimens (ZRC 42741) from the Segama River Sabah and4 specimens (part of ZRC 42743) from the KinabatanganRiver Sabah (Table S1) All of these specimenswere identifiedas H sabanus by NampK (Table S1) Finally H baramensisforms a distant subclade (147 mean sequence divergencebetween H baramensis and H sabanus) composed of 4sequences (Figure 6) shared by 8 specimens all sampled inthe Segama River Danum Valley Conservation Area SabahSix of these specimens (ZRC 40523 (2 specimens) ZRC40527 (2 specimens) and ZRC 40526 (2 specimens) Table S1)comprise the paratypes of H baramensis (furcatus) (NampK)

32 The H nemurus Species Group In both NJ and ML trees(Figures 2 and 3) the H nemurus species group is strongly

supported (bootstrap support 98 in NJ and 99 in ML)indicating that it forms a distinct and widespread geneticentity within the genus Hemibagrus However relationshipswithin the group are not so clear In general the H nemurusspecies group is composed of 4 clades The first clade may beconsidered as a species group nested within the H nemurusspecies group (85 and 93 bootstrap support in NJ andML resp) encompassing three clades H hoevenii (clade9) the H capitulum Sundaic clade (clade 10 including Hnemurus) and the H capitulum Indochinese clade (clade 11)(Figures 2 and 3) An additional three clades (Figures 2 and3) are formed by an unidentified species group (clade 6) Hspilopterus (clade 7) andH fortis (clade 8)These three latterclades are distinct at the congeneric species level (unspecifiedversus H spilopterus 119901 = 77 (07) unspecified versus Hfortis 119901 = 85 (07) and H spilopterus versus H fortis119901 = 75 (08)) The distribution of these three clades issomewhat disparate H fortis and the unspecified speciesgroup are principally found in Borneo whereasH spilopterusis widely distributed on the Indochinese mainland

mtDNA Clade 6 Unspecified Species Group This geneticallydiverse group encompassing at least 3 subclades (H spp AB and C Figure 7 within-group mean sequence divergence119901 = 68(06)) is confoundedwith theH baramensis speciesgroup byNampK It is composed of sequences fromfish sampledin Sarawak Sabah and eastern PeninsularMalaysia Subclade119867 spp A (Figure 7) is particularly interesting as it contains2 sequences (CHSARA20 CHRAJ0401) that characterize

Advances in Evolutionary Biology 11

110∘

XXPAD0303XXPAD0601

XXPAD0401XXBEL0202

XXBRM0202XXRAJ0511

XXPAD0501

XXEND0410CHSARA20CHRAJ0401

XXBEL0101XXSEG0101

100∘

10∘

0∘

120∘

0

0 250 500

250 500

750(km)

(mi)

91

86

83

99

98

47

81

001

91

72

99

80

H spp C

H spp B

H spp A

Shelf minus75mShelf minus120 m

Figure 7 Phylogenetic relationships of the individual sequences comprising the unspecified species group containing three subcladesidentified as H spp A B and C The subtree is extracted from the taxon identification phenogram inferred using the Neighbor-Joiningmethod (see Figure 2) The percentage of replicate trees in which the associated sequences clustered together in the bootstrap test (500replicates) is shown next to the branches The distribution of these sequences is illustrated in the right panel

H chrysops a new species identified by Ng and Dodson [11]However specimens of H chrysops from the Rajang Riverwere subsequently synonymized by NampK with H capitulumof the H nemurus species group (ZRC 23008ndash23014 TableS1) However an additional 14 fish from the Sadong Riverat Serian bearing genotype SARA20 (Table S1) and thusgenetically identical to the Rajang H chrysops samples wereidentified by NampK as H spilopterus (ZRC 29583ndash96 TableS1) These specimens however were erroneously identifiedin NampK as originating in the Chao Phraya River (Thailand)Subclade A also contains one sequence obtained from 10 fishsampled on the Endau River (Mersing) in eastern PeninsularMalaysia These fish were not morphologically examinedFinally subclade A also contains one sequence from oneunidentified fish sampled in the Segaliud River Sabah andanother unidentified fish sampled in the Belait River BruneiSubclade H spp B encompasses 2 sequences one of whichcomes from a fish sampled in the Padas River (XXPAD0501)and part of the ZRC 46122 fish lot identified as H semotusby NampK (Table S1) The second sequence (XXRAJ0511) wasshared by 11 unidentified fish sampled at Katibas on theRajang River Sarawak (Table S1) Finally subclade 119867 sppC encompasses 5 sequences Three sequences (XXPAD0303XXPAD0401 and XXPAD0601) were obtained from thePadas River and identified byNampKasH semotus (ZRC46122Table S1) The fourth sequence (XXBRM0202) was obtainedfrom 2 fish sampled in the Baram River and was identified

as H baramensis by NampK (ZRC 42742) The 5th sequencecomposing this group was obtained from 2 unidentified fishsampled in the Belait River Brunei (Table S1)

mtDNA clade 6 is thus problematical within the contextof the revision of the genus Hemibagrus Two highlydivergent mtDNA clades (clade 5 and clade 6 Figures 2and 3) occur in sympatry principally in the Padas River ofSabah North Borneo (Figures 6 and 7) Specimens of theunspecified species group are morphologically similar tothe H baramensis species group where they are assigned inthe analysis of NampK However the mean genetic distancebetween the unspecified species group and theH baramensisspecies group is 119901 = 193 (28) close to genus leveldivergence Furthermore subclade 119867 spp A (Figure 7)contains not only H chrysops but also an unidentifiedpopulation from eastern Peninsular Malaysia (XXEND0410Table S1) and two additional Sabah sequences On theother hand the unspecified species group clusters with allother members of the H nemurus species group (meangenetic divergence of unspecified group versus H capitulum(Sundaic clade) 119901 = 76 (07) (Figure 2)) with strongbootstrap support Such genetic clustering may justify theinclusion of H chrysops and associated sequences in theH nemurus species group but its synonymization with Hcapitulum (NampK) is difficult to defend on genetic groundsWe tentatively conclude that H chrysops is a valid speciesbased on genetic identity [11] forming part of an unknown

12 Advances in Evolutionary Biology

110∘

SPCPCHMK12

SPMEK0202SPMEK0401

SPMEK0301SPMEK0103

SPMEK1401SPMEK0501

SPMEK1201SPMEK1301

SPMEK1601SPMEK1502

SPMEK0701SPMEK0801SPMEK1701

SPCHP0501SPCHA0201

SPCHA0304SPPAT0101

100∘

10∘

0∘

120∘

0

0 250 500

250 500

750(km)

(mi)

000499

99 98

60

47

48

54

96

7091

38

86

87

99

80

58

H spilopterus ldquoCrdquo

H spilopterus ldquoBrdquo

H spilopterus ldquoArdquo

Shelf minus75mShelf minus120 m

Figure 8 Phylogenetic relationships within H spilopterus comprised of 3 subclades identified as A B and C The subtree is extracted fromthe taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicate trees in whichthe associated sequences clustered together in the bootstrap test (500 replicates) is shown next to the branches The distribution of thesesequences is illustrated in the right panel

species group found throughout North Borneo (and toa lesser degree in eastern Peninsular Malaysia) that maybe considered as part of the H nemurus species groupHowever the morphological confusion of the unspecifiedspecies group with the sympatric highly divergent Hbaramensis species group suggests the existence of a crypticspecies assemblage (see Section 4)

mtDNA Clade 7 Clade 7 (Figures 2 and 3) is composed of atleast 3 distinct subclades that appear to reflect 3 geneticallydivergent populations ofH spilopterus Subclade A (Figure 8)encompasses 7 sequences obtained from 8 fish sampled inthe Mekong River Four fish bearing 3 of these sequenceswere identified as paratypes (ZRC 43324) of H spilopterusby NampK (Table S1) One additional fish was identified asH spilopterus by NampK (ZRC 46157 Table S1) Subclade Bencompasses 6 sequences obtained from 9 fish all sampledfrom the Mekong River (Table S1) The fish sampled at ThatPhanom (ZRC 46012) was identified as H spilopterus inthe analysis of HampK (Table S1) Subclade C encompasses 5sequences obtained from 19 fish sampled in the Chao PhrayaMae Khlong and Chanthaburi Rivers Thailand Onesequence (CPCHMK12) was found in all of these drainages(Table S1) A closely allied sequence was found in one fishsampled at Phatthalung just south of the Isthmus of Kra onthe Thai-Malay Peninsula None of these fish were examined

by NampK and thus we are unable to definitively identifythe species However given the widespread occurrence ofH spilopterus in the Chao Phraya and Mae Klong Riversreported by NampK we conclude that these fish are part of awidespread H spilopterus population complex The meanwithin-group (clade 7) genetic divergence (119901 = 29 (04))suggests intraspecific population-level divergence

mtDNA Clade 8 Clade 8 encompasses 3 sequencesobtained from 9 fish sampled in east Sabah specifically theKinabatangan Segama and Umas Umas Rivers (Figure 9)The Kinabatangan sequence is somewhat divergent withinthe clade and the specimen was not identified by NampK Onesequence (SGM0606) characterized 6 fish from the SegamaRiver 2 of which were identified as H fortis (ZEC 4052140522 Table S1) by NampK (Table S1) Finally one fish bearinga closely related genotype from the Umas Umas River wasidentified as H baramensis by NampK (Table S1)

mtDNA Clade 9 Clade 9 is a sister group to the remaining2 clades composing the H nemurus species group Thiswell-defined clade (Figure 10) clusters 6 sequences from42 fish that are clearly identified as H hoevenii (NampK)Some of these haplotypes are geographically widespread onesequence (JOMSKA11 Table S1) was found in the Johor River(East Peninsular Malaysia) the Musi River (Sumatra) and

Advances in Evolutionary Biology 13

100∘ 110∘

0∘

120∘

00 250

250500

500750

(km)

(mi)

FOKIN0102FOSGM0606

FOUMA0201H fortis

10∘

99

86

001

Shelf minus75mShelf minus120 m

Figure 9 Phylogenetic relationships within H fortis comprised of 3 sequences The subtree is extracted from the taxon identificationphenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicate trees in which the associated sequencesclustered together in the bootstrap test (500 replicates) is shown next to the branches The distribution of these sequences is illustrated in theright panel

the Kapuas River (West Borneo) and a second sequence(HMUBHKABM23 Table S1) was found inMuarRiver (WestPeninsular Malaysia) and the Batang Hari (Sumatra) theKapuas (Sarawak) and the Baram Rivers (Sarawak) Many ofthese fish were identified as H hoevenii by NampK (Table S1)The existence of twodistinct but very closely related subclades(Figure 10 119901 = 06 (03)) that are not geographicallysegregated suggests some recent isolation event followed bywidespread dispersal and secondary contact

33 The H capitulum Group The final two monophyleticgroups of the Hemibagrus gene tree are approximatelyequivalent to the remaining species composing the Hnemurus species group as defined by NampK Dodson etal [10] and Ng and Dodson [11] have previously referredto clade 10 as the Sundaic clade as most sequences wereobtained from fish sampled on the Greater Sunda Islandsand clade 11 as the Indochinese clade asmany sequences wereobtained from fish sampled on the Indochinese mainland(Figure 2 Table S1) Although most of these sequencesare assigned to H capitulum by NampK there are somesignificant phylogeographic differences between the 2 cladesIn particular there appears to be an east-west division in thedistribution of the two clades on the Thai-Malay Peninsulawith the Sundaic clade (clade 10) found on the west coast andthe Indochinese clade found on the east coast with sympatricoccurrence in the Muar River Nevertheless mean sequencedivergence between the two clades is 119901 = 29 (05) avalue that is indicative of population-level or subspecies

divergence The two clades are thus best considered ascomprising one species

mtDNA Clade 10 The Sundaic clade includes a number ofclosely related sequences that comprise part of H capitulumand H nemurus Very little genetic structure is apparentand bootstrap support for tree topology within this group isgenerally very weak (Figure 11) A widely spread genotype(NCLCTBA20) characterized 6 of 17 specimens from CiTarum at the Cirata reservoir (West Java) (Table S1) and5 were identified as H nemurus in the analysis of NampK(ZRC 42564) (Table S1) The remaining fish characterized bythis sequence were obtained from Ci Liwong at Sawangan(West Java) and one fish was obtained from the Barito Riverat Banjarmassin south Borneo This latter specimen (ZRC40553) was identified by NampK as H capitulum (Table S1)Another common genotype (NBHKPBR09) was identified aseither H capitulum H hoevenii or H nemurus dependingon the river of origin (Table S1) Many of the remainingsequences of the Sundaic clade appear to characterize fish thatNampK identified asH capitulum A geographically widespreadgenotype (CPEBEMUKA36) characterized 9 fish sampledon the Perak River at Gerik that were identified by NampKas H capitulum (ZRC 41150 Table S1) Another commongenotype within this clade (CABHMS20) included 2 fishfrom the Batang Hari at Jambi and identified by NampK asH capitulum (ZRC40534 Table S1) Another 2 sequences(CABAR0203 CABAR0305) characterized 8 fish sampledon the Barito River at Banjarmasin and were identified as

14 Advances in Evolutionary Biology

100∘

10∘

110∘

0∘

120∘

0

0 250

250

500

500

750(km)

(mi)

53

64

99

85

64

0002

HJOMSKA11

HOBAH0601

HOBHMS05

HMUBHKABM23

HOBAH0701

HOKAP0501

H hoevenii ldquoBrdquo

H hoevenii ldquoArdquo

Shelf minus75mShelf minus120 m

Figure 10 Phylogenetic relationships withinH hoevenii comprised of 2 subclades and 6 sequences The subtree is extracted from the taxonidentification phenogram inferred using theNeighbor-Joiningmethod (see Figure 2)The percentage of replicate trees in which the associatedsequences clustered together in the bootstrap test (500 replicates) is shown next to the branches The distribution of these sequences isillustrated in the right panel

110∘

XXMAH0110

CABAR0305

96 CAKPR0201

74CAKAP0401

5931 CAKAP0601

35 CPEBEMUKA36

646 CAKAP0903

33 CAKAP080114 CAKAP0701

CARAJ0210

4 CABAR0203CABRA0204

20

CBHKPBR0919

CABRA0301

15CABHMS20

24 NCLCTBA20

63 NECIT0201

H capitulumldquoSundaic claderdquo

H nemurus

0002 120∘

0 250 500 750

(km)

0 250 500

(mi)100

10∘

0∘

Shelf minus75mShelf minus120 m

Figure 11 Phylogenetic relationships within theH capitulum ldquoSundaicrdquo clade including 2 sequences associated withH nemurusThe subtreeis extracted from the taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicatetrees in which the associated sequences clustered together in the bootstrap test (500 replicates) is shown next to the branchesThe distributionof these sequences is illustrated in the right panel

Advances in Evolutionary Biology 15

H capitulum

0∘

10∘

100∘

110∘

120∘

66

63

88

59

38

39 68

71

6991

57

9754

88 4944

39

53

89

0002

ldquoIndochineserdquogroup ldquoErdquo

H capitulumldquoIndochineserdquo

group ldquoDrdquoH capitulumldquoIndochineserdquo

group ldquoCrdquo

H capitulumldquoIndochineserdquo

group ldquoBrdquo

H capitulumldquoIndochineserdquo

group ldquoArdquo0

0

250

250

500

500

750

(km)

(mi)

CATER0301CATERR0401

CKETEMU23

CAMEK0601CACHP0301

CAMKPA03

CAMEK0910CAMEK1003

XXMUBM02

CABMTU09

CABRM0601

CAKAP1201CAKAP1001

CAKAP1101CAKAP1301

CABRM0501

CASAD0102

CASAD0220

CAEND0301

CABAH0801

CAPABH05CATEEN03

CATER0701CATERR0801

CAKETE04CATER0501

CAPAH0303

Shelf minus75mShelf minus120 m

Figure 12 Phylogenetic relationships within theH capitulum ldquoIndochineserdquo clade encompassing 5 subclades identified as A B C D and EThe subtree is extracted from the taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2)The percentageof replicate trees in which the associated sequences clustered together in the bootstrap test (500 replicates) is shown next to the branchesThedistribution of these sequences is illustrated in the right panel

H capitulum by NampK (ZRC 40553 Table S1) Although notincluded in the analysis of NampK samples from the Sundaicclade were also found in the Kapuas River (West Borneo)Finally 10 fish sampled on the Mahakam River at Samarindaeast Borneo are all characterized by one relatively divergentgenotype (CAMAH0110) butwere considered asH capitulumin the analysis of NampK (ZRC 40533 Table S1)TheMahakampopulation sample is the most genetically distinct of theSundaic clade (119901 = 17 (04)) but nevertheless indicativeof intraspecific population-level divergence (Figure 12)

The topology of the Sundaic clade does not permit usto draw a clear distinction between H nemurus and theSundaic H capitulum The mean within-group divergenceof the Sundaic clade is only 119901 = 08 (02) well withinpopulation-level divergence values We postulate that Hnemurus may be one of several regional variants of awidespread Sundaic H capitulum clade that has radiatedrelatively recently This may explain the existence of manyshared sequences throughout the Sundaic islands assigned todifferent species in different regions

mtDNA Clade 11 Finally the Indochinese clade of Hcapitulum represents a geographically widespread andweakly diversified clade (within-group mean sequencedivergence 119901 = 11 (02) Figure 12) Although few fishfrom this genetic clade were included in the morphological

analysis of NampK most may be considered as H capitulumgiven their geographic distribution and close geneticrelationship to the Sundaic H capitulum Four sequencesin this clade were identified as H capitulum in the analysisof NampK 3 specimens characterized by one sequence(CABMTU09) found in the Tutong River of westernBorneo (ZRC 40482) 6 characterized by the same sequencefrom the Baram River (ZRC 40498) another 2 sequencesfrom the Baram River (ZRC 40498) and 20 specimenscharacterized by an abundant genotype (CASAD0220)found at 2 sites in the Sadong River also in western Borneo(ZRC 39526 5 specimens 39527 3 specimens and 38753 4specimens) (Table S1) Unfortunately none of the specimenscollected on the Indochinese mainland for genetic analysiswere examined by NampK Nevertheless the distribution ofspecimens analyzed for mtDNA resembles the distribution ofspecimens examined by NampK Although weakly structuredwe may recognize 5 subclades One genotype (CAPAH0303)is basal to the phenogram Subclade A encompasses 8sequences mainly found in eastern Peninsular Malaysia(Table S1 Figure 12) These rivers furnished many of thespecimens NampK examined to define H capitulum Howeversome sequences in this group occur in sympatry withsequences identified within the Sundaic H capitulum groupTwo sequences from subclade A occurred in the Batang HariSumatra (Table S1) Subclade E also encompasses sequences

16 Advances in Evolutionary Biology

found in Peninsular Malaysia as well as in the Mekong ChaoPhraya and Mea Khlong Rivers (Figure 12) In addition3 subclades encompass sequences found principally inwestern Borneo subclade B in the Baram and Sadong RiversSarawak subclade C in the Kapuas River West Kalimantanand subclade D in the Baram and Tutong Rivers of Sarawakand Brunei respectively (Figure 12) This latter subclade alsoincludes one genotype found in the Muar River of westernpeninsular Malaysia Finally subclade E encompasses 5sequences from the Mekong Chao Praya and Mae KhlongRivers all within the distribution area of H spilopterus

EvolutionaryHistoryThe evolutionary history of theHemiba-grus species groups analyzed here dates from at least theMiocene 15ndash20Ma (Figure 13) when considering the widelyused molecular clock of 2 sequence divergence per millionyears Bootstrap support for the sister group relationships ofH wyckioides theH guttatus species group andH peguensisare all weak (clearly due to undersampling) and little can besaid of their evolutionary history other than the fact that theyoccupy basal positions in the rooted trees The remainder ofboth the NJ and ML trees is composed of two major sistergroups the H planiceps + H baramensis group and the Hnemurus species group (Figures 2 3 and 13)TheH planiceps+ H baramensis group is strongly supported in both NJ andML analyses (84 and 90 bootstrap support resp Figures2 and 3) The H nemurus species group is also stronglysupported in both analyses (98 and 99 bootstrap supportFigures 2 and 3) These two major phylogenetic groups areestimated to have diverged at least 8 to 12Ma ago during themid to late Miocene (Figure 13)

Much of the evolutionary history of Hemibagrus hasoccurred during the Quaternary starting approximately26Ma ago This is particularly true of the H planicepsand H nemurus species groups The radiation of the 5species included in the H planiceps species group appearsto coincide with the middle Pleistocene transition period ofapproximately 1Ma ago Their high-elevation distributionhowever appears to have largely isolated them from theeffects of lowered sea levels during glacial maxima and theattendant opportunities for widespread dispersal (Figure 5)The evolutionary histories of theH baramensis species groupand the unspecified species group date back to the latePliocene In contrast the low-elevation distribution of theHnemurus species group in Sundaland appears to have favoredtheir widespread distribution during the Quaternary ice ageIn particular H hoevenii appears to be the most recentlydispersed of the species under study with diversification anddispersal occurring approximately 03Ma ago (Figure 13)

4 Discussion

41 The Congruence of Morphological and Genetic AnalysesCombining genetic and morphological analyses to elucidatetaxonomy allowed us to better understand the diversificationand evolutionary relationships within this speciose group offishes Given the rapid and relatively constant rate of mtDNAevolution the deeper the branches within the phylogenetictree are the greater our ability to distinguish valid taxa

should be Conversely the lack of complete congruencebetween morphological- and genetic-based identificationsis expected to increase among the shallower branches of thephylogenetic tree resulting in the appearance of polyphyleticspecies Although 2 of the 8 species groups defined by Ngand Kottelat [7] were not sampled in this study the validityof the remaining 6 species groups and their componentspecies defined on the basis of morphological similaritywas largely confirmed by the analysis of the mitochondrialCyt 119887 bar code As expected congruence is strongest at thebase of the NJ and ML trees where the H guttatus speciesgroup formed a well-defined monophyletic group The Hmenoda and H wyckii species group were each representedby only one species but they were clearly distinct relativeto sequences characterizing the neighboring species groupsThe H planiceps H baramensis and H nemurus speciesgroups and their component species were also generallycongruent in morphological and genetic analyses Also asexpected most cases of discrepancy between morphological-based species identification and genetic-based speciesidentification occurred on relatively shallow branches of thephylogenetic tree These involve principally H spilopterusand the two mtDNA subclades composing H capitulumHowever a far more unexpected lack of congruence involvesthe definition of a deeply divergent unspecified genetic groupconfounding members of theH baramensis species group inthe morphological analysis of Ng and Kottelat [7]

Interpreting Polyphyly The apparent polyphyletic origin ofa species may be due to incomplete lineage sorting amongrecently diverged species the introgression of mtDNA dueto species hybridization or the erroneous identificationof species due to the lumping of morphologically crypticspecies In the absence of nuclear DNA analyses it isoften difficult to distinguish between introgression andincomplete lineage sorting For example several sequencescharacterizing the Indochinese clade of H capitulum(subclade E) are found in the Mekong and Chao PhrayaRivers well within the distributional area of H spilopterusH spilopterus and H capitulum first diverged at least 3MaOn the one hand this apparent case of polyphyly mayhave resulted from incomplete lineage sorting betweenrecently divergedH spilopterus andH capitulum We wouldhowever have expected similar cases of polyphyly amongpopulations of the Indochinese clade of H capitulum butthis was not detected On the other hand the sympatricoccurrence of H spilopterus subclade A and subcladeB sequences with H capitulum Indochinese subclade Esequences in the Mekong River may represent a migrationevent and local introgression of H capitulum with Hspilopterus A similar event may have occurred in theChao Phraya and Mae Khlong Rivers where H spilopterussequences cooccur withH capitulum sequences Computingthe divergence of H capitulum sequences (Indochinesesubclade E) cooccurring with H spilopterus and Hcapitulum sequences occurring in eastern PeninsularMalaysia (subclade A) reveals a mean sequence divergence119901 = 15 (04) Thus the H capitulum sequences occurringin sympatry with H spilopterus diverged at most 750 ka ago

Advances in Evolutionary Biology 17

H fortis

XXRAJ0604H furcatus

H sabanusH semotusH divaricatus

H planicepsGREND0206

VEBAH0907

H bonganPESAL0102

H wyckioidesH macropterus

H guttatusXXRAJ0301Leiocassis longirostris

Ma

4971

92

47

51

9999

99

9999

99

99

99

9999

99

99

99

99

65

96

96

4946

93

79

78

70

91

93

98

86

87

91

95

97

71

85

9999

05101520

005010015020 000

H capitulum Indochinese clades AndashE

H capitulum Sundaic clade

H hoevenii B

H hoevenii A

H spilopterus C

H spilopterus B

H spilopterus AH spilopterus A

H spp CH spp B

H spp A

Figure 13 Linearized tree with branch lengths estimated with the molecular clock using a mutation rate of 1 per Ma (=divergence rate of2 per Ma) The tree topology is identical to the ML reconstruction illustrated in Figure 3 (see the specificities of the tree reconstruction inthe caption of Figure 3) Boxes represent 95 confidence intervals of divergence time estimates for each node Vertical dashed lines redirectto dates indicated on the timescale (Ma) Numbers below timescale indicate the estimated number of substitution changes using Hasegawa-Kishino-Yanomodel (HKY)with gamma distribution (+119866 = 13290) after linearizing the treeH furcatus is synonymizedwithH baramensis

from eastern Peninsular Malaysia populations suggestinga recent introgression event Similarly H capitulum in theKapuas River is characterized by sequences of both theSundaic and the Indochinese clades As both sequences arebroadly sympatric it is not possible to distinguish betweenincomplete lineage sorting and introgression of these closelyrelated intraspecific lineages

A Possible Case of Morphological Stasis Clade 6 of thepresent analysis defines a well-supported monophyletic cladefound in Sabah Sarawak and the Endau River of easternpeninsula Malaysia that has no equivalent taxonomic statusin the revision of Ng and Kottelat [7] In Borneo they occursympatrically with the H baramensis species group and areconfounded as either H baramensis or H semotus These

two clades are genetically highly divergent having divergedbetween 8 and 12Ma ago It thus seems highly unlikely thatthey represent retained polymorphisms due to incompletelineage sorting Sequences previously identified as character-izing H chrysops [11] are part of clade 6 and extend to thesouth (Rajang and Sadong Rivers of Sarawak West Borneo)with one closely related genotype found in eastern peninsularMalaysia (Endau River) where it may well be confoundedwith H capitulum H chrysops was synonymized with Hcapitulum by Ng and Kottelat [7] based on their analysis ofspecimens ofH chrysops from the Rajang RiverThus despitea long and apparently independent phylogenetic historythe unspecified species group appears largely cryptic andconfounded principally with members of the H baramensisspecies group

18 Advances in Evolutionary Biology

We suggest that this situation is an example of mor-phological stasis involving the coexistence of a number ofgenetically divergent yet morphologically cryptic species thatare lumped together [24] A striking example of morpho-logical and ecological stasis among fishes is provided by thecoastal marine bonefishes (Albula spp) Distinct evolution-ary lineages (separated by sequence divergence between 5 and30 [25]) of bonefish coexist in several sample locations yetthey show little morphological or ecological differentiationin sympatry [25] Similarly a proportion of the grey mulletspecies (Mugilidae) also appears to consist of cryptic species[26] For exampleMugil cephalus in Taiwan is characterizedby three divergent lineages that have been shown to charac-terize 3 reproductively isolated but morphologically crypticspecies [26 27]

Here we have discovered 2 highly distinct and structuredclades (each composed of 3 mtDNA subclades) that aremorphologically confounded within different species Forexample H spp C of the unspecified species group isidentified as H baramensis in the Baram River and as Hsemotus in the Padas River We speculate that North Borneoharbors a morphologically cryptic species complex possiblycomposed of several species pairs inhabiting different riversystems The reconstruction of this phylogenetic history andthe taxonomic status of its components remains a majorchallenge to the revision of the Hemibagrus genus

42 Phylogeographic Hypotheses The phylogeography ofthe genus appears to have been principally influenced bytwo geological histories one encompassing the formationof North Borneo the second the emergence of the SundaIslands and their connectivity with the Indochinesemainlandassociated with recurrent sea-level changes North Borneoappears to have acted as a species sink promoting speciationand locally high biodiversity within the genus but with littleevidence of subsequent dispersal out of North Borneo Incontrast the geological events influencing the Indochinesemainland and the Sunda Islands have permitted bothspeciation and differential dispersal depending on thebiology of the species For example the H planiceps speciesgroup is found throughout the Greater Sunda Islands andtheThai-Malay Peninsula as is theH nemurus species groupWhereas the H nemurus species group generally occupiesthe low altitude lower reaches of large rivers the speciescomposing the H planiceps group are found in the upperreaches of large rivers The H nemurus species group iscomposed of a number of widely distributed closely relatedspecies whereas theH planiceps group is composed of a largenumber of species that are highly localized geographicallyIt thus appears that sea-level changes have had little impacton the dispersal history of the H planiceps species group buthave deeply influenced that of the H nemurus species group

The Case of North Borneo From 15Ma to 5Ma greater areasof Borneo became emergent and the central mountainson the Sarawak-Kalimantan border extending into Sabahbecame wider and higher which may have represented newbarriers to dispersal [28 29] Borneo gradually developedinto the present large island by emergence of land in the

north and east The H baramensis species group is restrictedto North Borneo as is the unspecified species group of theHnemurus species group H fortis is also distributed in NorthBorneo The isolation of the freshwater fish communities ofNorth Borneo (particularly in the Labuk-Segama watershed)has long been recognized based on the number of endemicspecies and the concentration of relict species [30] The latePliocene-early Pleistocene diversification of H fortis theunspecified species group and the H baramensis speciesgroup may thus be associated with their dispersal to andisolation within the watersheds of North Borneo Only onesequence of the unspecified species group is found outsideof North Borneo and H fortis appears to be distributedthroughout central and east Borneo [7]

ThePhylogeography of the H nemurus Species Group Periodicmarine transgressions on the Sundaland platform during theearly and middle Miocene [2 29] may have been sufficientto isolate populations of freshwater organisms and promotecladogenesis [31] This may have been responsible for theinitial mid to late Miocene divergence of the H nemurusspecies group The majority of sequences assigned to thisspecies group belong to 3 species (H spilopterus H capitulum(including H nemurus) and H hoevenii) all of which havebeen influenced by the more recent geological history of theSunda Islands and their connectivity with the Indochinesemainland H spilopterus is known from river drainages inIndochina including the Mekong Dong Nai Ban PakongChao Phraya and Mae Klong where it is found in a widevariety of lotic and lentic habitats [7] H spilopterus divergedin the late Pliocene-early Pleistocene possibly isolated tothe north of the Kra Peninsula during the late Pliocenemarine transgression that is purported to have created amajor biogeographic transition zone between the Sundaicand Indochinese biotas ([32] but see [33] for a critique ofthis biogeographic scenario) The existence of 2 subclades(A and B) in the Mekong River separated by 2 sequencedivergence is consistent with intraspecific divergence Thetwo subclades possibly represent 2 historical mtDNA lineagesthat are retained within the species reflecting a Pleistoceneisolation event followed by secondary contact within thecontemporary Mekong River Subclade C from the ChaoPhraya and Mae Khlong Rivers exhibits a level of geneticdivergence consistent with sibling species-level divergenceshowing a 5 sequence divergence relative to Mekong RiverH spilopterus

H capitulum may be considered as part of the Sundaicbiota located principally to the south of the Kra marinetransgression zone that may have isolated H spilopterus tothe north on the Indochinese mainlandThe biogeographicalevent leading to the subsequent formation of two intraspecificgroups within H capitulum is not at all clear The onlyarea where the two clades are clearly geographically separateis on the Thai-Malay Peninsula with the ldquoSundaicrdquo cladeon the west coast and the ldquoIndochineserdquo clade on theeast coast separated by the central Titiwangsa mountainrange The genetic distinctiveness of east and west coastpopulations of H capitulum has also been documented fornuclear DNA [34] A similar pattern of separation has been

Advances in Evolutionary Biology 19

reported for 5 additional freshwater fishes within this region[35] However as the divergence of the two H capitulumclades postdates the formation of the mountain range byapproximately 200Ma the geographic separation of the twoclades most likely represents differential dispersal rather thanancient vicariance The presence of the Sundaic clade on thewest coast of the Thai-Malay Peninsula as well as on theSundaic Islands reflects the historical connections betweeneast Sumatra and west Peninsular Malaysia across the Straitof Malacca This area encompasses the Northern CentralSumatra-Western Malaysia freshwater ecoregion [36] TheMuar River forms its southern border coincident with thesouthernmost distribution of the Sundaic clade on the Thai-Malay Peninsula It is important to note however that mostof Sumatra and Java emerged to achieve their present size andelevation only 5Ma ago and much of East Java continuedto be the site of marine deposition until late Pliocene-earlyPleistocene [29] We may speculate that the Sundaic clade ofH capitulum resulted from the relatively recent colonizationof these islands and Borneo The radiation within the cladeonly dates back to approximately 1Ma ago H nemurus fromWest Java may thus best be considered an isolated regionalpopulation of very recent origin

The diversification of the Indochinese clade dates fromat least 12Ma ago coincident with the mid-Pleistocenetransition at which time low-frequency high-amplitude andquasiperiodic (100 ka) glacial variability emerged It began125Ma ago and was complete by 07Ma ago [37] The tran-sition marked the beginning of major changes in sea levelsduring which the Sunda Shelf periodically emerged and wastraversed by enormous fluvial systems [3] The contributionof these events to the diversification of this clade cannotbe satisfactorily reconstructed One possible explanation isthat high sea levels produced barriers to gene flow and thepopulations of West Borneo (subclades B C and D) wereisolated from those of east Peninsular Malaysia This couldbe due to one or more dispersal events from Borneo via theGreat Sunda River (Figure 1) to east Peninsular Malaysia orlineage sorting whereby sequences of subclades A and E havebeen lost from West Borneo As in the case of the sympatricoccurrence of H spilopterus and H capitulum subclade E(see above) in the Mekong River we are unable to clearlydefine the relative roles of incomplete lineage sorting andintrogression followingmigration Given the recent origins ofthe Indochinese H capitulum sequences incomplete lineagesorting most probably blurs patterns of recent isolation

The last species of the H nemurus species group is Hhoevenii The species is widespread throughout PeninsularMalaysia and the Sundaic Islands It is found primarily inthe lower reaches of rivers and is apparently capable ofwithstanding low salt concentration [7] H hoevenii thusappears to be the most liable of all members of the Hnemurus species group to be impacted by Pleistocene sea-level changes This is clearly reflected in the widespreaddistribution of the sequences of this species Two lineages areevident having diverged no more than 03Ma ago suggest-ing a late Pleistocene isolation event followed by extensivedispersal and admixture in recent times This represents theclearest example of the dispersal opportunities afforded by

Pleistocene sea level changes on the Sunda Shelf In additionthe relatively low genetic diversity detected (only 6 sequencesin 42 fish) suggests a population bottleneck in the recent pastpossibly related to population declines in the lower reaches ofrivers flooded by increasing sea levels

43 Future Directions We have provided a global picture ofthe evolutionary taxonomy of the genus in Southeast AsiaClearly a more rigorous phylogenetic reconstruction of thegenus Hemibagrus requiring the construction of multiplegene trees based on the analysis of both mitochondrial andnuclear DNA markers from a wider taxonomic array ofhemibagrids will involve an even greater effort of samplingthroughout the region In particular we require samplingat smaller spatial and temporal scales For example smallerscale sampling in the Padas River and the Segama Riverof North Borneo using both mitochondrial and nucleargenetic markers would contribute to clarifying the natureof the cryptic species complex believed to inhabit theserivers The demonstration of reproductive isolation andormicroallopatric distribution among morphologically crypticspecies may result in the description of greater speciesrichness within the genus than presently described Thespecies status of H chrysops merits reconsideration givenits highly divergent evolutionary history Nuclear geneticmarkers in conjunction with mtDNA markers could revealcases of historical introgression particularly in the KapuasRiver and the Mekong River The hemibagrid catfishes con-tinue to provide multiple opportunities for the study of theevolutionary taxonomy and biogeography of a major faunalcomponent of the fluvial ecosystems of Southeast Asia

Disclosure

Ethanol-preserved tissue samples of many of the specimensidentified in Table S1 are freely available upon request fromJJD

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Goh Yan Yih for assistance in collectingspecimens and molecular analyses Mrs Francoise Colom-bani for molecular analyses and Dr Heok Hee Ng forspecies identification This work was funded by the NationalUniversity of Singapore grant to P K L Ng and a CanadianNatural Sciences and Engineering Research Council grant toJ J Dodson

References

[1] D J Lohman M de Bruyn T Page et al ldquoBiogeography ofthe Indo-Australian archipelagordquo Annual Review of EcologyEvolution and Systematics vol 42 pp 205ndash226 2011

20 Advances in Evolutionary Biology

[2] D S Woodruff ldquoNeogene marine transgressions palaeogeog-raphy and biogeographic transitions on the Thai-Malay Penin-sulardquo Journal of Biogeography vol 30 no 4 pp 551ndash567 2003

[3] H K Voris ldquoMaps of Pleistocene sea levels in SoutheastAsia shorelines river systems and time durationsrdquo Journal ofBiogeography vol 27 no 5 pp 1153ndash1167 2000

[4] S Y Yap ldquoOn the distributional patterns of Southeast-EastAsian freshwater fish and their historyrdquo Journal of Biogeographyvol 29 no 9 pp 1187ndash1199 2002

[5] S K J McConnell ldquoMapping aquatic faunal exchanges acrossthe Sunda shelf South-East Asia using distributional andgenetic data sets from the cyprinid fish Barbodes gonionotus(Bleeker 1850)rdquo Journal of Natural History vol 38 no 5 pp651ndash670 2004

[6] T Mo Anatomy Relationships and Systematics of the Bagridae(Teleostei Siluroidei) with a Hypothesis of Siluroid Phylogenyvol 17 ofTheses Zoologicae Koeltz Scientific Books 1991

[7] H H Ng and M Kottelat ldquoRevision of the Asian catfish genusHemibagrus bleeker 1862 (Teleostei Siluriformes Bagridae)rdquoRaffles Bulletin of Zoology vol 61 no 1 pp 205ndash291 2013

[8] P K L Ng and H H Ng ldquoHemibagrus gracilis a new speciesof large riverine catfish (Teleostei Bagridae) from PeninsularMalaysiardquo Raffles Bulletin of Zoology vol 43 pp 133ndash142 1995

[9] H H Tan and M Kottelat ldquoRedescription of Betta picta(Teleostei Osphronemidae) and description of Betta falx sp nfrom central Sumatrardquo Revue Suisse de Zoologie vol 105 no 3pp 557ndash568 1998

[10] J J Dodson F Colombani and P K Ng ldquoPhylogeographicstructure in mitochondrial DNA of a South-east Asian fresh-water fish Hemibagrus nemurus (Siluroidei Bagridae) andPleistocene sea-level changes on the Sunda shelfrdquo MolecularEcology vol 4 no 3 pp 331ndash346 1995

[11] H H Ng and J J Dodson ldquoMorphological and genetic descrip-tions of a new species of catfish Hemibagrus chrysops fromSarawak East Malaysia with an assessment of phylogeneticrelationships (Teleostei Bagridae)rdquo Raffles Bulletin of Zoologyvol 47 no 1 pp 45ndash57 1999

[12] P D N Hebert A Cywinska S L Ball and J R DeWaardldquoBiological identifications through DNA barcodesrdquo Proceedingsof the Royal Society B Biological Sciences vol 270 no 1512 pp313ndash321 2003

[13] J C Carolan T EMurray U Fitzpatrick et al ldquoColour patternsdo not diagnose species quantitative evaluation of a DNAbarcoded cryptic bumblebee complexrdquo PLoS ONE vol 7 no 1Article ID e29251 2012

[14] M Kekkonen and P D N Hebert ldquoDNA barcode-baseddelineation of putative species efficient start for taxonomicworkflowsrdquoMolecular Ecology Resources vol 14 no 4 pp 706ndash715 2014

[15] DM Hillis CMoritz and B KMarbleMolecular SystematicsSinauer Sunderland Mass USA 2nd edition 1996

[16] T D Kocher W K Thomas A Meyer et al ldquoDynamics ofmitochondrial DNA evolution in animals amplification andsequencingwith conserved primersrdquoProceedings of theNationalAcademy of Sciences of the United States of America vol 86 no16 pp 6196ndash6200 1989

[17] T A Hall ldquoBioEdit a user-friendly biological sequence align-ment editor and analysis program for Windows 9598NTrdquoNucleic Acids Symposium Series vol 41 pp 95ndash98 1999

[18] D A Benson I Karsch-Mizrachi K Clark D J Lipman JOstell and EW Sayers ldquoGenBankrdquoNucleic Acids Research vol40 no D1 pp D48ndashD53 2012

[19] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[20] X Ku Z Peng R Diogo and S He ldquoMtDNA phylogenyprovides evidence of generic polyphyleticism for East Asianbagrid catfishesrdquoHydrobiologia vol 579 no 1 pp 147ndash159 2007

[21] L Yang and S He ldquoPhylogeography of the freshwater catfishHemibagrus guttatus (Siluriformes Bagridae) implications forSouth China biogeography and influence of sea-level changesrdquoMolecular Phylogenetics and Evolution vol 49 no 1 pp 393ndash398 2008

[22] B W Bowen A L Bass L A Rocha W S Grant and DR Robertson ldquoPhylogeography of the trumpetfishes (Aulosto-mus) ring species complex on a global scalerdquo Evolution vol 55no 5 pp 1029ndash1039 2001

[23] Y P Kartavtsev ldquoDivergence at Cyt-b and Co-1 mtDNA geneson different taxonomic levels and genetics of speciation inanimalsrdquoMitochondrial DNA vol 22 no 3 pp 55ndash65 2011

[24] SWada Y Kameda and S Chiba ldquoLong-term stasis and short-term divergence in the phenotypes of microsnails on oceanicislandsrdquoMolecular Ecology vol 22 no 18 pp 4801ndash4810 2013

[25] J Colborn R E Crabtree J B Shaklee E Pfeiler and B WBowen ldquoThe evolutionary enigma of bonefishes (Albula spp)cryptic species and ancient separations in a globally distributedshorefishrdquo Evolution vol 55 no 4 pp 807ndash820 2001

[26] J-D Durand K-N Shen W-J Chen et al ldquoSystematics of thegrey mullets (Teleostei Mugiliformes Mugilidae) molecularphylogenetic evidence challenges two centuries ofmorphology-based taxonomyrdquo Molecular Phylogenetics and Evolution vol64 no 1 pp 73ndash92 2012

[27] K-N Shen B W Jamandre C-C Hsu W-N Tzeng and J-D Durand ldquoPlio-Pleistocene sea level and temperature fluc-tuations in the northwestern Pacific promoted speciation inthe globally-distributed flathead mullet Mugil cephalusrdquo BMCEvolutionary Biology vol 11 no 1 article 83 2011

[28] R Hall ldquoThe plate tectonics of Cenozoic SE Asia and thedistribution of land and seardquo in Biogeography and GeologicalEvolution of SE Asia R Hall and J D Holloway Eds pp 99ndash131Backhuys Publishers Leiden The Netherlands 1998

[29] R Hall ldquoSoutheast Asiarsquos changing palaeogeographyrdquo Blumeavol 54 no 1ndash3 pp 148ndash161 2009

[30] R F Inger and P K Chen ldquoThe freshwater fishes of NorthBorneordquo Fieldiana Zoology vol 45 pp 1ndash268 1962

[31] J Bohlen V Slechtova H H Tan and R Britz ldquoPhylogenyof the Southeast Asian freshwater fish genus Pangio (Cyprini-formes Cobitidae)rdquoMolecular Phylogenetics and Evolution vol61 no 3 pp 854ndash865 2011

[32] M de Bruyn E Nugroho M M Hossain J C Wilson andP B Mather ldquoPhylogeographic evidence for the existence ofan ancient biogeographic barrier the Isthmus of Kra SeawayrdquoHeredity vol 94 no 3 pp 370ndash378 2005

[33] J Parnell ldquoThe biogeography of the Isthmus of Kra region areviewrdquoNordic Journal of Botany vol 31 no 1 pp 001ndash015 2013

[34] S Usmani S G Tan S S Siraj and K Yusoff ldquoPopulationstructure of the Southeast Asian river catfishMystus nemurusrdquoAnimal Genetics vol 34 no 6 pp 462ndash464 2003

[35] M P Tan A F J Jamsari and M N Siti Azizah ldquoPhylo-geographic pattern of the striped snakehead Channa striatain Sundaland ancient river connectivity geographical and

Advances in Evolutionary Biology 21

anthropogenic singnaturesrdquo PLoS ONE vol 7 no 12 Article IDe52089 2012

[36] R Abell M L Thieme C Revenga et al ldquoFreshwater ecore-gions of the world a new map of biogeographic units forfreshwater biodiversity conservationrdquo BioScience vol 58 no 5pp 403ndash414 2008

[37] P U Clark D Archer D Pollard et al ldquoThemiddle Pleistocenetransition characteristics mechanisms and implications forlong-term changes in atmospheric pCO

2rdquo Quaternary Science

Reviews vol 25 no 23-24 pp 3150ndash3184 2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 5: ResearchArticle - Université Laval Julian... · ResearchArticle A DNA Barcode-Based Evaluation of the Southeast Asian Catfish Genus Hemibagrus Bleeker, 1862 (Teleostei: Siluriformes;

Advances in Evolutionary Biology 5

(1) Mekong(2) Chao Phraya(3) Chanthaburi(4) Mea Khlong(5) Phatthalung(6) Kelatan(7) Terengannu(8) Pahang(9) Endau(10) Johor(11) Perak(12) Bernam

(13) Muar(14) Batang Hari(15) Musi(16) Ci Liwong(17) Ci Sadane(18) Ci Tarum(19) Ci Manuk(20) Kali Progo(21) Brantas(22) Mahakam(23) Barito(24) Kapuas(25) Sadong(26) Rajang

(27) Baram(28) Tutong(29) Belait(30) Padas(31) Segaliud(32) Segama(33) Kinabatangan(34) Umas-Umas(35) Salween(36) Red(37) Pearl

0 250 500 750

(km)

0 250 500

(mi)100

∘110

∘120

0∘

10∘

Siam RiverNor

th Su

nda R

iver

East Sunda River

35 36

37

1

5

11

12

17

8 9

10

14

16

17

18

19

25

24

23

20

21

22

27

26

28

29 3031

32

33

34

15

6

13

2

3

4

Shelf minus75mShelf minus120 m

Figure 1 Location map of Southeast Asia illustrating the 37 rivers sampled in this study Black dots along rivers represent the sampling sitesas identified in Table 1 and Table S1 Dark grey shading illustrates the extent of the emerging Sunda Shelf at sea levels 75m below present daylevels light grey denotes emerging shelf at sea levels 120m below present day levels (as illustrated by Voris 2000) Rivers illustrated on theSunda Shelf represent drowned Pleistocene river drainage systems according to Voris (2000)

accession numbers corresponding to sequence codes andreported in Supplementary Material Table S1

Data Analysis We first used the Neighbor-Joining (NJ)algorithm in MEGA 52 (Tamura et al 2011) to develop ataxon identification phenogram ofHemibagrusThe Tamura-Nei distance matrix considering invariable site contribution

(+119868) was selected according to the model selection algo-rithm implemented in MEGA 52 [19] The rate variationamong sites was modeled with a gamma distribution (shapeparameter = 1) Recognizing the limitations of single-locusanalyses in phylogeny reconstruction and the objectionsof cladists to distance-based methodology we neverthelesssought to provide a tentative evaluation of phylogenetic

6 Advances in Evolutionary Biology

relationships among taxa To do so the Cyt 119887 sequence of theChinese longsnout catfish (Leiocassis longirostris) obtainedfrom GenBank (accession number DQ3217551) was usedas an outgroup to root the phenogram Chinese speciescurrently assigned to Leiocassismay be considered membersof the genus Pseudobagrus [20] Furthermore we employeda maximum likelihood (ML) phylogenetic reconstructionbased on the comparison of nucleotides within sites to assessthe congruence of the taxon identification phenogram andthe ML phylogenetic reconstruction In the latter case theevolutionary model used was the Hasegawa-Kishino-Yanomodel (HKY)with gammadistribution (+119866 = 13290) Initialtree(s) for the heuristic search were obtained by applyingthe Neighbor-Joining (NJ) method to a matrix of pairwisedistances estimated using the maximum composite likeli-hood (MCL) approach The rate-variation model allowedfor some sites to be evolutionarily invariable (+119868 4644of sites) The ML tree was rooted with the Cyt 119887 sequenceof the Chinese longsnout catfish (Leiocassis longirostris) andconstructed using MEGA52 [19]

TheNJ phenogram classifiedmtDNA sequences based onoverall similarity Ng and Kottelat [7] also defined speciesgroups based on overall similarity but according to mor-phological criteria Of the 8 species groups defined by Ngand Kottelat [7] two groups (H olyroides (1 species) and Hpluriradiatus (5 species)) were not included in our samplesWe thus evaluated the congruence of the two methods indefining the remaining six species groups (H baramensisH guttatus H menoda H nemurus H planiceps and Hwyckii species groups) When possible mtDNA sequenceswere linked to ZRC accession numbers identifying specimensexamined by Ng and Kottelat [7] in their morphologicalrevision of the genus (Table S1)

To provide time estimates for the major phylogeneticevents we present a calibrated linearized tree obtained fromour ML reconstruction (MEGA 52) Time of divergencebetween Hemibagrus lineages has been approximated byapplying a rate of between 10 and 15 genetic divergence permillion years as reported for bagrid fish by Yang and He [21]However the justification for this rate is not clear We thusapplied the more widely used rate of 2 sequence divergenceper million years [22] As there is undoubtedly a wide errorassociated with any such molecular clock calibrations esti-mated divergence times are used to discriminate more recentfrom more ancient cladogenetic events while providing veryapproximate dates for such events

Genetic divergence estimates using 119901-distances (119901) wereused in the present study to identify the different taxonomiclevels observed among the Hemibagrus spp analyzed Sincesuch information is readily available for thousands of animalgroups [23] we used 119901-distance (119901) as a benchmark for taxo-nomic levelsThe survey of 20 731 vertebrate and invertebratespecies reported by Kartavtsev [23] revealed increasing levelsof 119901-distances () of the mtDNA Cyt 119887 gene with taxonomiclevel populations within species 119901 = 138 plusmn 030 (SD)subspecies or sibling species119901 = 510plusmn091 morphologicallydistinct species within genera 119901 = 1031 plusmn 093 generawithin a family 119901 = 1786 plusmn 136 and families within anorder 119901 = 2636 plusmn 388 Although heterogeneity in the

gene evolution rate differs amongdifferent taxonomic groupsthese estimates provide an order of magnitude evaluationof intraspecific versus interspecific divergence and permittedus to discriminate between population- and species-leveldivergences However as 119901-distances underestimate truegenetic distance (eg site saturation and back-mutation)divergence times based on119901-distances greater than about 10are generally underestimated We calculated 119901-distances inMEGA 52 and report them as percentages (standard error)

3 Results

The 452 samples yielded a total of 118 distinct sequences(not including the outgroup taxon) The Neighbor-Joiningphenogram clustered the 119 sequences into a large numberof hierarchically organized monophyletic clades To facilitatepresentation we first present a compressed NJ tree andidentify 11 major mtDNA clades (Figure 2) One unidentifiedfish from the Rajang River was characterized by a genotypethat clustered with the outgroup taxon (XXRAJ0604GenBank accession number KP322872 Figures 2 and3) A second fish from the Rajang was characterized byan isolated genotype (XXRAJ0301 GenBank accessionnumber KP322869) of unknown affiliation (Figures 2and 3) To assess the congruence of the species groupsdefined by Ng and Kottelat ([7] hereafter referred toas NampK) and the mtDNA groupings revealed by thepresent analysis we provide a direct comparison of thetwo methods following the order determined by the taxonidentification phenogram (Figure 2 Table S1) Table S1 alsoprovides additional information about the geographicaldistribution of sequences Gene trees of individual cladesincluding all sequences are presented at each step in theanalysis along with their geographical distributions We alsopresent a compressed maximum likelihood phylogenetictree (Figure 3) The phenogram and the ML tree providedcongruent phylogenetic reconstructions However both treesfailed to resolve the phylogenetic relationships of H wyckiiH guttatus and H menoda species groups most likelydue to undersampling of these lineages The relationshipsamong an unspecified species group H spilopterus and Hfortis are also not well resolved Their position in the NJtree receives weak bootstrap support and they appear as amultifurcation along with a clade composed of H capitulumand H hoevenii in the ML phylogenetic tree (Figures 2 and3) To facilitate comparisons with the taxonomic scheme ofNampK we follow the sequence of taxa as identified in Figure 2

mtDNAClade 1The H wyckii Species GroupThe first clade isclearly consistent with theH wyckii species group (Figure 4)This species group is composed of 4 valid speciesHmaydelliH microphthalmus H wyckii andH wyckioides (NampK)Thelatter species is known from the Mekong and Chao Phrayadrainage Only specimens of H wyckioides obtained fromthe Mekong and the Chao Phraya were available for DNAanalysis Three specimens from the Chao Phraya bearingsequence WYCHP0203 (Table S1) were identified as Hwyckioides (H H Ng personal communication) although

Advances in Evolutionary Biology 7

8811

10

9

8

7

6

5

4

3

1

2

66

85

44

32

98

91

81

8432

57

99

99

99

49

002

34

15

43

86

99

96

H capitulum (Indochinese clade)

H capitulum (Sundaic clade)

H hoeveniiH fortis

H spilopterus

H baramensis species group

H planiceps species groupH menoda species group

H guttatus species group

H wyckii species group

XXRAJ0604

PESAL0102

XXRAJ0301Leiocassis longirostris

mtDNAclade

H nemurusspecies group

Unspecified species group

Figure 2 The taxon identification phenogram of Hemibagrus inferred using the Neighbor-Joining method in MEGA 52 The optimal treewith the sumof branch length = 186668598 is shown Bootstrap values are indicated on each node of the tree (500 replicates) Bar indicates thenumber of substitutions changes estimated using Tamura-Nei distance considering invariable site contribution (+I)The analysis involved 119nucleotide sequences All positions containing gaps and missing data were eliminatedThere were a total of 597 positions in the final dataset

mtDNAclade

H capitulum (Indochinese clade) 11

93

97H capitulum (Sundaic clade)H hoevenii

9999

84H fortis 8

9

10

H spilopterus 773 92

Unspecified species group 678

XXRAJ060454 34

90 H baramensis species group 5

49 H planiceps species group 4

H menoda species groupPESAL0102 3

H wyckii species group 199

99

99H guttatus species group 2

XXRAJ030149

47

Leiocassis longirostris

005

H nemurusspecies group

94

72

Figure 3 Evolutionary history of Hemibagrus inferred by using the maximum likelihood method based on the Hasegawa-Kishino-YanomodelThe tree with the highest log likelihood (minus56774825) is shown Bootstrap values are indicated on each node of the tree (500 replicates)Bar indicates the number of substitution changes estimated using Hasegawa-Kishino-Yano model (HKY) with gamma distribution (+119866 =13290) The analysis involved 119 nucleotide sequences All positions containing gaps and missing data were eliminated There were a totalof 597 positions in the final dataset

their ZRC numbers do not appear in the analysis of NampK Asno specimens of the other 3 species comprising the H wyckiispecies group were available for analysis we cannot confirmif this species group is monophyletic

mtDNA Clade 2 The H guttatus Species Group The secondclade of the Hemibagrus gene tree (Figure 2) is composed oftwo reciprocally monophyletic subclades clearly reflectingthe H guttatus species group as defined by NampK (Figure 4)

8 Advances in Evolutionary Biology

100∘

10∘

110∘

0∘

120∘

0

0 250

250

500

500

750(km)

(mi)

002

98

43

84

34

99

99

79

96

51

59

99

78

49

H peguensis

H guttatus

H macropterus

H wyckioides

PESAL0102

XXRAJ0301Leiocassis longirostris

MAPEA0201MAPEA0302MAPEA0101

GUPEA0501GURED0102

GUPEA0401GUPEA0602

WYCHP0401WYMEK1101

WYCHP0203

See Figures 7ndash12

See Figures 5 and 6

Shelf minus75mShelf minus120 m

Figure 4 Phylogenetic relationships of the individual sequences comprising four species H guttatus H macropterus (together forming theH guttatus species group) H wyckioides and H peguensisThe subtree is extracted from the taxon identification phenogram inferred usingthe Neighbor-Joining method (see Figure 2) The percentage of replicate trees in which the associated sequences clustered together in thebootstrap test (500 replicates) is shown next to the branches The distribution of these sequences is illustrated in the right panel

The group comprises three valid species H guttatus Hmacropterus andH vietnamicus (NampK) Specimens availablefor DNA analysis were obtained from the Pearl River andRed River (11 fish 7 sequences Table 1 and Table S1) Thetwo reciprocally monophyletic subclades comprising theH guttatus species group represent H guttatus and Hmacropterus (Figure 4) Although no ZRC specimens wereexamined by NampK species identification was confirmed(H H Ng personal communication) No H vietnamicusspecimens were available for DNA analysis The twospecies composing the group are closely related (119901 = 51(SE = 08))

mtDNA ldquoCladerdquo 3 The H menoda Species Group Thisisolated genotype (Figure 4) most likely identifies a cladeencompassing the H menoda species group although the

single sequence (Table S1) is shared by only 2 specimensof H peguensis from the Salween River Myanmar alsoincluded in the analysis of NampK (where its provenance wasidentified as the neighboring Irrawaddy River) The otherspecies comprising this group (H menoda H caveatusand H punctatus) were not sampled for DNA analysis Themonophyletic nature of this species group thus remains to bedemonstrated

31 The H planiceps + H baramensis Group The remainderof the gene tree is split into two large sister groups withmarginal bootstrap support (73 in ML and 43 in NJ)the H planiceps + H baramensis group and the H nemurusspecies group (Figures 2 and 3) The H planiceps + Hbaramensis group is strongly supported in both NJ and MLanalyses (84 and 90 bootstrap support respectively

Advances in Evolutionary Biology 9

110∘

47

53

69

81

69

5199

52

95

97

78

001

H bongan

H velox

H gracilis

H planiceps

H divaricatus

BOKAP1801

BOKAP1601BOKAP1701

BOKAP1402BOKAP1503

VEBAH0907GREND0206

PLCIM0114PLCIS0101

DIPER0401DIPER0301

DIPER0201

100∘

10∘

0∘

120∘

0

0 250 500

250 500

750(km)

(mi)

Shelf minus75mShelf minus120 m

Figure 5 Phylogenetic relationships of the individual sequences and species comprising theH planiceps species groupH bongan H veloxH gracilis H planiceps and H divaricatus The subtree is extracted from the taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicate trees in which the associated sequences clustered together in the bootstrap test(500 replicates) is shown next to the branches The distribution of these sequences is illustrated in the right panel

Figures 2 and 3) The two groups are genetically similarwith a genetic divergence estimate of 119901 = 121 (SE = 09)Although theH planiceps species group is strongly supportedin both trees (99) bootstrap support of the H baramensisspecies group is weak (Figure 2 32 Figure 3 34) Thegeographical distribution of the two groups differs Membersof the H planiceps species group are found throughout theGreater Sunda Islands and Peninsular Malaysia but they areisolated in the upper reaches of large rivers (Figure 5) Incontrast members of theH baramensis species group appearto be restricted to Sabah North Borneo (Figure 6)

mtDNA Clade 4 The H planiceps Species Group The Hplaniceps species group (Figure 5) is comprised of 5 closelyrelated but genetically distinct species (mean within-groupdivergence 119901 = 20 (SD = 033) H planiceps (Java)H gracilis (eastern Peninsular Malaysia) H divaricatus(western Peninsular Malaysia) H bongan (Borneo) andH velox (Sumatra)) H lacustrinus the sixth species of thisgroup (NampK) was not sampled in the present study WithintheH planiceps species groupH planiceps formed a distinctspecies (Figure 5) comprising two sequences One sequence(PLCIM0114 Table S1) characterized 14 specimens obtainedfrom Ci Manuk West Java 9 of which were identified as Hplaniceps by NampK (Table S1) A second sequence (PLCIS0101

Table S1) was obtained from one specimen sampled atCi Sadane West Java that we classified as H planicepsH bongan is represented by 5 sequences obtained from 8specimens sampled in the Kapuas River (Figure 5) Two ofthese specimens (ZRC 43013 and 43015) were identified asH bongan by NampK (Table S1) H divaricatus is composedof 3 sequences from three specimens obtained at Gerik onthe Perak River (western Peninsular Malaysia) One of thesespecimens (ZRC 41151) is the holotype of this new species(NampK) H gracilis is represented by only one sequence(GREND0206 Table S1) shared by 6 specimens sampledat Ulu Endau Jasin River eastern Peninsular Malaysiafour of which were identified by NampK (Table S1) ZRC21484 comprises the holotype and ZRC 21482 21486 and21487 comprise the paratypes (NampK) of this species (TableS1) Finally H velox is represented by only one sequence(VEBAH0907) from 7 specimens sampled at Kerinci BatangHari River Sumatra Three of these specimens (ZRC 41503Table S1) represent 3 paratypes of the species (NampK)

mtDNA Clade 5 The H baramensis Species Group The threespecies comprising the H baramensis species group (Figures2 and 3) form three monophyletic subclades (mean within-group divergence 119901 = 106 (08) Figure 6) H semotus iscomposed of two sequences (SEPAD0101 SEPAD0202) that

10 Advances in Evolutionary Biology

110∘

002

H sabanus

H semotus

H baramensis

SAKIN0204SASGM0706

BASGM0402BASGM0301

BASGM0201BASGM0104

SESGM0502SEUMA0101

SEPAD0202SEPAD0101

See Figure 5

100∘

10∘

0∘

120∘

0

0 250 500

250 500

750(km)

(mi)

99

57

32

83

99

72

99

56

66

54

99

Shelf minus75mShelf minus120 m

Figure 6 Phylogenetic relationships of the individual sequences and three species comprising the H baramensis species group H semotusH sabanus and H baramensis (furcatus) The subtree is extracted from the taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicate trees in which the associated sequences clustered together in the bootstrap test(500 replicates) is shown next to the branches The distribution of these sequences is illustrated in the right panel

characterize fish that are the holotype and paratypes of Hsemotus (ZRC 46122 Table S1) along with two somewhatmore divergent sequences (Figure 6) One of these sequences(SGM0502) was found in the Segama River (Danum Valley)and identified as H baramensis by NampK (ZRC 40525)(Table S1) The second sequence (UMA0101) was from afish sampled at Tawau Umas Umas River (ZRC 46123) alsoidentified as H baramensis by NampK These sequences mostlikely reflect distinct lineages within H semotus inhabitingdifferent rivers as the mean intraspecies sequence divergence(119901 = 16) indicates population-level divergence

H sabanus and H furcatus (synonymized with H bara-mensis by NampK) cluster together to form a distinct subcladebut clearly diverge at the species level (Figure 6) H sabanuscomprises 2 sequences that are shared respectively by 6specimens (ZRC 42741) from the Segama River Sabah and4 specimens (part of ZRC 42743) from the KinabatanganRiver Sabah (Table S1) All of these specimenswere identifiedas H sabanus by NampK (Table S1) Finally H baramensisforms a distant subclade (147 mean sequence divergencebetween H baramensis and H sabanus) composed of 4sequences (Figure 6) shared by 8 specimens all sampled inthe Segama River Danum Valley Conservation Area SabahSix of these specimens (ZRC 40523 (2 specimens) ZRC40527 (2 specimens) and ZRC 40526 (2 specimens) Table S1)comprise the paratypes of H baramensis (furcatus) (NampK)

32 The H nemurus Species Group In both NJ and ML trees(Figures 2 and 3) the H nemurus species group is strongly

supported (bootstrap support 98 in NJ and 99 in ML)indicating that it forms a distinct and widespread geneticentity within the genus Hemibagrus However relationshipswithin the group are not so clear In general the H nemurusspecies group is composed of 4 clades The first clade may beconsidered as a species group nested within the H nemurusspecies group (85 and 93 bootstrap support in NJ andML resp) encompassing three clades H hoevenii (clade9) the H capitulum Sundaic clade (clade 10 including Hnemurus) and the H capitulum Indochinese clade (clade 11)(Figures 2 and 3) An additional three clades (Figures 2 and3) are formed by an unidentified species group (clade 6) Hspilopterus (clade 7) andH fortis (clade 8)These three latterclades are distinct at the congeneric species level (unspecifiedversus H spilopterus 119901 = 77 (07) unspecified versus Hfortis 119901 = 85 (07) and H spilopterus versus H fortis119901 = 75 (08)) The distribution of these three clades issomewhat disparate H fortis and the unspecified speciesgroup are principally found in Borneo whereasH spilopterusis widely distributed on the Indochinese mainland

mtDNA Clade 6 Unspecified Species Group This geneticallydiverse group encompassing at least 3 subclades (H spp AB and C Figure 7 within-group mean sequence divergence119901 = 68(06)) is confoundedwith theH baramensis speciesgroup byNampK It is composed of sequences fromfish sampledin Sarawak Sabah and eastern PeninsularMalaysia Subclade119867 spp A (Figure 7) is particularly interesting as it contains2 sequences (CHSARA20 CHRAJ0401) that characterize

Advances in Evolutionary Biology 11

110∘

XXPAD0303XXPAD0601

XXPAD0401XXBEL0202

XXBRM0202XXRAJ0511

XXPAD0501

XXEND0410CHSARA20CHRAJ0401

XXBEL0101XXSEG0101

100∘

10∘

0∘

120∘

0

0 250 500

250 500

750(km)

(mi)

91

86

83

99

98

47

81

001

91

72

99

80

H spp C

H spp B

H spp A

Shelf minus75mShelf minus120 m

Figure 7 Phylogenetic relationships of the individual sequences comprising the unspecified species group containing three subcladesidentified as H spp A B and C The subtree is extracted from the taxon identification phenogram inferred using the Neighbor-Joiningmethod (see Figure 2) The percentage of replicate trees in which the associated sequences clustered together in the bootstrap test (500replicates) is shown next to the branches The distribution of these sequences is illustrated in the right panel

H chrysops a new species identified by Ng and Dodson [11]However specimens of H chrysops from the Rajang Riverwere subsequently synonymized by NampK with H capitulumof the H nemurus species group (ZRC 23008ndash23014 TableS1) However an additional 14 fish from the Sadong Riverat Serian bearing genotype SARA20 (Table S1) and thusgenetically identical to the Rajang H chrysops samples wereidentified by NampK as H spilopterus (ZRC 29583ndash96 TableS1) These specimens however were erroneously identifiedin NampK as originating in the Chao Phraya River (Thailand)Subclade A also contains one sequence obtained from 10 fishsampled on the Endau River (Mersing) in eastern PeninsularMalaysia These fish were not morphologically examinedFinally subclade A also contains one sequence from oneunidentified fish sampled in the Segaliud River Sabah andanother unidentified fish sampled in the Belait River BruneiSubclade H spp B encompasses 2 sequences one of whichcomes from a fish sampled in the Padas River (XXPAD0501)and part of the ZRC 46122 fish lot identified as H semotusby NampK (Table S1) The second sequence (XXRAJ0511) wasshared by 11 unidentified fish sampled at Katibas on theRajang River Sarawak (Table S1) Finally subclade 119867 sppC encompasses 5 sequences Three sequences (XXPAD0303XXPAD0401 and XXPAD0601) were obtained from thePadas River and identified byNampKasH semotus (ZRC46122Table S1) The fourth sequence (XXBRM0202) was obtainedfrom 2 fish sampled in the Baram River and was identified

as H baramensis by NampK (ZRC 42742) The 5th sequencecomposing this group was obtained from 2 unidentified fishsampled in the Belait River Brunei (Table S1)

mtDNA clade 6 is thus problematical within the contextof the revision of the genus Hemibagrus Two highlydivergent mtDNA clades (clade 5 and clade 6 Figures 2and 3) occur in sympatry principally in the Padas River ofSabah North Borneo (Figures 6 and 7) Specimens of theunspecified species group are morphologically similar tothe H baramensis species group where they are assigned inthe analysis of NampK However the mean genetic distancebetween the unspecified species group and theH baramensisspecies group is 119901 = 193 (28) close to genus leveldivergence Furthermore subclade 119867 spp A (Figure 7)contains not only H chrysops but also an unidentifiedpopulation from eastern Peninsular Malaysia (XXEND0410Table S1) and two additional Sabah sequences On theother hand the unspecified species group clusters with allother members of the H nemurus species group (meangenetic divergence of unspecified group versus H capitulum(Sundaic clade) 119901 = 76 (07) (Figure 2)) with strongbootstrap support Such genetic clustering may justify theinclusion of H chrysops and associated sequences in theH nemurus species group but its synonymization with Hcapitulum (NampK) is difficult to defend on genetic groundsWe tentatively conclude that H chrysops is a valid speciesbased on genetic identity [11] forming part of an unknown

12 Advances in Evolutionary Biology

110∘

SPCPCHMK12

SPMEK0202SPMEK0401

SPMEK0301SPMEK0103

SPMEK1401SPMEK0501

SPMEK1201SPMEK1301

SPMEK1601SPMEK1502

SPMEK0701SPMEK0801SPMEK1701

SPCHP0501SPCHA0201

SPCHA0304SPPAT0101

100∘

10∘

0∘

120∘

0

0 250 500

250 500

750(km)

(mi)

000499

99 98

60

47

48

54

96

7091

38

86

87

99

80

58

H spilopterus ldquoCrdquo

H spilopterus ldquoBrdquo

H spilopterus ldquoArdquo

Shelf minus75mShelf minus120 m

Figure 8 Phylogenetic relationships within H spilopterus comprised of 3 subclades identified as A B and C The subtree is extracted fromthe taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicate trees in whichthe associated sequences clustered together in the bootstrap test (500 replicates) is shown next to the branches The distribution of thesesequences is illustrated in the right panel

species group found throughout North Borneo (and toa lesser degree in eastern Peninsular Malaysia) that maybe considered as part of the H nemurus species groupHowever the morphological confusion of the unspecifiedspecies group with the sympatric highly divergent Hbaramensis species group suggests the existence of a crypticspecies assemblage (see Section 4)

mtDNA Clade 7 Clade 7 (Figures 2 and 3) is composed of atleast 3 distinct subclades that appear to reflect 3 geneticallydivergent populations ofH spilopterus Subclade A (Figure 8)encompasses 7 sequences obtained from 8 fish sampled inthe Mekong River Four fish bearing 3 of these sequenceswere identified as paratypes (ZRC 43324) of H spilopterusby NampK (Table S1) One additional fish was identified asH spilopterus by NampK (ZRC 46157 Table S1) Subclade Bencompasses 6 sequences obtained from 9 fish all sampledfrom the Mekong River (Table S1) The fish sampled at ThatPhanom (ZRC 46012) was identified as H spilopterus inthe analysis of HampK (Table S1) Subclade C encompasses 5sequences obtained from 19 fish sampled in the Chao PhrayaMae Khlong and Chanthaburi Rivers Thailand Onesequence (CPCHMK12) was found in all of these drainages(Table S1) A closely allied sequence was found in one fishsampled at Phatthalung just south of the Isthmus of Kra onthe Thai-Malay Peninsula None of these fish were examined

by NampK and thus we are unable to definitively identifythe species However given the widespread occurrence ofH spilopterus in the Chao Phraya and Mae Klong Riversreported by NampK we conclude that these fish are part of awidespread H spilopterus population complex The meanwithin-group (clade 7) genetic divergence (119901 = 29 (04))suggests intraspecific population-level divergence

mtDNA Clade 8 Clade 8 encompasses 3 sequencesobtained from 9 fish sampled in east Sabah specifically theKinabatangan Segama and Umas Umas Rivers (Figure 9)The Kinabatangan sequence is somewhat divergent withinthe clade and the specimen was not identified by NampK Onesequence (SGM0606) characterized 6 fish from the SegamaRiver 2 of which were identified as H fortis (ZEC 4052140522 Table S1) by NampK (Table S1) Finally one fish bearinga closely related genotype from the Umas Umas River wasidentified as H baramensis by NampK (Table S1)

mtDNA Clade 9 Clade 9 is a sister group to the remaining2 clades composing the H nemurus species group Thiswell-defined clade (Figure 10) clusters 6 sequences from42 fish that are clearly identified as H hoevenii (NampK)Some of these haplotypes are geographically widespread onesequence (JOMSKA11 Table S1) was found in the Johor River(East Peninsular Malaysia) the Musi River (Sumatra) and

Advances in Evolutionary Biology 13

100∘ 110∘

0∘

120∘

00 250

250500

500750

(km)

(mi)

FOKIN0102FOSGM0606

FOUMA0201H fortis

10∘

99

86

001

Shelf minus75mShelf minus120 m

Figure 9 Phylogenetic relationships within H fortis comprised of 3 sequences The subtree is extracted from the taxon identificationphenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicate trees in which the associated sequencesclustered together in the bootstrap test (500 replicates) is shown next to the branches The distribution of these sequences is illustrated in theright panel

the Kapuas River (West Borneo) and a second sequence(HMUBHKABM23 Table S1) was found inMuarRiver (WestPeninsular Malaysia) and the Batang Hari (Sumatra) theKapuas (Sarawak) and the Baram Rivers (Sarawak) Many ofthese fish were identified as H hoevenii by NampK (Table S1)The existence of twodistinct but very closely related subclades(Figure 10 119901 = 06 (03)) that are not geographicallysegregated suggests some recent isolation event followed bywidespread dispersal and secondary contact

33 The H capitulum Group The final two monophyleticgroups of the Hemibagrus gene tree are approximatelyequivalent to the remaining species composing the Hnemurus species group as defined by NampK Dodson etal [10] and Ng and Dodson [11] have previously referredto clade 10 as the Sundaic clade as most sequences wereobtained from fish sampled on the Greater Sunda Islandsand clade 11 as the Indochinese clade asmany sequences wereobtained from fish sampled on the Indochinese mainland(Figure 2 Table S1) Although most of these sequencesare assigned to H capitulum by NampK there are somesignificant phylogeographic differences between the 2 cladesIn particular there appears to be an east-west division in thedistribution of the two clades on the Thai-Malay Peninsulawith the Sundaic clade (clade 10) found on the west coast andthe Indochinese clade found on the east coast with sympatricoccurrence in the Muar River Nevertheless mean sequencedivergence between the two clades is 119901 = 29 (05) avalue that is indicative of population-level or subspecies

divergence The two clades are thus best considered ascomprising one species

mtDNA Clade 10 The Sundaic clade includes a number ofclosely related sequences that comprise part of H capitulumand H nemurus Very little genetic structure is apparentand bootstrap support for tree topology within this group isgenerally very weak (Figure 11) A widely spread genotype(NCLCTBA20) characterized 6 of 17 specimens from CiTarum at the Cirata reservoir (West Java) (Table S1) and5 were identified as H nemurus in the analysis of NampK(ZRC 42564) (Table S1) The remaining fish characterized bythis sequence were obtained from Ci Liwong at Sawangan(West Java) and one fish was obtained from the Barito Riverat Banjarmassin south Borneo This latter specimen (ZRC40553) was identified by NampK as H capitulum (Table S1)Another common genotype (NBHKPBR09) was identified aseither H capitulum H hoevenii or H nemurus dependingon the river of origin (Table S1) Many of the remainingsequences of the Sundaic clade appear to characterize fish thatNampK identified asH capitulum A geographically widespreadgenotype (CPEBEMUKA36) characterized 9 fish sampledon the Perak River at Gerik that were identified by NampKas H capitulum (ZRC 41150 Table S1) Another commongenotype within this clade (CABHMS20) included 2 fishfrom the Batang Hari at Jambi and identified by NampK asH capitulum (ZRC40534 Table S1) Another 2 sequences(CABAR0203 CABAR0305) characterized 8 fish sampledon the Barito River at Banjarmasin and were identified as

14 Advances in Evolutionary Biology

100∘

10∘

110∘

0∘

120∘

0

0 250

250

500

500

750(km)

(mi)

53

64

99

85

64

0002

HJOMSKA11

HOBAH0601

HOBHMS05

HMUBHKABM23

HOBAH0701

HOKAP0501

H hoevenii ldquoBrdquo

H hoevenii ldquoArdquo

Shelf minus75mShelf minus120 m

Figure 10 Phylogenetic relationships withinH hoevenii comprised of 2 subclades and 6 sequences The subtree is extracted from the taxonidentification phenogram inferred using theNeighbor-Joiningmethod (see Figure 2)The percentage of replicate trees in which the associatedsequences clustered together in the bootstrap test (500 replicates) is shown next to the branches The distribution of these sequences isillustrated in the right panel

110∘

XXMAH0110

CABAR0305

96 CAKPR0201

74CAKAP0401

5931 CAKAP0601

35 CPEBEMUKA36

646 CAKAP0903

33 CAKAP080114 CAKAP0701

CARAJ0210

4 CABAR0203CABRA0204

20

CBHKPBR0919

CABRA0301

15CABHMS20

24 NCLCTBA20

63 NECIT0201

H capitulumldquoSundaic claderdquo

H nemurus

0002 120∘

0 250 500 750

(km)

0 250 500

(mi)100

10∘

0∘

Shelf minus75mShelf minus120 m

Figure 11 Phylogenetic relationships within theH capitulum ldquoSundaicrdquo clade including 2 sequences associated withH nemurusThe subtreeis extracted from the taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicatetrees in which the associated sequences clustered together in the bootstrap test (500 replicates) is shown next to the branchesThe distributionof these sequences is illustrated in the right panel

Advances in Evolutionary Biology 15

H capitulum

0∘

10∘

100∘

110∘

120∘

66

63

88

59

38

39 68

71

6991

57

9754

88 4944

39

53

89

0002

ldquoIndochineserdquogroup ldquoErdquo

H capitulumldquoIndochineserdquo

group ldquoDrdquoH capitulumldquoIndochineserdquo

group ldquoCrdquo

H capitulumldquoIndochineserdquo

group ldquoBrdquo

H capitulumldquoIndochineserdquo

group ldquoArdquo0

0

250

250

500

500

750

(km)

(mi)

CATER0301CATERR0401

CKETEMU23

CAMEK0601CACHP0301

CAMKPA03

CAMEK0910CAMEK1003

XXMUBM02

CABMTU09

CABRM0601

CAKAP1201CAKAP1001

CAKAP1101CAKAP1301

CABRM0501

CASAD0102

CASAD0220

CAEND0301

CABAH0801

CAPABH05CATEEN03

CATER0701CATERR0801

CAKETE04CATER0501

CAPAH0303

Shelf minus75mShelf minus120 m

Figure 12 Phylogenetic relationships within theH capitulum ldquoIndochineserdquo clade encompassing 5 subclades identified as A B C D and EThe subtree is extracted from the taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2)The percentageof replicate trees in which the associated sequences clustered together in the bootstrap test (500 replicates) is shown next to the branchesThedistribution of these sequences is illustrated in the right panel

H capitulum by NampK (ZRC 40553 Table S1) Although notincluded in the analysis of NampK samples from the Sundaicclade were also found in the Kapuas River (West Borneo)Finally 10 fish sampled on the Mahakam River at Samarindaeast Borneo are all characterized by one relatively divergentgenotype (CAMAH0110) butwere considered asH capitulumin the analysis of NampK (ZRC 40533 Table S1)TheMahakampopulation sample is the most genetically distinct of theSundaic clade (119901 = 17 (04)) but nevertheless indicativeof intraspecific population-level divergence (Figure 12)

The topology of the Sundaic clade does not permit usto draw a clear distinction between H nemurus and theSundaic H capitulum The mean within-group divergenceof the Sundaic clade is only 119901 = 08 (02) well withinpopulation-level divergence values We postulate that Hnemurus may be one of several regional variants of awidespread Sundaic H capitulum clade that has radiatedrelatively recently This may explain the existence of manyshared sequences throughout the Sundaic islands assigned todifferent species in different regions

mtDNA Clade 11 Finally the Indochinese clade of Hcapitulum represents a geographically widespread andweakly diversified clade (within-group mean sequencedivergence 119901 = 11 (02) Figure 12) Although few fishfrom this genetic clade were included in the morphological

analysis of NampK most may be considered as H capitulumgiven their geographic distribution and close geneticrelationship to the Sundaic H capitulum Four sequencesin this clade were identified as H capitulum in the analysisof NampK 3 specimens characterized by one sequence(CABMTU09) found in the Tutong River of westernBorneo (ZRC 40482) 6 characterized by the same sequencefrom the Baram River (ZRC 40498) another 2 sequencesfrom the Baram River (ZRC 40498) and 20 specimenscharacterized by an abundant genotype (CASAD0220)found at 2 sites in the Sadong River also in western Borneo(ZRC 39526 5 specimens 39527 3 specimens and 38753 4specimens) (Table S1) Unfortunately none of the specimenscollected on the Indochinese mainland for genetic analysiswere examined by NampK Nevertheless the distribution ofspecimens analyzed for mtDNA resembles the distribution ofspecimens examined by NampK Although weakly structuredwe may recognize 5 subclades One genotype (CAPAH0303)is basal to the phenogram Subclade A encompasses 8sequences mainly found in eastern Peninsular Malaysia(Table S1 Figure 12) These rivers furnished many of thespecimens NampK examined to define H capitulum Howeversome sequences in this group occur in sympatry withsequences identified within the Sundaic H capitulum groupTwo sequences from subclade A occurred in the Batang HariSumatra (Table S1) Subclade E also encompasses sequences

16 Advances in Evolutionary Biology

found in Peninsular Malaysia as well as in the Mekong ChaoPhraya and Mea Khlong Rivers (Figure 12) In addition3 subclades encompass sequences found principally inwestern Borneo subclade B in the Baram and Sadong RiversSarawak subclade C in the Kapuas River West Kalimantanand subclade D in the Baram and Tutong Rivers of Sarawakand Brunei respectively (Figure 12) This latter subclade alsoincludes one genotype found in the Muar River of westernpeninsular Malaysia Finally subclade E encompasses 5sequences from the Mekong Chao Praya and Mae KhlongRivers all within the distribution area of H spilopterus

EvolutionaryHistoryThe evolutionary history of theHemiba-grus species groups analyzed here dates from at least theMiocene 15ndash20Ma (Figure 13) when considering the widelyused molecular clock of 2 sequence divergence per millionyears Bootstrap support for the sister group relationships ofH wyckioides theH guttatus species group andH peguensisare all weak (clearly due to undersampling) and little can besaid of their evolutionary history other than the fact that theyoccupy basal positions in the rooted trees The remainder ofboth the NJ and ML trees is composed of two major sistergroups the H planiceps + H baramensis group and the Hnemurus species group (Figures 2 3 and 13)TheH planiceps+ H baramensis group is strongly supported in both NJ andML analyses (84 and 90 bootstrap support resp Figures2 and 3) The H nemurus species group is also stronglysupported in both analyses (98 and 99 bootstrap supportFigures 2 and 3) These two major phylogenetic groups areestimated to have diverged at least 8 to 12Ma ago during themid to late Miocene (Figure 13)

Much of the evolutionary history of Hemibagrus hasoccurred during the Quaternary starting approximately26Ma ago This is particularly true of the H planicepsand H nemurus species groups The radiation of the 5species included in the H planiceps species group appearsto coincide with the middle Pleistocene transition period ofapproximately 1Ma ago Their high-elevation distributionhowever appears to have largely isolated them from theeffects of lowered sea levels during glacial maxima and theattendant opportunities for widespread dispersal (Figure 5)The evolutionary histories of theH baramensis species groupand the unspecified species group date back to the latePliocene In contrast the low-elevation distribution of theHnemurus species group in Sundaland appears to have favoredtheir widespread distribution during the Quaternary ice ageIn particular H hoevenii appears to be the most recentlydispersed of the species under study with diversification anddispersal occurring approximately 03Ma ago (Figure 13)

4 Discussion

41 The Congruence of Morphological and Genetic AnalysesCombining genetic and morphological analyses to elucidatetaxonomy allowed us to better understand the diversificationand evolutionary relationships within this speciose group offishes Given the rapid and relatively constant rate of mtDNAevolution the deeper the branches within the phylogenetictree are the greater our ability to distinguish valid taxa

should be Conversely the lack of complete congruencebetween morphological- and genetic-based identificationsis expected to increase among the shallower branches of thephylogenetic tree resulting in the appearance of polyphyleticspecies Although 2 of the 8 species groups defined by Ngand Kottelat [7] were not sampled in this study the validityof the remaining 6 species groups and their componentspecies defined on the basis of morphological similaritywas largely confirmed by the analysis of the mitochondrialCyt 119887 bar code As expected congruence is strongest at thebase of the NJ and ML trees where the H guttatus speciesgroup formed a well-defined monophyletic group The Hmenoda and H wyckii species group were each representedby only one species but they were clearly distinct relativeto sequences characterizing the neighboring species groupsThe H planiceps H baramensis and H nemurus speciesgroups and their component species were also generallycongruent in morphological and genetic analyses Also asexpected most cases of discrepancy between morphological-based species identification and genetic-based speciesidentification occurred on relatively shallow branches of thephylogenetic tree These involve principally H spilopterusand the two mtDNA subclades composing H capitulumHowever a far more unexpected lack of congruence involvesthe definition of a deeply divergent unspecified genetic groupconfounding members of theH baramensis species group inthe morphological analysis of Ng and Kottelat [7]

Interpreting Polyphyly The apparent polyphyletic origin ofa species may be due to incomplete lineage sorting amongrecently diverged species the introgression of mtDNA dueto species hybridization or the erroneous identificationof species due to the lumping of morphologically crypticspecies In the absence of nuclear DNA analyses it isoften difficult to distinguish between introgression andincomplete lineage sorting For example several sequencescharacterizing the Indochinese clade of H capitulum(subclade E) are found in the Mekong and Chao PhrayaRivers well within the distributional area of H spilopterusH spilopterus and H capitulum first diverged at least 3MaOn the one hand this apparent case of polyphyly mayhave resulted from incomplete lineage sorting betweenrecently divergedH spilopterus andH capitulum We wouldhowever have expected similar cases of polyphyly amongpopulations of the Indochinese clade of H capitulum butthis was not detected On the other hand the sympatricoccurrence of H spilopterus subclade A and subcladeB sequences with H capitulum Indochinese subclade Esequences in the Mekong River may represent a migrationevent and local introgression of H capitulum with Hspilopterus A similar event may have occurred in theChao Phraya and Mae Khlong Rivers where H spilopterussequences cooccur withH capitulum sequences Computingthe divergence of H capitulum sequences (Indochinesesubclade E) cooccurring with H spilopterus and Hcapitulum sequences occurring in eastern PeninsularMalaysia (subclade A) reveals a mean sequence divergence119901 = 15 (04) Thus the H capitulum sequences occurringin sympatry with H spilopterus diverged at most 750 ka ago

Advances in Evolutionary Biology 17

H fortis

XXRAJ0604H furcatus

H sabanusH semotusH divaricatus

H planicepsGREND0206

VEBAH0907

H bonganPESAL0102

H wyckioidesH macropterus

H guttatusXXRAJ0301Leiocassis longirostris

Ma

4971

92

47

51

9999

99

9999

99

99

99

9999

99

99

99

99

65

96

96

4946

93

79

78

70

91

93

98

86

87

91

95

97

71

85

9999

05101520

005010015020 000

H capitulum Indochinese clades AndashE

H capitulum Sundaic clade

H hoevenii B

H hoevenii A

H spilopterus C

H spilopterus B

H spilopterus AH spilopterus A

H spp CH spp B

H spp A

Figure 13 Linearized tree with branch lengths estimated with the molecular clock using a mutation rate of 1 per Ma (=divergence rate of2 per Ma) The tree topology is identical to the ML reconstruction illustrated in Figure 3 (see the specificities of the tree reconstruction inthe caption of Figure 3) Boxes represent 95 confidence intervals of divergence time estimates for each node Vertical dashed lines redirectto dates indicated on the timescale (Ma) Numbers below timescale indicate the estimated number of substitution changes using Hasegawa-Kishino-Yanomodel (HKY)with gamma distribution (+119866 = 13290) after linearizing the treeH furcatus is synonymizedwithH baramensis

from eastern Peninsular Malaysia populations suggestinga recent introgression event Similarly H capitulum in theKapuas River is characterized by sequences of both theSundaic and the Indochinese clades As both sequences arebroadly sympatric it is not possible to distinguish betweenincomplete lineage sorting and introgression of these closelyrelated intraspecific lineages

A Possible Case of Morphological Stasis Clade 6 of thepresent analysis defines a well-supported monophyletic cladefound in Sabah Sarawak and the Endau River of easternpeninsula Malaysia that has no equivalent taxonomic statusin the revision of Ng and Kottelat [7] In Borneo they occursympatrically with the H baramensis species group and areconfounded as either H baramensis or H semotus These

two clades are genetically highly divergent having divergedbetween 8 and 12Ma ago It thus seems highly unlikely thatthey represent retained polymorphisms due to incompletelineage sorting Sequences previously identified as character-izing H chrysops [11] are part of clade 6 and extend to thesouth (Rajang and Sadong Rivers of Sarawak West Borneo)with one closely related genotype found in eastern peninsularMalaysia (Endau River) where it may well be confoundedwith H capitulum H chrysops was synonymized with Hcapitulum by Ng and Kottelat [7] based on their analysis ofspecimens ofH chrysops from the Rajang RiverThus despitea long and apparently independent phylogenetic historythe unspecified species group appears largely cryptic andconfounded principally with members of the H baramensisspecies group

18 Advances in Evolutionary Biology

We suggest that this situation is an example of mor-phological stasis involving the coexistence of a number ofgenetically divergent yet morphologically cryptic species thatare lumped together [24] A striking example of morpho-logical and ecological stasis among fishes is provided by thecoastal marine bonefishes (Albula spp) Distinct evolution-ary lineages (separated by sequence divergence between 5 and30 [25]) of bonefish coexist in several sample locations yetthey show little morphological or ecological differentiationin sympatry [25] Similarly a proportion of the grey mulletspecies (Mugilidae) also appears to consist of cryptic species[26] For exampleMugil cephalus in Taiwan is characterizedby three divergent lineages that have been shown to charac-terize 3 reproductively isolated but morphologically crypticspecies [26 27]

Here we have discovered 2 highly distinct and structuredclades (each composed of 3 mtDNA subclades) that aremorphologically confounded within different species Forexample H spp C of the unspecified species group isidentified as H baramensis in the Baram River and as Hsemotus in the Padas River We speculate that North Borneoharbors a morphologically cryptic species complex possiblycomposed of several species pairs inhabiting different riversystems The reconstruction of this phylogenetic history andthe taxonomic status of its components remains a majorchallenge to the revision of the Hemibagrus genus

42 Phylogeographic Hypotheses The phylogeography ofthe genus appears to have been principally influenced bytwo geological histories one encompassing the formationof North Borneo the second the emergence of the SundaIslands and their connectivity with the Indochinesemainlandassociated with recurrent sea-level changes North Borneoappears to have acted as a species sink promoting speciationand locally high biodiversity within the genus but with littleevidence of subsequent dispersal out of North Borneo Incontrast the geological events influencing the Indochinesemainland and the Sunda Islands have permitted bothspeciation and differential dispersal depending on thebiology of the species For example the H planiceps speciesgroup is found throughout the Greater Sunda Islands andtheThai-Malay Peninsula as is theH nemurus species groupWhereas the H nemurus species group generally occupiesthe low altitude lower reaches of large rivers the speciescomposing the H planiceps group are found in the upperreaches of large rivers The H nemurus species group iscomposed of a number of widely distributed closely relatedspecies whereas theH planiceps group is composed of a largenumber of species that are highly localized geographicallyIt thus appears that sea-level changes have had little impacton the dispersal history of the H planiceps species group buthave deeply influenced that of the H nemurus species group

The Case of North Borneo From 15Ma to 5Ma greater areasof Borneo became emergent and the central mountainson the Sarawak-Kalimantan border extending into Sabahbecame wider and higher which may have represented newbarriers to dispersal [28 29] Borneo gradually developedinto the present large island by emergence of land in the

north and east The H baramensis species group is restrictedto North Borneo as is the unspecified species group of theHnemurus species group H fortis is also distributed in NorthBorneo The isolation of the freshwater fish communities ofNorth Borneo (particularly in the Labuk-Segama watershed)has long been recognized based on the number of endemicspecies and the concentration of relict species [30] The latePliocene-early Pleistocene diversification of H fortis theunspecified species group and the H baramensis speciesgroup may thus be associated with their dispersal to andisolation within the watersheds of North Borneo Only onesequence of the unspecified species group is found outsideof North Borneo and H fortis appears to be distributedthroughout central and east Borneo [7]

ThePhylogeography of the H nemurus Species Group Periodicmarine transgressions on the Sundaland platform during theearly and middle Miocene [2 29] may have been sufficientto isolate populations of freshwater organisms and promotecladogenesis [31] This may have been responsible for theinitial mid to late Miocene divergence of the H nemurusspecies group The majority of sequences assigned to thisspecies group belong to 3 species (H spilopterus H capitulum(including H nemurus) and H hoevenii) all of which havebeen influenced by the more recent geological history of theSunda Islands and their connectivity with the Indochinesemainland H spilopterus is known from river drainages inIndochina including the Mekong Dong Nai Ban PakongChao Phraya and Mae Klong where it is found in a widevariety of lotic and lentic habitats [7] H spilopterus divergedin the late Pliocene-early Pleistocene possibly isolated tothe north of the Kra Peninsula during the late Pliocenemarine transgression that is purported to have created amajor biogeographic transition zone between the Sundaicand Indochinese biotas ([32] but see [33] for a critique ofthis biogeographic scenario) The existence of 2 subclades(A and B) in the Mekong River separated by 2 sequencedivergence is consistent with intraspecific divergence Thetwo subclades possibly represent 2 historical mtDNA lineagesthat are retained within the species reflecting a Pleistoceneisolation event followed by secondary contact within thecontemporary Mekong River Subclade C from the ChaoPhraya and Mae Khlong Rivers exhibits a level of geneticdivergence consistent with sibling species-level divergenceshowing a 5 sequence divergence relative to Mekong RiverH spilopterus

H capitulum may be considered as part of the Sundaicbiota located principally to the south of the Kra marinetransgression zone that may have isolated H spilopterus tothe north on the Indochinese mainlandThe biogeographicalevent leading to the subsequent formation of two intraspecificgroups within H capitulum is not at all clear The onlyarea where the two clades are clearly geographically separateis on the Thai-Malay Peninsula with the ldquoSundaicrdquo cladeon the west coast and the ldquoIndochineserdquo clade on theeast coast separated by the central Titiwangsa mountainrange The genetic distinctiveness of east and west coastpopulations of H capitulum has also been documented fornuclear DNA [34] A similar pattern of separation has been

Advances in Evolutionary Biology 19

reported for 5 additional freshwater fishes within this region[35] However as the divergence of the two H capitulumclades postdates the formation of the mountain range byapproximately 200Ma the geographic separation of the twoclades most likely represents differential dispersal rather thanancient vicariance The presence of the Sundaic clade on thewest coast of the Thai-Malay Peninsula as well as on theSundaic Islands reflects the historical connections betweeneast Sumatra and west Peninsular Malaysia across the Straitof Malacca This area encompasses the Northern CentralSumatra-Western Malaysia freshwater ecoregion [36] TheMuar River forms its southern border coincident with thesouthernmost distribution of the Sundaic clade on the Thai-Malay Peninsula It is important to note however that mostof Sumatra and Java emerged to achieve their present size andelevation only 5Ma ago and much of East Java continuedto be the site of marine deposition until late Pliocene-earlyPleistocene [29] We may speculate that the Sundaic clade ofH capitulum resulted from the relatively recent colonizationof these islands and Borneo The radiation within the cladeonly dates back to approximately 1Ma ago H nemurus fromWest Java may thus best be considered an isolated regionalpopulation of very recent origin

The diversification of the Indochinese clade dates fromat least 12Ma ago coincident with the mid-Pleistocenetransition at which time low-frequency high-amplitude andquasiperiodic (100 ka) glacial variability emerged It began125Ma ago and was complete by 07Ma ago [37] The tran-sition marked the beginning of major changes in sea levelsduring which the Sunda Shelf periodically emerged and wastraversed by enormous fluvial systems [3] The contributionof these events to the diversification of this clade cannotbe satisfactorily reconstructed One possible explanation isthat high sea levels produced barriers to gene flow and thepopulations of West Borneo (subclades B C and D) wereisolated from those of east Peninsular Malaysia This couldbe due to one or more dispersal events from Borneo via theGreat Sunda River (Figure 1) to east Peninsular Malaysia orlineage sorting whereby sequences of subclades A and E havebeen lost from West Borneo As in the case of the sympatricoccurrence of H spilopterus and H capitulum subclade E(see above) in the Mekong River we are unable to clearlydefine the relative roles of incomplete lineage sorting andintrogression followingmigration Given the recent origins ofthe Indochinese H capitulum sequences incomplete lineagesorting most probably blurs patterns of recent isolation

The last species of the H nemurus species group is Hhoevenii The species is widespread throughout PeninsularMalaysia and the Sundaic Islands It is found primarily inthe lower reaches of rivers and is apparently capable ofwithstanding low salt concentration [7] H hoevenii thusappears to be the most liable of all members of the Hnemurus species group to be impacted by Pleistocene sea-level changes This is clearly reflected in the widespreaddistribution of the sequences of this species Two lineages areevident having diverged no more than 03Ma ago suggest-ing a late Pleistocene isolation event followed by extensivedispersal and admixture in recent times This represents theclearest example of the dispersal opportunities afforded by

Pleistocene sea level changes on the Sunda Shelf In additionthe relatively low genetic diversity detected (only 6 sequencesin 42 fish) suggests a population bottleneck in the recent pastpossibly related to population declines in the lower reaches ofrivers flooded by increasing sea levels

43 Future Directions We have provided a global picture ofthe evolutionary taxonomy of the genus in Southeast AsiaClearly a more rigorous phylogenetic reconstruction of thegenus Hemibagrus requiring the construction of multiplegene trees based on the analysis of both mitochondrial andnuclear DNA markers from a wider taxonomic array ofhemibagrids will involve an even greater effort of samplingthroughout the region In particular we require samplingat smaller spatial and temporal scales For example smallerscale sampling in the Padas River and the Segama Riverof North Borneo using both mitochondrial and nucleargenetic markers would contribute to clarifying the natureof the cryptic species complex believed to inhabit theserivers The demonstration of reproductive isolation andormicroallopatric distribution among morphologically crypticspecies may result in the description of greater speciesrichness within the genus than presently described Thespecies status of H chrysops merits reconsideration givenits highly divergent evolutionary history Nuclear geneticmarkers in conjunction with mtDNA markers could revealcases of historical introgression particularly in the KapuasRiver and the Mekong River The hemibagrid catfishes con-tinue to provide multiple opportunities for the study of theevolutionary taxonomy and biogeography of a major faunalcomponent of the fluvial ecosystems of Southeast Asia

Disclosure

Ethanol-preserved tissue samples of many of the specimensidentified in Table S1 are freely available upon request fromJJD

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Goh Yan Yih for assistance in collectingspecimens and molecular analyses Mrs Francoise Colom-bani for molecular analyses and Dr Heok Hee Ng forspecies identification This work was funded by the NationalUniversity of Singapore grant to P K L Ng and a CanadianNatural Sciences and Engineering Research Council grant toJ J Dodson

References

[1] D J Lohman M de Bruyn T Page et al ldquoBiogeography ofthe Indo-Australian archipelagordquo Annual Review of EcologyEvolution and Systematics vol 42 pp 205ndash226 2011

20 Advances in Evolutionary Biology

[2] D S Woodruff ldquoNeogene marine transgressions palaeogeog-raphy and biogeographic transitions on the Thai-Malay Penin-sulardquo Journal of Biogeography vol 30 no 4 pp 551ndash567 2003

[3] H K Voris ldquoMaps of Pleistocene sea levels in SoutheastAsia shorelines river systems and time durationsrdquo Journal ofBiogeography vol 27 no 5 pp 1153ndash1167 2000

[4] S Y Yap ldquoOn the distributional patterns of Southeast-EastAsian freshwater fish and their historyrdquo Journal of Biogeographyvol 29 no 9 pp 1187ndash1199 2002

[5] S K J McConnell ldquoMapping aquatic faunal exchanges acrossthe Sunda shelf South-East Asia using distributional andgenetic data sets from the cyprinid fish Barbodes gonionotus(Bleeker 1850)rdquo Journal of Natural History vol 38 no 5 pp651ndash670 2004

[6] T Mo Anatomy Relationships and Systematics of the Bagridae(Teleostei Siluroidei) with a Hypothesis of Siluroid Phylogenyvol 17 ofTheses Zoologicae Koeltz Scientific Books 1991

[7] H H Ng and M Kottelat ldquoRevision of the Asian catfish genusHemibagrus bleeker 1862 (Teleostei Siluriformes Bagridae)rdquoRaffles Bulletin of Zoology vol 61 no 1 pp 205ndash291 2013

[8] P K L Ng and H H Ng ldquoHemibagrus gracilis a new speciesof large riverine catfish (Teleostei Bagridae) from PeninsularMalaysiardquo Raffles Bulletin of Zoology vol 43 pp 133ndash142 1995

[9] H H Tan and M Kottelat ldquoRedescription of Betta picta(Teleostei Osphronemidae) and description of Betta falx sp nfrom central Sumatrardquo Revue Suisse de Zoologie vol 105 no 3pp 557ndash568 1998

[10] J J Dodson F Colombani and P K Ng ldquoPhylogeographicstructure in mitochondrial DNA of a South-east Asian fresh-water fish Hemibagrus nemurus (Siluroidei Bagridae) andPleistocene sea-level changes on the Sunda shelfrdquo MolecularEcology vol 4 no 3 pp 331ndash346 1995

[11] H H Ng and J J Dodson ldquoMorphological and genetic descrip-tions of a new species of catfish Hemibagrus chrysops fromSarawak East Malaysia with an assessment of phylogeneticrelationships (Teleostei Bagridae)rdquo Raffles Bulletin of Zoologyvol 47 no 1 pp 45ndash57 1999

[12] P D N Hebert A Cywinska S L Ball and J R DeWaardldquoBiological identifications through DNA barcodesrdquo Proceedingsof the Royal Society B Biological Sciences vol 270 no 1512 pp313ndash321 2003

[13] J C Carolan T EMurray U Fitzpatrick et al ldquoColour patternsdo not diagnose species quantitative evaluation of a DNAbarcoded cryptic bumblebee complexrdquo PLoS ONE vol 7 no 1Article ID e29251 2012

[14] M Kekkonen and P D N Hebert ldquoDNA barcode-baseddelineation of putative species efficient start for taxonomicworkflowsrdquoMolecular Ecology Resources vol 14 no 4 pp 706ndash715 2014

[15] DM Hillis CMoritz and B KMarbleMolecular SystematicsSinauer Sunderland Mass USA 2nd edition 1996

[16] T D Kocher W K Thomas A Meyer et al ldquoDynamics ofmitochondrial DNA evolution in animals amplification andsequencingwith conserved primersrdquoProceedings of theNationalAcademy of Sciences of the United States of America vol 86 no16 pp 6196ndash6200 1989

[17] T A Hall ldquoBioEdit a user-friendly biological sequence align-ment editor and analysis program for Windows 9598NTrdquoNucleic Acids Symposium Series vol 41 pp 95ndash98 1999

[18] D A Benson I Karsch-Mizrachi K Clark D J Lipman JOstell and EW Sayers ldquoGenBankrdquoNucleic Acids Research vol40 no D1 pp D48ndashD53 2012

[19] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[20] X Ku Z Peng R Diogo and S He ldquoMtDNA phylogenyprovides evidence of generic polyphyleticism for East Asianbagrid catfishesrdquoHydrobiologia vol 579 no 1 pp 147ndash159 2007

[21] L Yang and S He ldquoPhylogeography of the freshwater catfishHemibagrus guttatus (Siluriformes Bagridae) implications forSouth China biogeography and influence of sea-level changesrdquoMolecular Phylogenetics and Evolution vol 49 no 1 pp 393ndash398 2008

[22] B W Bowen A L Bass L A Rocha W S Grant and DR Robertson ldquoPhylogeography of the trumpetfishes (Aulosto-mus) ring species complex on a global scalerdquo Evolution vol 55no 5 pp 1029ndash1039 2001

[23] Y P Kartavtsev ldquoDivergence at Cyt-b and Co-1 mtDNA geneson different taxonomic levels and genetics of speciation inanimalsrdquoMitochondrial DNA vol 22 no 3 pp 55ndash65 2011

[24] SWada Y Kameda and S Chiba ldquoLong-term stasis and short-term divergence in the phenotypes of microsnails on oceanicislandsrdquoMolecular Ecology vol 22 no 18 pp 4801ndash4810 2013

[25] J Colborn R E Crabtree J B Shaklee E Pfeiler and B WBowen ldquoThe evolutionary enigma of bonefishes (Albula spp)cryptic species and ancient separations in a globally distributedshorefishrdquo Evolution vol 55 no 4 pp 807ndash820 2001

[26] J-D Durand K-N Shen W-J Chen et al ldquoSystematics of thegrey mullets (Teleostei Mugiliformes Mugilidae) molecularphylogenetic evidence challenges two centuries ofmorphology-based taxonomyrdquo Molecular Phylogenetics and Evolution vol64 no 1 pp 73ndash92 2012

[27] K-N Shen B W Jamandre C-C Hsu W-N Tzeng and J-D Durand ldquoPlio-Pleistocene sea level and temperature fluc-tuations in the northwestern Pacific promoted speciation inthe globally-distributed flathead mullet Mugil cephalusrdquo BMCEvolutionary Biology vol 11 no 1 article 83 2011

[28] R Hall ldquoThe plate tectonics of Cenozoic SE Asia and thedistribution of land and seardquo in Biogeography and GeologicalEvolution of SE Asia R Hall and J D Holloway Eds pp 99ndash131Backhuys Publishers Leiden The Netherlands 1998

[29] R Hall ldquoSoutheast Asiarsquos changing palaeogeographyrdquo Blumeavol 54 no 1ndash3 pp 148ndash161 2009

[30] R F Inger and P K Chen ldquoThe freshwater fishes of NorthBorneordquo Fieldiana Zoology vol 45 pp 1ndash268 1962

[31] J Bohlen V Slechtova H H Tan and R Britz ldquoPhylogenyof the Southeast Asian freshwater fish genus Pangio (Cyprini-formes Cobitidae)rdquoMolecular Phylogenetics and Evolution vol61 no 3 pp 854ndash865 2011

[32] M de Bruyn E Nugroho M M Hossain J C Wilson andP B Mather ldquoPhylogeographic evidence for the existence ofan ancient biogeographic barrier the Isthmus of Kra SeawayrdquoHeredity vol 94 no 3 pp 370ndash378 2005

[33] J Parnell ldquoThe biogeography of the Isthmus of Kra region areviewrdquoNordic Journal of Botany vol 31 no 1 pp 001ndash015 2013

[34] S Usmani S G Tan S S Siraj and K Yusoff ldquoPopulationstructure of the Southeast Asian river catfishMystus nemurusrdquoAnimal Genetics vol 34 no 6 pp 462ndash464 2003

[35] M P Tan A F J Jamsari and M N Siti Azizah ldquoPhylo-geographic pattern of the striped snakehead Channa striatain Sundaland ancient river connectivity geographical and

Advances in Evolutionary Biology 21

anthropogenic singnaturesrdquo PLoS ONE vol 7 no 12 Article IDe52089 2012

[36] R Abell M L Thieme C Revenga et al ldquoFreshwater ecore-gions of the world a new map of biogeographic units forfreshwater biodiversity conservationrdquo BioScience vol 58 no 5pp 403ndash414 2008

[37] P U Clark D Archer D Pollard et al ldquoThemiddle Pleistocenetransition characteristics mechanisms and implications forlong-term changes in atmospheric pCO

2rdquo Quaternary Science

Reviews vol 25 no 23-24 pp 3150ndash3184 2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 6: ResearchArticle - Université Laval Julian... · ResearchArticle A DNA Barcode-Based Evaluation of the Southeast Asian Catfish Genus Hemibagrus Bleeker, 1862 (Teleostei: Siluriformes;

6 Advances in Evolutionary Biology

relationships among taxa To do so the Cyt 119887 sequence of theChinese longsnout catfish (Leiocassis longirostris) obtainedfrom GenBank (accession number DQ3217551) was usedas an outgroup to root the phenogram Chinese speciescurrently assigned to Leiocassismay be considered membersof the genus Pseudobagrus [20] Furthermore we employeda maximum likelihood (ML) phylogenetic reconstructionbased on the comparison of nucleotides within sites to assessthe congruence of the taxon identification phenogram andthe ML phylogenetic reconstruction In the latter case theevolutionary model used was the Hasegawa-Kishino-Yanomodel (HKY)with gammadistribution (+119866 = 13290) Initialtree(s) for the heuristic search were obtained by applyingthe Neighbor-Joining (NJ) method to a matrix of pairwisedistances estimated using the maximum composite likeli-hood (MCL) approach The rate-variation model allowedfor some sites to be evolutionarily invariable (+119868 4644of sites) The ML tree was rooted with the Cyt 119887 sequenceof the Chinese longsnout catfish (Leiocassis longirostris) andconstructed using MEGA52 [19]

TheNJ phenogram classifiedmtDNA sequences based onoverall similarity Ng and Kottelat [7] also defined speciesgroups based on overall similarity but according to mor-phological criteria Of the 8 species groups defined by Ngand Kottelat [7] two groups (H olyroides (1 species) and Hpluriradiatus (5 species)) were not included in our samplesWe thus evaluated the congruence of the two methods indefining the remaining six species groups (H baramensisH guttatus H menoda H nemurus H planiceps and Hwyckii species groups) When possible mtDNA sequenceswere linked to ZRC accession numbers identifying specimensexamined by Ng and Kottelat [7] in their morphologicalrevision of the genus (Table S1)

To provide time estimates for the major phylogeneticevents we present a calibrated linearized tree obtained fromour ML reconstruction (MEGA 52) Time of divergencebetween Hemibagrus lineages has been approximated byapplying a rate of between 10 and 15 genetic divergence permillion years as reported for bagrid fish by Yang and He [21]However the justification for this rate is not clear We thusapplied the more widely used rate of 2 sequence divergenceper million years [22] As there is undoubtedly a wide errorassociated with any such molecular clock calibrations esti-mated divergence times are used to discriminate more recentfrom more ancient cladogenetic events while providing veryapproximate dates for such events

Genetic divergence estimates using 119901-distances (119901) wereused in the present study to identify the different taxonomiclevels observed among the Hemibagrus spp analyzed Sincesuch information is readily available for thousands of animalgroups [23] we used 119901-distance (119901) as a benchmark for taxo-nomic levelsThe survey of 20 731 vertebrate and invertebratespecies reported by Kartavtsev [23] revealed increasing levelsof 119901-distances () of the mtDNA Cyt 119887 gene with taxonomiclevel populations within species 119901 = 138 plusmn 030 (SD)subspecies or sibling species119901 = 510plusmn091 morphologicallydistinct species within genera 119901 = 1031 plusmn 093 generawithin a family 119901 = 1786 plusmn 136 and families within anorder 119901 = 2636 plusmn 388 Although heterogeneity in the

gene evolution rate differs amongdifferent taxonomic groupsthese estimates provide an order of magnitude evaluationof intraspecific versus interspecific divergence and permittedus to discriminate between population- and species-leveldivergences However as 119901-distances underestimate truegenetic distance (eg site saturation and back-mutation)divergence times based on119901-distances greater than about 10are generally underestimated We calculated 119901-distances inMEGA 52 and report them as percentages (standard error)

3 Results

The 452 samples yielded a total of 118 distinct sequences(not including the outgroup taxon) The Neighbor-Joiningphenogram clustered the 119 sequences into a large numberof hierarchically organized monophyletic clades To facilitatepresentation we first present a compressed NJ tree andidentify 11 major mtDNA clades (Figure 2) One unidentifiedfish from the Rajang River was characterized by a genotypethat clustered with the outgroup taxon (XXRAJ0604GenBank accession number KP322872 Figures 2 and3) A second fish from the Rajang was characterized byan isolated genotype (XXRAJ0301 GenBank accessionnumber KP322869) of unknown affiliation (Figures 2and 3) To assess the congruence of the species groupsdefined by Ng and Kottelat ([7] hereafter referred toas NampK) and the mtDNA groupings revealed by thepresent analysis we provide a direct comparison of thetwo methods following the order determined by the taxonidentification phenogram (Figure 2 Table S1) Table S1 alsoprovides additional information about the geographicaldistribution of sequences Gene trees of individual cladesincluding all sequences are presented at each step in theanalysis along with their geographical distributions We alsopresent a compressed maximum likelihood phylogenetictree (Figure 3) The phenogram and the ML tree providedcongruent phylogenetic reconstructions However both treesfailed to resolve the phylogenetic relationships of H wyckiiH guttatus and H menoda species groups most likelydue to undersampling of these lineages The relationshipsamong an unspecified species group H spilopterus and Hfortis are also not well resolved Their position in the NJtree receives weak bootstrap support and they appear as amultifurcation along with a clade composed of H capitulumand H hoevenii in the ML phylogenetic tree (Figures 2 and3) To facilitate comparisons with the taxonomic scheme ofNampK we follow the sequence of taxa as identified in Figure 2

mtDNAClade 1The H wyckii Species GroupThe first clade isclearly consistent with theH wyckii species group (Figure 4)This species group is composed of 4 valid speciesHmaydelliH microphthalmus H wyckii andH wyckioides (NampK)Thelatter species is known from the Mekong and Chao Phrayadrainage Only specimens of H wyckioides obtained fromthe Mekong and the Chao Phraya were available for DNAanalysis Three specimens from the Chao Phraya bearingsequence WYCHP0203 (Table S1) were identified as Hwyckioides (H H Ng personal communication) although

Advances in Evolutionary Biology 7

8811

10

9

8

7

6

5

4

3

1

2

66

85

44

32

98

91

81

8432

57

99

99

99

49

002

34

15

43

86

99

96

H capitulum (Indochinese clade)

H capitulum (Sundaic clade)

H hoeveniiH fortis

H spilopterus

H baramensis species group

H planiceps species groupH menoda species group

H guttatus species group

H wyckii species group

XXRAJ0604

PESAL0102

XXRAJ0301Leiocassis longirostris

mtDNAclade

H nemurusspecies group

Unspecified species group

Figure 2 The taxon identification phenogram of Hemibagrus inferred using the Neighbor-Joining method in MEGA 52 The optimal treewith the sumof branch length = 186668598 is shown Bootstrap values are indicated on each node of the tree (500 replicates) Bar indicates thenumber of substitutions changes estimated using Tamura-Nei distance considering invariable site contribution (+I)The analysis involved 119nucleotide sequences All positions containing gaps and missing data were eliminatedThere were a total of 597 positions in the final dataset

mtDNAclade

H capitulum (Indochinese clade) 11

93

97H capitulum (Sundaic clade)H hoevenii

9999

84H fortis 8

9

10

H spilopterus 773 92

Unspecified species group 678

XXRAJ060454 34

90 H baramensis species group 5

49 H planiceps species group 4

H menoda species groupPESAL0102 3

H wyckii species group 199

99

99H guttatus species group 2

XXRAJ030149

47

Leiocassis longirostris

005

H nemurusspecies group

94

72

Figure 3 Evolutionary history of Hemibagrus inferred by using the maximum likelihood method based on the Hasegawa-Kishino-YanomodelThe tree with the highest log likelihood (minus56774825) is shown Bootstrap values are indicated on each node of the tree (500 replicates)Bar indicates the number of substitution changes estimated using Hasegawa-Kishino-Yano model (HKY) with gamma distribution (+119866 =13290) The analysis involved 119 nucleotide sequences All positions containing gaps and missing data were eliminated There were a totalof 597 positions in the final dataset

their ZRC numbers do not appear in the analysis of NampK Asno specimens of the other 3 species comprising the H wyckiispecies group were available for analysis we cannot confirmif this species group is monophyletic

mtDNA Clade 2 The H guttatus Species Group The secondclade of the Hemibagrus gene tree (Figure 2) is composed oftwo reciprocally monophyletic subclades clearly reflectingthe H guttatus species group as defined by NampK (Figure 4)

8 Advances in Evolutionary Biology

100∘

10∘

110∘

0∘

120∘

0

0 250

250

500

500

750(km)

(mi)

002

98

43

84

34

99

99

79

96

51

59

99

78

49

H peguensis

H guttatus

H macropterus

H wyckioides

PESAL0102

XXRAJ0301Leiocassis longirostris

MAPEA0201MAPEA0302MAPEA0101

GUPEA0501GURED0102

GUPEA0401GUPEA0602

WYCHP0401WYMEK1101

WYCHP0203

See Figures 7ndash12

See Figures 5 and 6

Shelf minus75mShelf minus120 m

Figure 4 Phylogenetic relationships of the individual sequences comprising four species H guttatus H macropterus (together forming theH guttatus species group) H wyckioides and H peguensisThe subtree is extracted from the taxon identification phenogram inferred usingthe Neighbor-Joining method (see Figure 2) The percentage of replicate trees in which the associated sequences clustered together in thebootstrap test (500 replicates) is shown next to the branches The distribution of these sequences is illustrated in the right panel

The group comprises three valid species H guttatus Hmacropterus andH vietnamicus (NampK) Specimens availablefor DNA analysis were obtained from the Pearl River andRed River (11 fish 7 sequences Table 1 and Table S1) Thetwo reciprocally monophyletic subclades comprising theH guttatus species group represent H guttatus and Hmacropterus (Figure 4) Although no ZRC specimens wereexamined by NampK species identification was confirmed(H H Ng personal communication) No H vietnamicusspecimens were available for DNA analysis The twospecies composing the group are closely related (119901 = 51(SE = 08))

mtDNA ldquoCladerdquo 3 The H menoda Species Group Thisisolated genotype (Figure 4) most likely identifies a cladeencompassing the H menoda species group although the

single sequence (Table S1) is shared by only 2 specimensof H peguensis from the Salween River Myanmar alsoincluded in the analysis of NampK (where its provenance wasidentified as the neighboring Irrawaddy River) The otherspecies comprising this group (H menoda H caveatusand H punctatus) were not sampled for DNA analysis Themonophyletic nature of this species group thus remains to bedemonstrated

31 The H planiceps + H baramensis Group The remainderof the gene tree is split into two large sister groups withmarginal bootstrap support (73 in ML and 43 in NJ)the H planiceps + H baramensis group and the H nemurusspecies group (Figures 2 and 3) The H planiceps + Hbaramensis group is strongly supported in both NJ and MLanalyses (84 and 90 bootstrap support respectively

Advances in Evolutionary Biology 9

110∘

47

53

69

81

69

5199

52

95

97

78

001

H bongan

H velox

H gracilis

H planiceps

H divaricatus

BOKAP1801

BOKAP1601BOKAP1701

BOKAP1402BOKAP1503

VEBAH0907GREND0206

PLCIM0114PLCIS0101

DIPER0401DIPER0301

DIPER0201

100∘

10∘

0∘

120∘

0

0 250 500

250 500

750(km)

(mi)

Shelf minus75mShelf minus120 m

Figure 5 Phylogenetic relationships of the individual sequences and species comprising theH planiceps species groupH bongan H veloxH gracilis H planiceps and H divaricatus The subtree is extracted from the taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicate trees in which the associated sequences clustered together in the bootstrap test(500 replicates) is shown next to the branches The distribution of these sequences is illustrated in the right panel

Figures 2 and 3) The two groups are genetically similarwith a genetic divergence estimate of 119901 = 121 (SE = 09)Although theH planiceps species group is strongly supportedin both trees (99) bootstrap support of the H baramensisspecies group is weak (Figure 2 32 Figure 3 34) Thegeographical distribution of the two groups differs Membersof the H planiceps species group are found throughout theGreater Sunda Islands and Peninsular Malaysia but they areisolated in the upper reaches of large rivers (Figure 5) Incontrast members of theH baramensis species group appearto be restricted to Sabah North Borneo (Figure 6)

mtDNA Clade 4 The H planiceps Species Group The Hplaniceps species group (Figure 5) is comprised of 5 closelyrelated but genetically distinct species (mean within-groupdivergence 119901 = 20 (SD = 033) H planiceps (Java)H gracilis (eastern Peninsular Malaysia) H divaricatus(western Peninsular Malaysia) H bongan (Borneo) andH velox (Sumatra)) H lacustrinus the sixth species of thisgroup (NampK) was not sampled in the present study WithintheH planiceps species groupH planiceps formed a distinctspecies (Figure 5) comprising two sequences One sequence(PLCIM0114 Table S1) characterized 14 specimens obtainedfrom Ci Manuk West Java 9 of which were identified as Hplaniceps by NampK (Table S1) A second sequence (PLCIS0101

Table S1) was obtained from one specimen sampled atCi Sadane West Java that we classified as H planicepsH bongan is represented by 5 sequences obtained from 8specimens sampled in the Kapuas River (Figure 5) Two ofthese specimens (ZRC 43013 and 43015) were identified asH bongan by NampK (Table S1) H divaricatus is composedof 3 sequences from three specimens obtained at Gerik onthe Perak River (western Peninsular Malaysia) One of thesespecimens (ZRC 41151) is the holotype of this new species(NampK) H gracilis is represented by only one sequence(GREND0206 Table S1) shared by 6 specimens sampledat Ulu Endau Jasin River eastern Peninsular Malaysiafour of which were identified by NampK (Table S1) ZRC21484 comprises the holotype and ZRC 21482 21486 and21487 comprise the paratypes (NampK) of this species (TableS1) Finally H velox is represented by only one sequence(VEBAH0907) from 7 specimens sampled at Kerinci BatangHari River Sumatra Three of these specimens (ZRC 41503Table S1) represent 3 paratypes of the species (NampK)

mtDNA Clade 5 The H baramensis Species Group The threespecies comprising the H baramensis species group (Figures2 and 3) form three monophyletic subclades (mean within-group divergence 119901 = 106 (08) Figure 6) H semotus iscomposed of two sequences (SEPAD0101 SEPAD0202) that

10 Advances in Evolutionary Biology

110∘

002

H sabanus

H semotus

H baramensis

SAKIN0204SASGM0706

BASGM0402BASGM0301

BASGM0201BASGM0104

SESGM0502SEUMA0101

SEPAD0202SEPAD0101

See Figure 5

100∘

10∘

0∘

120∘

0

0 250 500

250 500

750(km)

(mi)

99

57

32

83

99

72

99

56

66

54

99

Shelf minus75mShelf minus120 m

Figure 6 Phylogenetic relationships of the individual sequences and three species comprising the H baramensis species group H semotusH sabanus and H baramensis (furcatus) The subtree is extracted from the taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicate trees in which the associated sequences clustered together in the bootstrap test(500 replicates) is shown next to the branches The distribution of these sequences is illustrated in the right panel

characterize fish that are the holotype and paratypes of Hsemotus (ZRC 46122 Table S1) along with two somewhatmore divergent sequences (Figure 6) One of these sequences(SGM0502) was found in the Segama River (Danum Valley)and identified as H baramensis by NampK (ZRC 40525)(Table S1) The second sequence (UMA0101) was from afish sampled at Tawau Umas Umas River (ZRC 46123) alsoidentified as H baramensis by NampK These sequences mostlikely reflect distinct lineages within H semotus inhabitingdifferent rivers as the mean intraspecies sequence divergence(119901 = 16) indicates population-level divergence

H sabanus and H furcatus (synonymized with H bara-mensis by NampK) cluster together to form a distinct subcladebut clearly diverge at the species level (Figure 6) H sabanuscomprises 2 sequences that are shared respectively by 6specimens (ZRC 42741) from the Segama River Sabah and4 specimens (part of ZRC 42743) from the KinabatanganRiver Sabah (Table S1) All of these specimenswere identifiedas H sabanus by NampK (Table S1) Finally H baramensisforms a distant subclade (147 mean sequence divergencebetween H baramensis and H sabanus) composed of 4sequences (Figure 6) shared by 8 specimens all sampled inthe Segama River Danum Valley Conservation Area SabahSix of these specimens (ZRC 40523 (2 specimens) ZRC40527 (2 specimens) and ZRC 40526 (2 specimens) Table S1)comprise the paratypes of H baramensis (furcatus) (NampK)

32 The H nemurus Species Group In both NJ and ML trees(Figures 2 and 3) the H nemurus species group is strongly

supported (bootstrap support 98 in NJ and 99 in ML)indicating that it forms a distinct and widespread geneticentity within the genus Hemibagrus However relationshipswithin the group are not so clear In general the H nemurusspecies group is composed of 4 clades The first clade may beconsidered as a species group nested within the H nemurusspecies group (85 and 93 bootstrap support in NJ andML resp) encompassing three clades H hoevenii (clade9) the H capitulum Sundaic clade (clade 10 including Hnemurus) and the H capitulum Indochinese clade (clade 11)(Figures 2 and 3) An additional three clades (Figures 2 and3) are formed by an unidentified species group (clade 6) Hspilopterus (clade 7) andH fortis (clade 8)These three latterclades are distinct at the congeneric species level (unspecifiedversus H spilopterus 119901 = 77 (07) unspecified versus Hfortis 119901 = 85 (07) and H spilopterus versus H fortis119901 = 75 (08)) The distribution of these three clades issomewhat disparate H fortis and the unspecified speciesgroup are principally found in Borneo whereasH spilopterusis widely distributed on the Indochinese mainland

mtDNA Clade 6 Unspecified Species Group This geneticallydiverse group encompassing at least 3 subclades (H spp AB and C Figure 7 within-group mean sequence divergence119901 = 68(06)) is confoundedwith theH baramensis speciesgroup byNampK It is composed of sequences fromfish sampledin Sarawak Sabah and eastern PeninsularMalaysia Subclade119867 spp A (Figure 7) is particularly interesting as it contains2 sequences (CHSARA20 CHRAJ0401) that characterize

Advances in Evolutionary Biology 11

110∘

XXPAD0303XXPAD0601

XXPAD0401XXBEL0202

XXBRM0202XXRAJ0511

XXPAD0501

XXEND0410CHSARA20CHRAJ0401

XXBEL0101XXSEG0101

100∘

10∘

0∘

120∘

0

0 250 500

250 500

750(km)

(mi)

91

86

83

99

98

47

81

001

91

72

99

80

H spp C

H spp B

H spp A

Shelf minus75mShelf minus120 m

Figure 7 Phylogenetic relationships of the individual sequences comprising the unspecified species group containing three subcladesidentified as H spp A B and C The subtree is extracted from the taxon identification phenogram inferred using the Neighbor-Joiningmethod (see Figure 2) The percentage of replicate trees in which the associated sequences clustered together in the bootstrap test (500replicates) is shown next to the branches The distribution of these sequences is illustrated in the right panel

H chrysops a new species identified by Ng and Dodson [11]However specimens of H chrysops from the Rajang Riverwere subsequently synonymized by NampK with H capitulumof the H nemurus species group (ZRC 23008ndash23014 TableS1) However an additional 14 fish from the Sadong Riverat Serian bearing genotype SARA20 (Table S1) and thusgenetically identical to the Rajang H chrysops samples wereidentified by NampK as H spilopterus (ZRC 29583ndash96 TableS1) These specimens however were erroneously identifiedin NampK as originating in the Chao Phraya River (Thailand)Subclade A also contains one sequence obtained from 10 fishsampled on the Endau River (Mersing) in eastern PeninsularMalaysia These fish were not morphologically examinedFinally subclade A also contains one sequence from oneunidentified fish sampled in the Segaliud River Sabah andanother unidentified fish sampled in the Belait River BruneiSubclade H spp B encompasses 2 sequences one of whichcomes from a fish sampled in the Padas River (XXPAD0501)and part of the ZRC 46122 fish lot identified as H semotusby NampK (Table S1) The second sequence (XXRAJ0511) wasshared by 11 unidentified fish sampled at Katibas on theRajang River Sarawak (Table S1) Finally subclade 119867 sppC encompasses 5 sequences Three sequences (XXPAD0303XXPAD0401 and XXPAD0601) were obtained from thePadas River and identified byNampKasH semotus (ZRC46122Table S1) The fourth sequence (XXBRM0202) was obtainedfrom 2 fish sampled in the Baram River and was identified

as H baramensis by NampK (ZRC 42742) The 5th sequencecomposing this group was obtained from 2 unidentified fishsampled in the Belait River Brunei (Table S1)

mtDNA clade 6 is thus problematical within the contextof the revision of the genus Hemibagrus Two highlydivergent mtDNA clades (clade 5 and clade 6 Figures 2and 3) occur in sympatry principally in the Padas River ofSabah North Borneo (Figures 6 and 7) Specimens of theunspecified species group are morphologically similar tothe H baramensis species group where they are assigned inthe analysis of NampK However the mean genetic distancebetween the unspecified species group and theH baramensisspecies group is 119901 = 193 (28) close to genus leveldivergence Furthermore subclade 119867 spp A (Figure 7)contains not only H chrysops but also an unidentifiedpopulation from eastern Peninsular Malaysia (XXEND0410Table S1) and two additional Sabah sequences On theother hand the unspecified species group clusters with allother members of the H nemurus species group (meangenetic divergence of unspecified group versus H capitulum(Sundaic clade) 119901 = 76 (07) (Figure 2)) with strongbootstrap support Such genetic clustering may justify theinclusion of H chrysops and associated sequences in theH nemurus species group but its synonymization with Hcapitulum (NampK) is difficult to defend on genetic groundsWe tentatively conclude that H chrysops is a valid speciesbased on genetic identity [11] forming part of an unknown

12 Advances in Evolutionary Biology

110∘

SPCPCHMK12

SPMEK0202SPMEK0401

SPMEK0301SPMEK0103

SPMEK1401SPMEK0501

SPMEK1201SPMEK1301

SPMEK1601SPMEK1502

SPMEK0701SPMEK0801SPMEK1701

SPCHP0501SPCHA0201

SPCHA0304SPPAT0101

100∘

10∘

0∘

120∘

0

0 250 500

250 500

750(km)

(mi)

000499

99 98

60

47

48

54

96

7091

38

86

87

99

80

58

H spilopterus ldquoCrdquo

H spilopterus ldquoBrdquo

H spilopterus ldquoArdquo

Shelf minus75mShelf minus120 m

Figure 8 Phylogenetic relationships within H spilopterus comprised of 3 subclades identified as A B and C The subtree is extracted fromthe taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicate trees in whichthe associated sequences clustered together in the bootstrap test (500 replicates) is shown next to the branches The distribution of thesesequences is illustrated in the right panel

species group found throughout North Borneo (and toa lesser degree in eastern Peninsular Malaysia) that maybe considered as part of the H nemurus species groupHowever the morphological confusion of the unspecifiedspecies group with the sympatric highly divergent Hbaramensis species group suggests the existence of a crypticspecies assemblage (see Section 4)

mtDNA Clade 7 Clade 7 (Figures 2 and 3) is composed of atleast 3 distinct subclades that appear to reflect 3 geneticallydivergent populations ofH spilopterus Subclade A (Figure 8)encompasses 7 sequences obtained from 8 fish sampled inthe Mekong River Four fish bearing 3 of these sequenceswere identified as paratypes (ZRC 43324) of H spilopterusby NampK (Table S1) One additional fish was identified asH spilopterus by NampK (ZRC 46157 Table S1) Subclade Bencompasses 6 sequences obtained from 9 fish all sampledfrom the Mekong River (Table S1) The fish sampled at ThatPhanom (ZRC 46012) was identified as H spilopterus inthe analysis of HampK (Table S1) Subclade C encompasses 5sequences obtained from 19 fish sampled in the Chao PhrayaMae Khlong and Chanthaburi Rivers Thailand Onesequence (CPCHMK12) was found in all of these drainages(Table S1) A closely allied sequence was found in one fishsampled at Phatthalung just south of the Isthmus of Kra onthe Thai-Malay Peninsula None of these fish were examined

by NampK and thus we are unable to definitively identifythe species However given the widespread occurrence ofH spilopterus in the Chao Phraya and Mae Klong Riversreported by NampK we conclude that these fish are part of awidespread H spilopterus population complex The meanwithin-group (clade 7) genetic divergence (119901 = 29 (04))suggests intraspecific population-level divergence

mtDNA Clade 8 Clade 8 encompasses 3 sequencesobtained from 9 fish sampled in east Sabah specifically theKinabatangan Segama and Umas Umas Rivers (Figure 9)The Kinabatangan sequence is somewhat divergent withinthe clade and the specimen was not identified by NampK Onesequence (SGM0606) characterized 6 fish from the SegamaRiver 2 of which were identified as H fortis (ZEC 4052140522 Table S1) by NampK (Table S1) Finally one fish bearinga closely related genotype from the Umas Umas River wasidentified as H baramensis by NampK (Table S1)

mtDNA Clade 9 Clade 9 is a sister group to the remaining2 clades composing the H nemurus species group Thiswell-defined clade (Figure 10) clusters 6 sequences from42 fish that are clearly identified as H hoevenii (NampK)Some of these haplotypes are geographically widespread onesequence (JOMSKA11 Table S1) was found in the Johor River(East Peninsular Malaysia) the Musi River (Sumatra) and

Advances in Evolutionary Biology 13

100∘ 110∘

0∘

120∘

00 250

250500

500750

(km)

(mi)

FOKIN0102FOSGM0606

FOUMA0201H fortis

10∘

99

86

001

Shelf minus75mShelf minus120 m

Figure 9 Phylogenetic relationships within H fortis comprised of 3 sequences The subtree is extracted from the taxon identificationphenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicate trees in which the associated sequencesclustered together in the bootstrap test (500 replicates) is shown next to the branches The distribution of these sequences is illustrated in theright panel

the Kapuas River (West Borneo) and a second sequence(HMUBHKABM23 Table S1) was found inMuarRiver (WestPeninsular Malaysia) and the Batang Hari (Sumatra) theKapuas (Sarawak) and the Baram Rivers (Sarawak) Many ofthese fish were identified as H hoevenii by NampK (Table S1)The existence of twodistinct but very closely related subclades(Figure 10 119901 = 06 (03)) that are not geographicallysegregated suggests some recent isolation event followed bywidespread dispersal and secondary contact

33 The H capitulum Group The final two monophyleticgroups of the Hemibagrus gene tree are approximatelyequivalent to the remaining species composing the Hnemurus species group as defined by NampK Dodson etal [10] and Ng and Dodson [11] have previously referredto clade 10 as the Sundaic clade as most sequences wereobtained from fish sampled on the Greater Sunda Islandsand clade 11 as the Indochinese clade asmany sequences wereobtained from fish sampled on the Indochinese mainland(Figure 2 Table S1) Although most of these sequencesare assigned to H capitulum by NampK there are somesignificant phylogeographic differences between the 2 cladesIn particular there appears to be an east-west division in thedistribution of the two clades on the Thai-Malay Peninsulawith the Sundaic clade (clade 10) found on the west coast andthe Indochinese clade found on the east coast with sympatricoccurrence in the Muar River Nevertheless mean sequencedivergence between the two clades is 119901 = 29 (05) avalue that is indicative of population-level or subspecies

divergence The two clades are thus best considered ascomprising one species

mtDNA Clade 10 The Sundaic clade includes a number ofclosely related sequences that comprise part of H capitulumand H nemurus Very little genetic structure is apparentand bootstrap support for tree topology within this group isgenerally very weak (Figure 11) A widely spread genotype(NCLCTBA20) characterized 6 of 17 specimens from CiTarum at the Cirata reservoir (West Java) (Table S1) and5 were identified as H nemurus in the analysis of NampK(ZRC 42564) (Table S1) The remaining fish characterized bythis sequence were obtained from Ci Liwong at Sawangan(West Java) and one fish was obtained from the Barito Riverat Banjarmassin south Borneo This latter specimen (ZRC40553) was identified by NampK as H capitulum (Table S1)Another common genotype (NBHKPBR09) was identified aseither H capitulum H hoevenii or H nemurus dependingon the river of origin (Table S1) Many of the remainingsequences of the Sundaic clade appear to characterize fish thatNampK identified asH capitulum A geographically widespreadgenotype (CPEBEMUKA36) characterized 9 fish sampledon the Perak River at Gerik that were identified by NampKas H capitulum (ZRC 41150 Table S1) Another commongenotype within this clade (CABHMS20) included 2 fishfrom the Batang Hari at Jambi and identified by NampK asH capitulum (ZRC40534 Table S1) Another 2 sequences(CABAR0203 CABAR0305) characterized 8 fish sampledon the Barito River at Banjarmasin and were identified as

14 Advances in Evolutionary Biology

100∘

10∘

110∘

0∘

120∘

0

0 250

250

500

500

750(km)

(mi)

53

64

99

85

64

0002

HJOMSKA11

HOBAH0601

HOBHMS05

HMUBHKABM23

HOBAH0701

HOKAP0501

H hoevenii ldquoBrdquo

H hoevenii ldquoArdquo

Shelf minus75mShelf minus120 m

Figure 10 Phylogenetic relationships withinH hoevenii comprised of 2 subclades and 6 sequences The subtree is extracted from the taxonidentification phenogram inferred using theNeighbor-Joiningmethod (see Figure 2)The percentage of replicate trees in which the associatedsequences clustered together in the bootstrap test (500 replicates) is shown next to the branches The distribution of these sequences isillustrated in the right panel

110∘

XXMAH0110

CABAR0305

96 CAKPR0201

74CAKAP0401

5931 CAKAP0601

35 CPEBEMUKA36

646 CAKAP0903

33 CAKAP080114 CAKAP0701

CARAJ0210

4 CABAR0203CABRA0204

20

CBHKPBR0919

CABRA0301

15CABHMS20

24 NCLCTBA20

63 NECIT0201

H capitulumldquoSundaic claderdquo

H nemurus

0002 120∘

0 250 500 750

(km)

0 250 500

(mi)100

10∘

0∘

Shelf minus75mShelf minus120 m

Figure 11 Phylogenetic relationships within theH capitulum ldquoSundaicrdquo clade including 2 sequences associated withH nemurusThe subtreeis extracted from the taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicatetrees in which the associated sequences clustered together in the bootstrap test (500 replicates) is shown next to the branchesThe distributionof these sequences is illustrated in the right panel

Advances in Evolutionary Biology 15

H capitulum

0∘

10∘

100∘

110∘

120∘

66

63

88

59

38

39 68

71

6991

57

9754

88 4944

39

53

89

0002

ldquoIndochineserdquogroup ldquoErdquo

H capitulumldquoIndochineserdquo

group ldquoDrdquoH capitulumldquoIndochineserdquo

group ldquoCrdquo

H capitulumldquoIndochineserdquo

group ldquoBrdquo

H capitulumldquoIndochineserdquo

group ldquoArdquo0

0

250

250

500

500

750

(km)

(mi)

CATER0301CATERR0401

CKETEMU23

CAMEK0601CACHP0301

CAMKPA03

CAMEK0910CAMEK1003

XXMUBM02

CABMTU09

CABRM0601

CAKAP1201CAKAP1001

CAKAP1101CAKAP1301

CABRM0501

CASAD0102

CASAD0220

CAEND0301

CABAH0801

CAPABH05CATEEN03

CATER0701CATERR0801

CAKETE04CATER0501

CAPAH0303

Shelf minus75mShelf minus120 m

Figure 12 Phylogenetic relationships within theH capitulum ldquoIndochineserdquo clade encompassing 5 subclades identified as A B C D and EThe subtree is extracted from the taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2)The percentageof replicate trees in which the associated sequences clustered together in the bootstrap test (500 replicates) is shown next to the branchesThedistribution of these sequences is illustrated in the right panel

H capitulum by NampK (ZRC 40553 Table S1) Although notincluded in the analysis of NampK samples from the Sundaicclade were also found in the Kapuas River (West Borneo)Finally 10 fish sampled on the Mahakam River at Samarindaeast Borneo are all characterized by one relatively divergentgenotype (CAMAH0110) butwere considered asH capitulumin the analysis of NampK (ZRC 40533 Table S1)TheMahakampopulation sample is the most genetically distinct of theSundaic clade (119901 = 17 (04)) but nevertheless indicativeof intraspecific population-level divergence (Figure 12)

The topology of the Sundaic clade does not permit usto draw a clear distinction between H nemurus and theSundaic H capitulum The mean within-group divergenceof the Sundaic clade is only 119901 = 08 (02) well withinpopulation-level divergence values We postulate that Hnemurus may be one of several regional variants of awidespread Sundaic H capitulum clade that has radiatedrelatively recently This may explain the existence of manyshared sequences throughout the Sundaic islands assigned todifferent species in different regions

mtDNA Clade 11 Finally the Indochinese clade of Hcapitulum represents a geographically widespread andweakly diversified clade (within-group mean sequencedivergence 119901 = 11 (02) Figure 12) Although few fishfrom this genetic clade were included in the morphological

analysis of NampK most may be considered as H capitulumgiven their geographic distribution and close geneticrelationship to the Sundaic H capitulum Four sequencesin this clade were identified as H capitulum in the analysisof NampK 3 specimens characterized by one sequence(CABMTU09) found in the Tutong River of westernBorneo (ZRC 40482) 6 characterized by the same sequencefrom the Baram River (ZRC 40498) another 2 sequencesfrom the Baram River (ZRC 40498) and 20 specimenscharacterized by an abundant genotype (CASAD0220)found at 2 sites in the Sadong River also in western Borneo(ZRC 39526 5 specimens 39527 3 specimens and 38753 4specimens) (Table S1) Unfortunately none of the specimenscollected on the Indochinese mainland for genetic analysiswere examined by NampK Nevertheless the distribution ofspecimens analyzed for mtDNA resembles the distribution ofspecimens examined by NampK Although weakly structuredwe may recognize 5 subclades One genotype (CAPAH0303)is basal to the phenogram Subclade A encompasses 8sequences mainly found in eastern Peninsular Malaysia(Table S1 Figure 12) These rivers furnished many of thespecimens NampK examined to define H capitulum Howeversome sequences in this group occur in sympatry withsequences identified within the Sundaic H capitulum groupTwo sequences from subclade A occurred in the Batang HariSumatra (Table S1) Subclade E also encompasses sequences

16 Advances in Evolutionary Biology

found in Peninsular Malaysia as well as in the Mekong ChaoPhraya and Mea Khlong Rivers (Figure 12) In addition3 subclades encompass sequences found principally inwestern Borneo subclade B in the Baram and Sadong RiversSarawak subclade C in the Kapuas River West Kalimantanand subclade D in the Baram and Tutong Rivers of Sarawakand Brunei respectively (Figure 12) This latter subclade alsoincludes one genotype found in the Muar River of westernpeninsular Malaysia Finally subclade E encompasses 5sequences from the Mekong Chao Praya and Mae KhlongRivers all within the distribution area of H spilopterus

EvolutionaryHistoryThe evolutionary history of theHemiba-grus species groups analyzed here dates from at least theMiocene 15ndash20Ma (Figure 13) when considering the widelyused molecular clock of 2 sequence divergence per millionyears Bootstrap support for the sister group relationships ofH wyckioides theH guttatus species group andH peguensisare all weak (clearly due to undersampling) and little can besaid of their evolutionary history other than the fact that theyoccupy basal positions in the rooted trees The remainder ofboth the NJ and ML trees is composed of two major sistergroups the H planiceps + H baramensis group and the Hnemurus species group (Figures 2 3 and 13)TheH planiceps+ H baramensis group is strongly supported in both NJ andML analyses (84 and 90 bootstrap support resp Figures2 and 3) The H nemurus species group is also stronglysupported in both analyses (98 and 99 bootstrap supportFigures 2 and 3) These two major phylogenetic groups areestimated to have diverged at least 8 to 12Ma ago during themid to late Miocene (Figure 13)

Much of the evolutionary history of Hemibagrus hasoccurred during the Quaternary starting approximately26Ma ago This is particularly true of the H planicepsand H nemurus species groups The radiation of the 5species included in the H planiceps species group appearsto coincide with the middle Pleistocene transition period ofapproximately 1Ma ago Their high-elevation distributionhowever appears to have largely isolated them from theeffects of lowered sea levels during glacial maxima and theattendant opportunities for widespread dispersal (Figure 5)The evolutionary histories of theH baramensis species groupand the unspecified species group date back to the latePliocene In contrast the low-elevation distribution of theHnemurus species group in Sundaland appears to have favoredtheir widespread distribution during the Quaternary ice ageIn particular H hoevenii appears to be the most recentlydispersed of the species under study with diversification anddispersal occurring approximately 03Ma ago (Figure 13)

4 Discussion

41 The Congruence of Morphological and Genetic AnalysesCombining genetic and morphological analyses to elucidatetaxonomy allowed us to better understand the diversificationand evolutionary relationships within this speciose group offishes Given the rapid and relatively constant rate of mtDNAevolution the deeper the branches within the phylogenetictree are the greater our ability to distinguish valid taxa

should be Conversely the lack of complete congruencebetween morphological- and genetic-based identificationsis expected to increase among the shallower branches of thephylogenetic tree resulting in the appearance of polyphyleticspecies Although 2 of the 8 species groups defined by Ngand Kottelat [7] were not sampled in this study the validityof the remaining 6 species groups and their componentspecies defined on the basis of morphological similaritywas largely confirmed by the analysis of the mitochondrialCyt 119887 bar code As expected congruence is strongest at thebase of the NJ and ML trees where the H guttatus speciesgroup formed a well-defined monophyletic group The Hmenoda and H wyckii species group were each representedby only one species but they were clearly distinct relativeto sequences characterizing the neighboring species groupsThe H planiceps H baramensis and H nemurus speciesgroups and their component species were also generallycongruent in morphological and genetic analyses Also asexpected most cases of discrepancy between morphological-based species identification and genetic-based speciesidentification occurred on relatively shallow branches of thephylogenetic tree These involve principally H spilopterusand the two mtDNA subclades composing H capitulumHowever a far more unexpected lack of congruence involvesthe definition of a deeply divergent unspecified genetic groupconfounding members of theH baramensis species group inthe morphological analysis of Ng and Kottelat [7]

Interpreting Polyphyly The apparent polyphyletic origin ofa species may be due to incomplete lineage sorting amongrecently diverged species the introgression of mtDNA dueto species hybridization or the erroneous identificationof species due to the lumping of morphologically crypticspecies In the absence of nuclear DNA analyses it isoften difficult to distinguish between introgression andincomplete lineage sorting For example several sequencescharacterizing the Indochinese clade of H capitulum(subclade E) are found in the Mekong and Chao PhrayaRivers well within the distributional area of H spilopterusH spilopterus and H capitulum first diverged at least 3MaOn the one hand this apparent case of polyphyly mayhave resulted from incomplete lineage sorting betweenrecently divergedH spilopterus andH capitulum We wouldhowever have expected similar cases of polyphyly amongpopulations of the Indochinese clade of H capitulum butthis was not detected On the other hand the sympatricoccurrence of H spilopterus subclade A and subcladeB sequences with H capitulum Indochinese subclade Esequences in the Mekong River may represent a migrationevent and local introgression of H capitulum with Hspilopterus A similar event may have occurred in theChao Phraya and Mae Khlong Rivers where H spilopterussequences cooccur withH capitulum sequences Computingthe divergence of H capitulum sequences (Indochinesesubclade E) cooccurring with H spilopterus and Hcapitulum sequences occurring in eastern PeninsularMalaysia (subclade A) reveals a mean sequence divergence119901 = 15 (04) Thus the H capitulum sequences occurringin sympatry with H spilopterus diverged at most 750 ka ago

Advances in Evolutionary Biology 17

H fortis

XXRAJ0604H furcatus

H sabanusH semotusH divaricatus

H planicepsGREND0206

VEBAH0907

H bonganPESAL0102

H wyckioidesH macropterus

H guttatusXXRAJ0301Leiocassis longirostris

Ma

4971

92

47

51

9999

99

9999

99

99

99

9999

99

99

99

99

65

96

96

4946

93

79

78

70

91

93

98

86

87

91

95

97

71

85

9999

05101520

005010015020 000

H capitulum Indochinese clades AndashE

H capitulum Sundaic clade

H hoevenii B

H hoevenii A

H spilopterus C

H spilopterus B

H spilopterus AH spilopterus A

H spp CH spp B

H spp A

Figure 13 Linearized tree with branch lengths estimated with the molecular clock using a mutation rate of 1 per Ma (=divergence rate of2 per Ma) The tree topology is identical to the ML reconstruction illustrated in Figure 3 (see the specificities of the tree reconstruction inthe caption of Figure 3) Boxes represent 95 confidence intervals of divergence time estimates for each node Vertical dashed lines redirectto dates indicated on the timescale (Ma) Numbers below timescale indicate the estimated number of substitution changes using Hasegawa-Kishino-Yanomodel (HKY)with gamma distribution (+119866 = 13290) after linearizing the treeH furcatus is synonymizedwithH baramensis

from eastern Peninsular Malaysia populations suggestinga recent introgression event Similarly H capitulum in theKapuas River is characterized by sequences of both theSundaic and the Indochinese clades As both sequences arebroadly sympatric it is not possible to distinguish betweenincomplete lineage sorting and introgression of these closelyrelated intraspecific lineages

A Possible Case of Morphological Stasis Clade 6 of thepresent analysis defines a well-supported monophyletic cladefound in Sabah Sarawak and the Endau River of easternpeninsula Malaysia that has no equivalent taxonomic statusin the revision of Ng and Kottelat [7] In Borneo they occursympatrically with the H baramensis species group and areconfounded as either H baramensis or H semotus These

two clades are genetically highly divergent having divergedbetween 8 and 12Ma ago It thus seems highly unlikely thatthey represent retained polymorphisms due to incompletelineage sorting Sequences previously identified as character-izing H chrysops [11] are part of clade 6 and extend to thesouth (Rajang and Sadong Rivers of Sarawak West Borneo)with one closely related genotype found in eastern peninsularMalaysia (Endau River) where it may well be confoundedwith H capitulum H chrysops was synonymized with Hcapitulum by Ng and Kottelat [7] based on their analysis ofspecimens ofH chrysops from the Rajang RiverThus despitea long and apparently independent phylogenetic historythe unspecified species group appears largely cryptic andconfounded principally with members of the H baramensisspecies group

18 Advances in Evolutionary Biology

We suggest that this situation is an example of mor-phological stasis involving the coexistence of a number ofgenetically divergent yet morphologically cryptic species thatare lumped together [24] A striking example of morpho-logical and ecological stasis among fishes is provided by thecoastal marine bonefishes (Albula spp) Distinct evolution-ary lineages (separated by sequence divergence between 5 and30 [25]) of bonefish coexist in several sample locations yetthey show little morphological or ecological differentiationin sympatry [25] Similarly a proportion of the grey mulletspecies (Mugilidae) also appears to consist of cryptic species[26] For exampleMugil cephalus in Taiwan is characterizedby three divergent lineages that have been shown to charac-terize 3 reproductively isolated but morphologically crypticspecies [26 27]

Here we have discovered 2 highly distinct and structuredclades (each composed of 3 mtDNA subclades) that aremorphologically confounded within different species Forexample H spp C of the unspecified species group isidentified as H baramensis in the Baram River and as Hsemotus in the Padas River We speculate that North Borneoharbors a morphologically cryptic species complex possiblycomposed of several species pairs inhabiting different riversystems The reconstruction of this phylogenetic history andthe taxonomic status of its components remains a majorchallenge to the revision of the Hemibagrus genus

42 Phylogeographic Hypotheses The phylogeography ofthe genus appears to have been principally influenced bytwo geological histories one encompassing the formationof North Borneo the second the emergence of the SundaIslands and their connectivity with the Indochinesemainlandassociated with recurrent sea-level changes North Borneoappears to have acted as a species sink promoting speciationand locally high biodiversity within the genus but with littleevidence of subsequent dispersal out of North Borneo Incontrast the geological events influencing the Indochinesemainland and the Sunda Islands have permitted bothspeciation and differential dispersal depending on thebiology of the species For example the H planiceps speciesgroup is found throughout the Greater Sunda Islands andtheThai-Malay Peninsula as is theH nemurus species groupWhereas the H nemurus species group generally occupiesthe low altitude lower reaches of large rivers the speciescomposing the H planiceps group are found in the upperreaches of large rivers The H nemurus species group iscomposed of a number of widely distributed closely relatedspecies whereas theH planiceps group is composed of a largenumber of species that are highly localized geographicallyIt thus appears that sea-level changes have had little impacton the dispersal history of the H planiceps species group buthave deeply influenced that of the H nemurus species group

The Case of North Borneo From 15Ma to 5Ma greater areasof Borneo became emergent and the central mountainson the Sarawak-Kalimantan border extending into Sabahbecame wider and higher which may have represented newbarriers to dispersal [28 29] Borneo gradually developedinto the present large island by emergence of land in the

north and east The H baramensis species group is restrictedto North Borneo as is the unspecified species group of theHnemurus species group H fortis is also distributed in NorthBorneo The isolation of the freshwater fish communities ofNorth Borneo (particularly in the Labuk-Segama watershed)has long been recognized based on the number of endemicspecies and the concentration of relict species [30] The latePliocene-early Pleistocene diversification of H fortis theunspecified species group and the H baramensis speciesgroup may thus be associated with their dispersal to andisolation within the watersheds of North Borneo Only onesequence of the unspecified species group is found outsideof North Borneo and H fortis appears to be distributedthroughout central and east Borneo [7]

ThePhylogeography of the H nemurus Species Group Periodicmarine transgressions on the Sundaland platform during theearly and middle Miocene [2 29] may have been sufficientto isolate populations of freshwater organisms and promotecladogenesis [31] This may have been responsible for theinitial mid to late Miocene divergence of the H nemurusspecies group The majority of sequences assigned to thisspecies group belong to 3 species (H spilopterus H capitulum(including H nemurus) and H hoevenii) all of which havebeen influenced by the more recent geological history of theSunda Islands and their connectivity with the Indochinesemainland H spilopterus is known from river drainages inIndochina including the Mekong Dong Nai Ban PakongChao Phraya and Mae Klong where it is found in a widevariety of lotic and lentic habitats [7] H spilopterus divergedin the late Pliocene-early Pleistocene possibly isolated tothe north of the Kra Peninsula during the late Pliocenemarine transgression that is purported to have created amajor biogeographic transition zone between the Sundaicand Indochinese biotas ([32] but see [33] for a critique ofthis biogeographic scenario) The existence of 2 subclades(A and B) in the Mekong River separated by 2 sequencedivergence is consistent with intraspecific divergence Thetwo subclades possibly represent 2 historical mtDNA lineagesthat are retained within the species reflecting a Pleistoceneisolation event followed by secondary contact within thecontemporary Mekong River Subclade C from the ChaoPhraya and Mae Khlong Rivers exhibits a level of geneticdivergence consistent with sibling species-level divergenceshowing a 5 sequence divergence relative to Mekong RiverH spilopterus

H capitulum may be considered as part of the Sundaicbiota located principally to the south of the Kra marinetransgression zone that may have isolated H spilopterus tothe north on the Indochinese mainlandThe biogeographicalevent leading to the subsequent formation of two intraspecificgroups within H capitulum is not at all clear The onlyarea where the two clades are clearly geographically separateis on the Thai-Malay Peninsula with the ldquoSundaicrdquo cladeon the west coast and the ldquoIndochineserdquo clade on theeast coast separated by the central Titiwangsa mountainrange The genetic distinctiveness of east and west coastpopulations of H capitulum has also been documented fornuclear DNA [34] A similar pattern of separation has been

Advances in Evolutionary Biology 19

reported for 5 additional freshwater fishes within this region[35] However as the divergence of the two H capitulumclades postdates the formation of the mountain range byapproximately 200Ma the geographic separation of the twoclades most likely represents differential dispersal rather thanancient vicariance The presence of the Sundaic clade on thewest coast of the Thai-Malay Peninsula as well as on theSundaic Islands reflects the historical connections betweeneast Sumatra and west Peninsular Malaysia across the Straitof Malacca This area encompasses the Northern CentralSumatra-Western Malaysia freshwater ecoregion [36] TheMuar River forms its southern border coincident with thesouthernmost distribution of the Sundaic clade on the Thai-Malay Peninsula It is important to note however that mostof Sumatra and Java emerged to achieve their present size andelevation only 5Ma ago and much of East Java continuedto be the site of marine deposition until late Pliocene-earlyPleistocene [29] We may speculate that the Sundaic clade ofH capitulum resulted from the relatively recent colonizationof these islands and Borneo The radiation within the cladeonly dates back to approximately 1Ma ago H nemurus fromWest Java may thus best be considered an isolated regionalpopulation of very recent origin

The diversification of the Indochinese clade dates fromat least 12Ma ago coincident with the mid-Pleistocenetransition at which time low-frequency high-amplitude andquasiperiodic (100 ka) glacial variability emerged It began125Ma ago and was complete by 07Ma ago [37] The tran-sition marked the beginning of major changes in sea levelsduring which the Sunda Shelf periodically emerged and wastraversed by enormous fluvial systems [3] The contributionof these events to the diversification of this clade cannotbe satisfactorily reconstructed One possible explanation isthat high sea levels produced barriers to gene flow and thepopulations of West Borneo (subclades B C and D) wereisolated from those of east Peninsular Malaysia This couldbe due to one or more dispersal events from Borneo via theGreat Sunda River (Figure 1) to east Peninsular Malaysia orlineage sorting whereby sequences of subclades A and E havebeen lost from West Borneo As in the case of the sympatricoccurrence of H spilopterus and H capitulum subclade E(see above) in the Mekong River we are unable to clearlydefine the relative roles of incomplete lineage sorting andintrogression followingmigration Given the recent origins ofthe Indochinese H capitulum sequences incomplete lineagesorting most probably blurs patterns of recent isolation

The last species of the H nemurus species group is Hhoevenii The species is widespread throughout PeninsularMalaysia and the Sundaic Islands It is found primarily inthe lower reaches of rivers and is apparently capable ofwithstanding low salt concentration [7] H hoevenii thusappears to be the most liable of all members of the Hnemurus species group to be impacted by Pleistocene sea-level changes This is clearly reflected in the widespreaddistribution of the sequences of this species Two lineages areevident having diverged no more than 03Ma ago suggest-ing a late Pleistocene isolation event followed by extensivedispersal and admixture in recent times This represents theclearest example of the dispersal opportunities afforded by

Pleistocene sea level changes on the Sunda Shelf In additionthe relatively low genetic diversity detected (only 6 sequencesin 42 fish) suggests a population bottleneck in the recent pastpossibly related to population declines in the lower reaches ofrivers flooded by increasing sea levels

43 Future Directions We have provided a global picture ofthe evolutionary taxonomy of the genus in Southeast AsiaClearly a more rigorous phylogenetic reconstruction of thegenus Hemibagrus requiring the construction of multiplegene trees based on the analysis of both mitochondrial andnuclear DNA markers from a wider taxonomic array ofhemibagrids will involve an even greater effort of samplingthroughout the region In particular we require samplingat smaller spatial and temporal scales For example smallerscale sampling in the Padas River and the Segama Riverof North Borneo using both mitochondrial and nucleargenetic markers would contribute to clarifying the natureof the cryptic species complex believed to inhabit theserivers The demonstration of reproductive isolation andormicroallopatric distribution among morphologically crypticspecies may result in the description of greater speciesrichness within the genus than presently described Thespecies status of H chrysops merits reconsideration givenits highly divergent evolutionary history Nuclear geneticmarkers in conjunction with mtDNA markers could revealcases of historical introgression particularly in the KapuasRiver and the Mekong River The hemibagrid catfishes con-tinue to provide multiple opportunities for the study of theevolutionary taxonomy and biogeography of a major faunalcomponent of the fluvial ecosystems of Southeast Asia

Disclosure

Ethanol-preserved tissue samples of many of the specimensidentified in Table S1 are freely available upon request fromJJD

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Goh Yan Yih for assistance in collectingspecimens and molecular analyses Mrs Francoise Colom-bani for molecular analyses and Dr Heok Hee Ng forspecies identification This work was funded by the NationalUniversity of Singapore grant to P K L Ng and a CanadianNatural Sciences and Engineering Research Council grant toJ J Dodson

References

[1] D J Lohman M de Bruyn T Page et al ldquoBiogeography ofthe Indo-Australian archipelagordquo Annual Review of EcologyEvolution and Systematics vol 42 pp 205ndash226 2011

20 Advances in Evolutionary Biology

[2] D S Woodruff ldquoNeogene marine transgressions palaeogeog-raphy and biogeographic transitions on the Thai-Malay Penin-sulardquo Journal of Biogeography vol 30 no 4 pp 551ndash567 2003

[3] H K Voris ldquoMaps of Pleistocene sea levels in SoutheastAsia shorelines river systems and time durationsrdquo Journal ofBiogeography vol 27 no 5 pp 1153ndash1167 2000

[4] S Y Yap ldquoOn the distributional patterns of Southeast-EastAsian freshwater fish and their historyrdquo Journal of Biogeographyvol 29 no 9 pp 1187ndash1199 2002

[5] S K J McConnell ldquoMapping aquatic faunal exchanges acrossthe Sunda shelf South-East Asia using distributional andgenetic data sets from the cyprinid fish Barbodes gonionotus(Bleeker 1850)rdquo Journal of Natural History vol 38 no 5 pp651ndash670 2004

[6] T Mo Anatomy Relationships and Systematics of the Bagridae(Teleostei Siluroidei) with a Hypothesis of Siluroid Phylogenyvol 17 ofTheses Zoologicae Koeltz Scientific Books 1991

[7] H H Ng and M Kottelat ldquoRevision of the Asian catfish genusHemibagrus bleeker 1862 (Teleostei Siluriformes Bagridae)rdquoRaffles Bulletin of Zoology vol 61 no 1 pp 205ndash291 2013

[8] P K L Ng and H H Ng ldquoHemibagrus gracilis a new speciesof large riverine catfish (Teleostei Bagridae) from PeninsularMalaysiardquo Raffles Bulletin of Zoology vol 43 pp 133ndash142 1995

[9] H H Tan and M Kottelat ldquoRedescription of Betta picta(Teleostei Osphronemidae) and description of Betta falx sp nfrom central Sumatrardquo Revue Suisse de Zoologie vol 105 no 3pp 557ndash568 1998

[10] J J Dodson F Colombani and P K Ng ldquoPhylogeographicstructure in mitochondrial DNA of a South-east Asian fresh-water fish Hemibagrus nemurus (Siluroidei Bagridae) andPleistocene sea-level changes on the Sunda shelfrdquo MolecularEcology vol 4 no 3 pp 331ndash346 1995

[11] H H Ng and J J Dodson ldquoMorphological and genetic descrip-tions of a new species of catfish Hemibagrus chrysops fromSarawak East Malaysia with an assessment of phylogeneticrelationships (Teleostei Bagridae)rdquo Raffles Bulletin of Zoologyvol 47 no 1 pp 45ndash57 1999

[12] P D N Hebert A Cywinska S L Ball and J R DeWaardldquoBiological identifications through DNA barcodesrdquo Proceedingsof the Royal Society B Biological Sciences vol 270 no 1512 pp313ndash321 2003

[13] J C Carolan T EMurray U Fitzpatrick et al ldquoColour patternsdo not diagnose species quantitative evaluation of a DNAbarcoded cryptic bumblebee complexrdquo PLoS ONE vol 7 no 1Article ID e29251 2012

[14] M Kekkonen and P D N Hebert ldquoDNA barcode-baseddelineation of putative species efficient start for taxonomicworkflowsrdquoMolecular Ecology Resources vol 14 no 4 pp 706ndash715 2014

[15] DM Hillis CMoritz and B KMarbleMolecular SystematicsSinauer Sunderland Mass USA 2nd edition 1996

[16] T D Kocher W K Thomas A Meyer et al ldquoDynamics ofmitochondrial DNA evolution in animals amplification andsequencingwith conserved primersrdquoProceedings of theNationalAcademy of Sciences of the United States of America vol 86 no16 pp 6196ndash6200 1989

[17] T A Hall ldquoBioEdit a user-friendly biological sequence align-ment editor and analysis program for Windows 9598NTrdquoNucleic Acids Symposium Series vol 41 pp 95ndash98 1999

[18] D A Benson I Karsch-Mizrachi K Clark D J Lipman JOstell and EW Sayers ldquoGenBankrdquoNucleic Acids Research vol40 no D1 pp D48ndashD53 2012

[19] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[20] X Ku Z Peng R Diogo and S He ldquoMtDNA phylogenyprovides evidence of generic polyphyleticism for East Asianbagrid catfishesrdquoHydrobiologia vol 579 no 1 pp 147ndash159 2007

[21] L Yang and S He ldquoPhylogeography of the freshwater catfishHemibagrus guttatus (Siluriformes Bagridae) implications forSouth China biogeography and influence of sea-level changesrdquoMolecular Phylogenetics and Evolution vol 49 no 1 pp 393ndash398 2008

[22] B W Bowen A L Bass L A Rocha W S Grant and DR Robertson ldquoPhylogeography of the trumpetfishes (Aulosto-mus) ring species complex on a global scalerdquo Evolution vol 55no 5 pp 1029ndash1039 2001

[23] Y P Kartavtsev ldquoDivergence at Cyt-b and Co-1 mtDNA geneson different taxonomic levels and genetics of speciation inanimalsrdquoMitochondrial DNA vol 22 no 3 pp 55ndash65 2011

[24] SWada Y Kameda and S Chiba ldquoLong-term stasis and short-term divergence in the phenotypes of microsnails on oceanicislandsrdquoMolecular Ecology vol 22 no 18 pp 4801ndash4810 2013

[25] J Colborn R E Crabtree J B Shaklee E Pfeiler and B WBowen ldquoThe evolutionary enigma of bonefishes (Albula spp)cryptic species and ancient separations in a globally distributedshorefishrdquo Evolution vol 55 no 4 pp 807ndash820 2001

[26] J-D Durand K-N Shen W-J Chen et al ldquoSystematics of thegrey mullets (Teleostei Mugiliformes Mugilidae) molecularphylogenetic evidence challenges two centuries ofmorphology-based taxonomyrdquo Molecular Phylogenetics and Evolution vol64 no 1 pp 73ndash92 2012

[27] K-N Shen B W Jamandre C-C Hsu W-N Tzeng and J-D Durand ldquoPlio-Pleistocene sea level and temperature fluc-tuations in the northwestern Pacific promoted speciation inthe globally-distributed flathead mullet Mugil cephalusrdquo BMCEvolutionary Biology vol 11 no 1 article 83 2011

[28] R Hall ldquoThe plate tectonics of Cenozoic SE Asia and thedistribution of land and seardquo in Biogeography and GeologicalEvolution of SE Asia R Hall and J D Holloway Eds pp 99ndash131Backhuys Publishers Leiden The Netherlands 1998

[29] R Hall ldquoSoutheast Asiarsquos changing palaeogeographyrdquo Blumeavol 54 no 1ndash3 pp 148ndash161 2009

[30] R F Inger and P K Chen ldquoThe freshwater fishes of NorthBorneordquo Fieldiana Zoology vol 45 pp 1ndash268 1962

[31] J Bohlen V Slechtova H H Tan and R Britz ldquoPhylogenyof the Southeast Asian freshwater fish genus Pangio (Cyprini-formes Cobitidae)rdquoMolecular Phylogenetics and Evolution vol61 no 3 pp 854ndash865 2011

[32] M de Bruyn E Nugroho M M Hossain J C Wilson andP B Mather ldquoPhylogeographic evidence for the existence ofan ancient biogeographic barrier the Isthmus of Kra SeawayrdquoHeredity vol 94 no 3 pp 370ndash378 2005

[33] J Parnell ldquoThe biogeography of the Isthmus of Kra region areviewrdquoNordic Journal of Botany vol 31 no 1 pp 001ndash015 2013

[34] S Usmani S G Tan S S Siraj and K Yusoff ldquoPopulationstructure of the Southeast Asian river catfishMystus nemurusrdquoAnimal Genetics vol 34 no 6 pp 462ndash464 2003

[35] M P Tan A F J Jamsari and M N Siti Azizah ldquoPhylo-geographic pattern of the striped snakehead Channa striatain Sundaland ancient river connectivity geographical and

Advances in Evolutionary Biology 21

anthropogenic singnaturesrdquo PLoS ONE vol 7 no 12 Article IDe52089 2012

[36] R Abell M L Thieme C Revenga et al ldquoFreshwater ecore-gions of the world a new map of biogeographic units forfreshwater biodiversity conservationrdquo BioScience vol 58 no 5pp 403ndash414 2008

[37] P U Clark D Archer D Pollard et al ldquoThemiddle Pleistocenetransition characteristics mechanisms and implications forlong-term changes in atmospheric pCO

2rdquo Quaternary Science

Reviews vol 25 no 23-24 pp 3150ndash3184 2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 7: ResearchArticle - Université Laval Julian... · ResearchArticle A DNA Barcode-Based Evaluation of the Southeast Asian Catfish Genus Hemibagrus Bleeker, 1862 (Teleostei: Siluriformes;

Advances in Evolutionary Biology 7

8811

10

9

8

7

6

5

4

3

1

2

66

85

44

32

98

91

81

8432

57

99

99

99

49

002

34

15

43

86

99

96

H capitulum (Indochinese clade)

H capitulum (Sundaic clade)

H hoeveniiH fortis

H spilopterus

H baramensis species group

H planiceps species groupH menoda species group

H guttatus species group

H wyckii species group

XXRAJ0604

PESAL0102

XXRAJ0301Leiocassis longirostris

mtDNAclade

H nemurusspecies group

Unspecified species group

Figure 2 The taxon identification phenogram of Hemibagrus inferred using the Neighbor-Joining method in MEGA 52 The optimal treewith the sumof branch length = 186668598 is shown Bootstrap values are indicated on each node of the tree (500 replicates) Bar indicates thenumber of substitutions changes estimated using Tamura-Nei distance considering invariable site contribution (+I)The analysis involved 119nucleotide sequences All positions containing gaps and missing data were eliminatedThere were a total of 597 positions in the final dataset

mtDNAclade

H capitulum (Indochinese clade) 11

93

97H capitulum (Sundaic clade)H hoevenii

9999

84H fortis 8

9

10

H spilopterus 773 92

Unspecified species group 678

XXRAJ060454 34

90 H baramensis species group 5

49 H planiceps species group 4

H menoda species groupPESAL0102 3

H wyckii species group 199

99

99H guttatus species group 2

XXRAJ030149

47

Leiocassis longirostris

005

H nemurusspecies group

94

72

Figure 3 Evolutionary history of Hemibagrus inferred by using the maximum likelihood method based on the Hasegawa-Kishino-YanomodelThe tree with the highest log likelihood (minus56774825) is shown Bootstrap values are indicated on each node of the tree (500 replicates)Bar indicates the number of substitution changes estimated using Hasegawa-Kishino-Yano model (HKY) with gamma distribution (+119866 =13290) The analysis involved 119 nucleotide sequences All positions containing gaps and missing data were eliminated There were a totalof 597 positions in the final dataset

their ZRC numbers do not appear in the analysis of NampK Asno specimens of the other 3 species comprising the H wyckiispecies group were available for analysis we cannot confirmif this species group is monophyletic

mtDNA Clade 2 The H guttatus Species Group The secondclade of the Hemibagrus gene tree (Figure 2) is composed oftwo reciprocally monophyletic subclades clearly reflectingthe H guttatus species group as defined by NampK (Figure 4)

8 Advances in Evolutionary Biology

100∘

10∘

110∘

0∘

120∘

0

0 250

250

500

500

750(km)

(mi)

002

98

43

84

34

99

99

79

96

51

59

99

78

49

H peguensis

H guttatus

H macropterus

H wyckioides

PESAL0102

XXRAJ0301Leiocassis longirostris

MAPEA0201MAPEA0302MAPEA0101

GUPEA0501GURED0102

GUPEA0401GUPEA0602

WYCHP0401WYMEK1101

WYCHP0203

See Figures 7ndash12

See Figures 5 and 6

Shelf minus75mShelf minus120 m

Figure 4 Phylogenetic relationships of the individual sequences comprising four species H guttatus H macropterus (together forming theH guttatus species group) H wyckioides and H peguensisThe subtree is extracted from the taxon identification phenogram inferred usingthe Neighbor-Joining method (see Figure 2) The percentage of replicate trees in which the associated sequences clustered together in thebootstrap test (500 replicates) is shown next to the branches The distribution of these sequences is illustrated in the right panel

The group comprises three valid species H guttatus Hmacropterus andH vietnamicus (NampK) Specimens availablefor DNA analysis were obtained from the Pearl River andRed River (11 fish 7 sequences Table 1 and Table S1) Thetwo reciprocally monophyletic subclades comprising theH guttatus species group represent H guttatus and Hmacropterus (Figure 4) Although no ZRC specimens wereexamined by NampK species identification was confirmed(H H Ng personal communication) No H vietnamicusspecimens were available for DNA analysis The twospecies composing the group are closely related (119901 = 51(SE = 08))

mtDNA ldquoCladerdquo 3 The H menoda Species Group Thisisolated genotype (Figure 4) most likely identifies a cladeencompassing the H menoda species group although the

single sequence (Table S1) is shared by only 2 specimensof H peguensis from the Salween River Myanmar alsoincluded in the analysis of NampK (where its provenance wasidentified as the neighboring Irrawaddy River) The otherspecies comprising this group (H menoda H caveatusand H punctatus) were not sampled for DNA analysis Themonophyletic nature of this species group thus remains to bedemonstrated

31 The H planiceps + H baramensis Group The remainderof the gene tree is split into two large sister groups withmarginal bootstrap support (73 in ML and 43 in NJ)the H planiceps + H baramensis group and the H nemurusspecies group (Figures 2 and 3) The H planiceps + Hbaramensis group is strongly supported in both NJ and MLanalyses (84 and 90 bootstrap support respectively

Advances in Evolutionary Biology 9

110∘

47

53

69

81

69

5199

52

95

97

78

001

H bongan

H velox

H gracilis

H planiceps

H divaricatus

BOKAP1801

BOKAP1601BOKAP1701

BOKAP1402BOKAP1503

VEBAH0907GREND0206

PLCIM0114PLCIS0101

DIPER0401DIPER0301

DIPER0201

100∘

10∘

0∘

120∘

0

0 250 500

250 500

750(km)

(mi)

Shelf minus75mShelf minus120 m

Figure 5 Phylogenetic relationships of the individual sequences and species comprising theH planiceps species groupH bongan H veloxH gracilis H planiceps and H divaricatus The subtree is extracted from the taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicate trees in which the associated sequences clustered together in the bootstrap test(500 replicates) is shown next to the branches The distribution of these sequences is illustrated in the right panel

Figures 2 and 3) The two groups are genetically similarwith a genetic divergence estimate of 119901 = 121 (SE = 09)Although theH planiceps species group is strongly supportedin both trees (99) bootstrap support of the H baramensisspecies group is weak (Figure 2 32 Figure 3 34) Thegeographical distribution of the two groups differs Membersof the H planiceps species group are found throughout theGreater Sunda Islands and Peninsular Malaysia but they areisolated in the upper reaches of large rivers (Figure 5) Incontrast members of theH baramensis species group appearto be restricted to Sabah North Borneo (Figure 6)

mtDNA Clade 4 The H planiceps Species Group The Hplaniceps species group (Figure 5) is comprised of 5 closelyrelated but genetically distinct species (mean within-groupdivergence 119901 = 20 (SD = 033) H planiceps (Java)H gracilis (eastern Peninsular Malaysia) H divaricatus(western Peninsular Malaysia) H bongan (Borneo) andH velox (Sumatra)) H lacustrinus the sixth species of thisgroup (NampK) was not sampled in the present study WithintheH planiceps species groupH planiceps formed a distinctspecies (Figure 5) comprising two sequences One sequence(PLCIM0114 Table S1) characterized 14 specimens obtainedfrom Ci Manuk West Java 9 of which were identified as Hplaniceps by NampK (Table S1) A second sequence (PLCIS0101

Table S1) was obtained from one specimen sampled atCi Sadane West Java that we classified as H planicepsH bongan is represented by 5 sequences obtained from 8specimens sampled in the Kapuas River (Figure 5) Two ofthese specimens (ZRC 43013 and 43015) were identified asH bongan by NampK (Table S1) H divaricatus is composedof 3 sequences from three specimens obtained at Gerik onthe Perak River (western Peninsular Malaysia) One of thesespecimens (ZRC 41151) is the holotype of this new species(NampK) H gracilis is represented by only one sequence(GREND0206 Table S1) shared by 6 specimens sampledat Ulu Endau Jasin River eastern Peninsular Malaysiafour of which were identified by NampK (Table S1) ZRC21484 comprises the holotype and ZRC 21482 21486 and21487 comprise the paratypes (NampK) of this species (TableS1) Finally H velox is represented by only one sequence(VEBAH0907) from 7 specimens sampled at Kerinci BatangHari River Sumatra Three of these specimens (ZRC 41503Table S1) represent 3 paratypes of the species (NampK)

mtDNA Clade 5 The H baramensis Species Group The threespecies comprising the H baramensis species group (Figures2 and 3) form three monophyletic subclades (mean within-group divergence 119901 = 106 (08) Figure 6) H semotus iscomposed of two sequences (SEPAD0101 SEPAD0202) that

10 Advances in Evolutionary Biology

110∘

002

H sabanus

H semotus

H baramensis

SAKIN0204SASGM0706

BASGM0402BASGM0301

BASGM0201BASGM0104

SESGM0502SEUMA0101

SEPAD0202SEPAD0101

See Figure 5

100∘

10∘

0∘

120∘

0

0 250 500

250 500

750(km)

(mi)

99

57

32

83

99

72

99

56

66

54

99

Shelf minus75mShelf minus120 m

Figure 6 Phylogenetic relationships of the individual sequences and three species comprising the H baramensis species group H semotusH sabanus and H baramensis (furcatus) The subtree is extracted from the taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicate trees in which the associated sequences clustered together in the bootstrap test(500 replicates) is shown next to the branches The distribution of these sequences is illustrated in the right panel

characterize fish that are the holotype and paratypes of Hsemotus (ZRC 46122 Table S1) along with two somewhatmore divergent sequences (Figure 6) One of these sequences(SGM0502) was found in the Segama River (Danum Valley)and identified as H baramensis by NampK (ZRC 40525)(Table S1) The second sequence (UMA0101) was from afish sampled at Tawau Umas Umas River (ZRC 46123) alsoidentified as H baramensis by NampK These sequences mostlikely reflect distinct lineages within H semotus inhabitingdifferent rivers as the mean intraspecies sequence divergence(119901 = 16) indicates population-level divergence

H sabanus and H furcatus (synonymized with H bara-mensis by NampK) cluster together to form a distinct subcladebut clearly diverge at the species level (Figure 6) H sabanuscomprises 2 sequences that are shared respectively by 6specimens (ZRC 42741) from the Segama River Sabah and4 specimens (part of ZRC 42743) from the KinabatanganRiver Sabah (Table S1) All of these specimenswere identifiedas H sabanus by NampK (Table S1) Finally H baramensisforms a distant subclade (147 mean sequence divergencebetween H baramensis and H sabanus) composed of 4sequences (Figure 6) shared by 8 specimens all sampled inthe Segama River Danum Valley Conservation Area SabahSix of these specimens (ZRC 40523 (2 specimens) ZRC40527 (2 specimens) and ZRC 40526 (2 specimens) Table S1)comprise the paratypes of H baramensis (furcatus) (NampK)

32 The H nemurus Species Group In both NJ and ML trees(Figures 2 and 3) the H nemurus species group is strongly

supported (bootstrap support 98 in NJ and 99 in ML)indicating that it forms a distinct and widespread geneticentity within the genus Hemibagrus However relationshipswithin the group are not so clear In general the H nemurusspecies group is composed of 4 clades The first clade may beconsidered as a species group nested within the H nemurusspecies group (85 and 93 bootstrap support in NJ andML resp) encompassing three clades H hoevenii (clade9) the H capitulum Sundaic clade (clade 10 including Hnemurus) and the H capitulum Indochinese clade (clade 11)(Figures 2 and 3) An additional three clades (Figures 2 and3) are formed by an unidentified species group (clade 6) Hspilopterus (clade 7) andH fortis (clade 8)These three latterclades are distinct at the congeneric species level (unspecifiedversus H spilopterus 119901 = 77 (07) unspecified versus Hfortis 119901 = 85 (07) and H spilopterus versus H fortis119901 = 75 (08)) The distribution of these three clades issomewhat disparate H fortis and the unspecified speciesgroup are principally found in Borneo whereasH spilopterusis widely distributed on the Indochinese mainland

mtDNA Clade 6 Unspecified Species Group This geneticallydiverse group encompassing at least 3 subclades (H spp AB and C Figure 7 within-group mean sequence divergence119901 = 68(06)) is confoundedwith theH baramensis speciesgroup byNampK It is composed of sequences fromfish sampledin Sarawak Sabah and eastern PeninsularMalaysia Subclade119867 spp A (Figure 7) is particularly interesting as it contains2 sequences (CHSARA20 CHRAJ0401) that characterize

Advances in Evolutionary Biology 11

110∘

XXPAD0303XXPAD0601

XXPAD0401XXBEL0202

XXBRM0202XXRAJ0511

XXPAD0501

XXEND0410CHSARA20CHRAJ0401

XXBEL0101XXSEG0101

100∘

10∘

0∘

120∘

0

0 250 500

250 500

750(km)

(mi)

91

86

83

99

98

47

81

001

91

72

99

80

H spp C

H spp B

H spp A

Shelf minus75mShelf minus120 m

Figure 7 Phylogenetic relationships of the individual sequences comprising the unspecified species group containing three subcladesidentified as H spp A B and C The subtree is extracted from the taxon identification phenogram inferred using the Neighbor-Joiningmethod (see Figure 2) The percentage of replicate trees in which the associated sequences clustered together in the bootstrap test (500replicates) is shown next to the branches The distribution of these sequences is illustrated in the right panel

H chrysops a new species identified by Ng and Dodson [11]However specimens of H chrysops from the Rajang Riverwere subsequently synonymized by NampK with H capitulumof the H nemurus species group (ZRC 23008ndash23014 TableS1) However an additional 14 fish from the Sadong Riverat Serian bearing genotype SARA20 (Table S1) and thusgenetically identical to the Rajang H chrysops samples wereidentified by NampK as H spilopterus (ZRC 29583ndash96 TableS1) These specimens however were erroneously identifiedin NampK as originating in the Chao Phraya River (Thailand)Subclade A also contains one sequence obtained from 10 fishsampled on the Endau River (Mersing) in eastern PeninsularMalaysia These fish were not morphologically examinedFinally subclade A also contains one sequence from oneunidentified fish sampled in the Segaliud River Sabah andanother unidentified fish sampled in the Belait River BruneiSubclade H spp B encompasses 2 sequences one of whichcomes from a fish sampled in the Padas River (XXPAD0501)and part of the ZRC 46122 fish lot identified as H semotusby NampK (Table S1) The second sequence (XXRAJ0511) wasshared by 11 unidentified fish sampled at Katibas on theRajang River Sarawak (Table S1) Finally subclade 119867 sppC encompasses 5 sequences Three sequences (XXPAD0303XXPAD0401 and XXPAD0601) were obtained from thePadas River and identified byNampKasH semotus (ZRC46122Table S1) The fourth sequence (XXBRM0202) was obtainedfrom 2 fish sampled in the Baram River and was identified

as H baramensis by NampK (ZRC 42742) The 5th sequencecomposing this group was obtained from 2 unidentified fishsampled in the Belait River Brunei (Table S1)

mtDNA clade 6 is thus problematical within the contextof the revision of the genus Hemibagrus Two highlydivergent mtDNA clades (clade 5 and clade 6 Figures 2and 3) occur in sympatry principally in the Padas River ofSabah North Borneo (Figures 6 and 7) Specimens of theunspecified species group are morphologically similar tothe H baramensis species group where they are assigned inthe analysis of NampK However the mean genetic distancebetween the unspecified species group and theH baramensisspecies group is 119901 = 193 (28) close to genus leveldivergence Furthermore subclade 119867 spp A (Figure 7)contains not only H chrysops but also an unidentifiedpopulation from eastern Peninsular Malaysia (XXEND0410Table S1) and two additional Sabah sequences On theother hand the unspecified species group clusters with allother members of the H nemurus species group (meangenetic divergence of unspecified group versus H capitulum(Sundaic clade) 119901 = 76 (07) (Figure 2)) with strongbootstrap support Such genetic clustering may justify theinclusion of H chrysops and associated sequences in theH nemurus species group but its synonymization with Hcapitulum (NampK) is difficult to defend on genetic groundsWe tentatively conclude that H chrysops is a valid speciesbased on genetic identity [11] forming part of an unknown

12 Advances in Evolutionary Biology

110∘

SPCPCHMK12

SPMEK0202SPMEK0401

SPMEK0301SPMEK0103

SPMEK1401SPMEK0501

SPMEK1201SPMEK1301

SPMEK1601SPMEK1502

SPMEK0701SPMEK0801SPMEK1701

SPCHP0501SPCHA0201

SPCHA0304SPPAT0101

100∘

10∘

0∘

120∘

0

0 250 500

250 500

750(km)

(mi)

000499

99 98

60

47

48

54

96

7091

38

86

87

99

80

58

H spilopterus ldquoCrdquo

H spilopterus ldquoBrdquo

H spilopterus ldquoArdquo

Shelf minus75mShelf minus120 m

Figure 8 Phylogenetic relationships within H spilopterus comprised of 3 subclades identified as A B and C The subtree is extracted fromthe taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicate trees in whichthe associated sequences clustered together in the bootstrap test (500 replicates) is shown next to the branches The distribution of thesesequences is illustrated in the right panel

species group found throughout North Borneo (and toa lesser degree in eastern Peninsular Malaysia) that maybe considered as part of the H nemurus species groupHowever the morphological confusion of the unspecifiedspecies group with the sympatric highly divergent Hbaramensis species group suggests the existence of a crypticspecies assemblage (see Section 4)

mtDNA Clade 7 Clade 7 (Figures 2 and 3) is composed of atleast 3 distinct subclades that appear to reflect 3 geneticallydivergent populations ofH spilopterus Subclade A (Figure 8)encompasses 7 sequences obtained from 8 fish sampled inthe Mekong River Four fish bearing 3 of these sequenceswere identified as paratypes (ZRC 43324) of H spilopterusby NampK (Table S1) One additional fish was identified asH spilopterus by NampK (ZRC 46157 Table S1) Subclade Bencompasses 6 sequences obtained from 9 fish all sampledfrom the Mekong River (Table S1) The fish sampled at ThatPhanom (ZRC 46012) was identified as H spilopterus inthe analysis of HampK (Table S1) Subclade C encompasses 5sequences obtained from 19 fish sampled in the Chao PhrayaMae Khlong and Chanthaburi Rivers Thailand Onesequence (CPCHMK12) was found in all of these drainages(Table S1) A closely allied sequence was found in one fishsampled at Phatthalung just south of the Isthmus of Kra onthe Thai-Malay Peninsula None of these fish were examined

by NampK and thus we are unable to definitively identifythe species However given the widespread occurrence ofH spilopterus in the Chao Phraya and Mae Klong Riversreported by NampK we conclude that these fish are part of awidespread H spilopterus population complex The meanwithin-group (clade 7) genetic divergence (119901 = 29 (04))suggests intraspecific population-level divergence

mtDNA Clade 8 Clade 8 encompasses 3 sequencesobtained from 9 fish sampled in east Sabah specifically theKinabatangan Segama and Umas Umas Rivers (Figure 9)The Kinabatangan sequence is somewhat divergent withinthe clade and the specimen was not identified by NampK Onesequence (SGM0606) characterized 6 fish from the SegamaRiver 2 of which were identified as H fortis (ZEC 4052140522 Table S1) by NampK (Table S1) Finally one fish bearinga closely related genotype from the Umas Umas River wasidentified as H baramensis by NampK (Table S1)

mtDNA Clade 9 Clade 9 is a sister group to the remaining2 clades composing the H nemurus species group Thiswell-defined clade (Figure 10) clusters 6 sequences from42 fish that are clearly identified as H hoevenii (NampK)Some of these haplotypes are geographically widespread onesequence (JOMSKA11 Table S1) was found in the Johor River(East Peninsular Malaysia) the Musi River (Sumatra) and

Advances in Evolutionary Biology 13

100∘ 110∘

0∘

120∘

00 250

250500

500750

(km)

(mi)

FOKIN0102FOSGM0606

FOUMA0201H fortis

10∘

99

86

001

Shelf minus75mShelf minus120 m

Figure 9 Phylogenetic relationships within H fortis comprised of 3 sequences The subtree is extracted from the taxon identificationphenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicate trees in which the associated sequencesclustered together in the bootstrap test (500 replicates) is shown next to the branches The distribution of these sequences is illustrated in theright panel

the Kapuas River (West Borneo) and a second sequence(HMUBHKABM23 Table S1) was found inMuarRiver (WestPeninsular Malaysia) and the Batang Hari (Sumatra) theKapuas (Sarawak) and the Baram Rivers (Sarawak) Many ofthese fish were identified as H hoevenii by NampK (Table S1)The existence of twodistinct but very closely related subclades(Figure 10 119901 = 06 (03)) that are not geographicallysegregated suggests some recent isolation event followed bywidespread dispersal and secondary contact

33 The H capitulum Group The final two monophyleticgroups of the Hemibagrus gene tree are approximatelyequivalent to the remaining species composing the Hnemurus species group as defined by NampK Dodson etal [10] and Ng and Dodson [11] have previously referredto clade 10 as the Sundaic clade as most sequences wereobtained from fish sampled on the Greater Sunda Islandsand clade 11 as the Indochinese clade asmany sequences wereobtained from fish sampled on the Indochinese mainland(Figure 2 Table S1) Although most of these sequencesare assigned to H capitulum by NampK there are somesignificant phylogeographic differences between the 2 cladesIn particular there appears to be an east-west division in thedistribution of the two clades on the Thai-Malay Peninsulawith the Sundaic clade (clade 10) found on the west coast andthe Indochinese clade found on the east coast with sympatricoccurrence in the Muar River Nevertheless mean sequencedivergence between the two clades is 119901 = 29 (05) avalue that is indicative of population-level or subspecies

divergence The two clades are thus best considered ascomprising one species

mtDNA Clade 10 The Sundaic clade includes a number ofclosely related sequences that comprise part of H capitulumand H nemurus Very little genetic structure is apparentand bootstrap support for tree topology within this group isgenerally very weak (Figure 11) A widely spread genotype(NCLCTBA20) characterized 6 of 17 specimens from CiTarum at the Cirata reservoir (West Java) (Table S1) and5 were identified as H nemurus in the analysis of NampK(ZRC 42564) (Table S1) The remaining fish characterized bythis sequence were obtained from Ci Liwong at Sawangan(West Java) and one fish was obtained from the Barito Riverat Banjarmassin south Borneo This latter specimen (ZRC40553) was identified by NampK as H capitulum (Table S1)Another common genotype (NBHKPBR09) was identified aseither H capitulum H hoevenii or H nemurus dependingon the river of origin (Table S1) Many of the remainingsequences of the Sundaic clade appear to characterize fish thatNampK identified asH capitulum A geographically widespreadgenotype (CPEBEMUKA36) characterized 9 fish sampledon the Perak River at Gerik that were identified by NampKas H capitulum (ZRC 41150 Table S1) Another commongenotype within this clade (CABHMS20) included 2 fishfrom the Batang Hari at Jambi and identified by NampK asH capitulum (ZRC40534 Table S1) Another 2 sequences(CABAR0203 CABAR0305) characterized 8 fish sampledon the Barito River at Banjarmasin and were identified as

14 Advances in Evolutionary Biology

100∘

10∘

110∘

0∘

120∘

0

0 250

250

500

500

750(km)

(mi)

53

64

99

85

64

0002

HJOMSKA11

HOBAH0601

HOBHMS05

HMUBHKABM23

HOBAH0701

HOKAP0501

H hoevenii ldquoBrdquo

H hoevenii ldquoArdquo

Shelf minus75mShelf minus120 m

Figure 10 Phylogenetic relationships withinH hoevenii comprised of 2 subclades and 6 sequences The subtree is extracted from the taxonidentification phenogram inferred using theNeighbor-Joiningmethod (see Figure 2)The percentage of replicate trees in which the associatedsequences clustered together in the bootstrap test (500 replicates) is shown next to the branches The distribution of these sequences isillustrated in the right panel

110∘

XXMAH0110

CABAR0305

96 CAKPR0201

74CAKAP0401

5931 CAKAP0601

35 CPEBEMUKA36

646 CAKAP0903

33 CAKAP080114 CAKAP0701

CARAJ0210

4 CABAR0203CABRA0204

20

CBHKPBR0919

CABRA0301

15CABHMS20

24 NCLCTBA20

63 NECIT0201

H capitulumldquoSundaic claderdquo

H nemurus

0002 120∘

0 250 500 750

(km)

0 250 500

(mi)100

10∘

0∘

Shelf minus75mShelf minus120 m

Figure 11 Phylogenetic relationships within theH capitulum ldquoSundaicrdquo clade including 2 sequences associated withH nemurusThe subtreeis extracted from the taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicatetrees in which the associated sequences clustered together in the bootstrap test (500 replicates) is shown next to the branchesThe distributionof these sequences is illustrated in the right panel

Advances in Evolutionary Biology 15

H capitulum

0∘

10∘

100∘

110∘

120∘

66

63

88

59

38

39 68

71

6991

57

9754

88 4944

39

53

89

0002

ldquoIndochineserdquogroup ldquoErdquo

H capitulumldquoIndochineserdquo

group ldquoDrdquoH capitulumldquoIndochineserdquo

group ldquoCrdquo

H capitulumldquoIndochineserdquo

group ldquoBrdquo

H capitulumldquoIndochineserdquo

group ldquoArdquo0

0

250

250

500

500

750

(km)

(mi)

CATER0301CATERR0401

CKETEMU23

CAMEK0601CACHP0301

CAMKPA03

CAMEK0910CAMEK1003

XXMUBM02

CABMTU09

CABRM0601

CAKAP1201CAKAP1001

CAKAP1101CAKAP1301

CABRM0501

CASAD0102

CASAD0220

CAEND0301

CABAH0801

CAPABH05CATEEN03

CATER0701CATERR0801

CAKETE04CATER0501

CAPAH0303

Shelf minus75mShelf minus120 m

Figure 12 Phylogenetic relationships within theH capitulum ldquoIndochineserdquo clade encompassing 5 subclades identified as A B C D and EThe subtree is extracted from the taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2)The percentageof replicate trees in which the associated sequences clustered together in the bootstrap test (500 replicates) is shown next to the branchesThedistribution of these sequences is illustrated in the right panel

H capitulum by NampK (ZRC 40553 Table S1) Although notincluded in the analysis of NampK samples from the Sundaicclade were also found in the Kapuas River (West Borneo)Finally 10 fish sampled on the Mahakam River at Samarindaeast Borneo are all characterized by one relatively divergentgenotype (CAMAH0110) butwere considered asH capitulumin the analysis of NampK (ZRC 40533 Table S1)TheMahakampopulation sample is the most genetically distinct of theSundaic clade (119901 = 17 (04)) but nevertheless indicativeof intraspecific population-level divergence (Figure 12)

The topology of the Sundaic clade does not permit usto draw a clear distinction between H nemurus and theSundaic H capitulum The mean within-group divergenceof the Sundaic clade is only 119901 = 08 (02) well withinpopulation-level divergence values We postulate that Hnemurus may be one of several regional variants of awidespread Sundaic H capitulum clade that has radiatedrelatively recently This may explain the existence of manyshared sequences throughout the Sundaic islands assigned todifferent species in different regions

mtDNA Clade 11 Finally the Indochinese clade of Hcapitulum represents a geographically widespread andweakly diversified clade (within-group mean sequencedivergence 119901 = 11 (02) Figure 12) Although few fishfrom this genetic clade were included in the morphological

analysis of NampK most may be considered as H capitulumgiven their geographic distribution and close geneticrelationship to the Sundaic H capitulum Four sequencesin this clade were identified as H capitulum in the analysisof NampK 3 specimens characterized by one sequence(CABMTU09) found in the Tutong River of westernBorneo (ZRC 40482) 6 characterized by the same sequencefrom the Baram River (ZRC 40498) another 2 sequencesfrom the Baram River (ZRC 40498) and 20 specimenscharacterized by an abundant genotype (CASAD0220)found at 2 sites in the Sadong River also in western Borneo(ZRC 39526 5 specimens 39527 3 specimens and 38753 4specimens) (Table S1) Unfortunately none of the specimenscollected on the Indochinese mainland for genetic analysiswere examined by NampK Nevertheless the distribution ofspecimens analyzed for mtDNA resembles the distribution ofspecimens examined by NampK Although weakly structuredwe may recognize 5 subclades One genotype (CAPAH0303)is basal to the phenogram Subclade A encompasses 8sequences mainly found in eastern Peninsular Malaysia(Table S1 Figure 12) These rivers furnished many of thespecimens NampK examined to define H capitulum Howeversome sequences in this group occur in sympatry withsequences identified within the Sundaic H capitulum groupTwo sequences from subclade A occurred in the Batang HariSumatra (Table S1) Subclade E also encompasses sequences

16 Advances in Evolutionary Biology

found in Peninsular Malaysia as well as in the Mekong ChaoPhraya and Mea Khlong Rivers (Figure 12) In addition3 subclades encompass sequences found principally inwestern Borneo subclade B in the Baram and Sadong RiversSarawak subclade C in the Kapuas River West Kalimantanand subclade D in the Baram and Tutong Rivers of Sarawakand Brunei respectively (Figure 12) This latter subclade alsoincludes one genotype found in the Muar River of westernpeninsular Malaysia Finally subclade E encompasses 5sequences from the Mekong Chao Praya and Mae KhlongRivers all within the distribution area of H spilopterus

EvolutionaryHistoryThe evolutionary history of theHemiba-grus species groups analyzed here dates from at least theMiocene 15ndash20Ma (Figure 13) when considering the widelyused molecular clock of 2 sequence divergence per millionyears Bootstrap support for the sister group relationships ofH wyckioides theH guttatus species group andH peguensisare all weak (clearly due to undersampling) and little can besaid of their evolutionary history other than the fact that theyoccupy basal positions in the rooted trees The remainder ofboth the NJ and ML trees is composed of two major sistergroups the H planiceps + H baramensis group and the Hnemurus species group (Figures 2 3 and 13)TheH planiceps+ H baramensis group is strongly supported in both NJ andML analyses (84 and 90 bootstrap support resp Figures2 and 3) The H nemurus species group is also stronglysupported in both analyses (98 and 99 bootstrap supportFigures 2 and 3) These two major phylogenetic groups areestimated to have diverged at least 8 to 12Ma ago during themid to late Miocene (Figure 13)

Much of the evolutionary history of Hemibagrus hasoccurred during the Quaternary starting approximately26Ma ago This is particularly true of the H planicepsand H nemurus species groups The radiation of the 5species included in the H planiceps species group appearsto coincide with the middle Pleistocene transition period ofapproximately 1Ma ago Their high-elevation distributionhowever appears to have largely isolated them from theeffects of lowered sea levels during glacial maxima and theattendant opportunities for widespread dispersal (Figure 5)The evolutionary histories of theH baramensis species groupand the unspecified species group date back to the latePliocene In contrast the low-elevation distribution of theHnemurus species group in Sundaland appears to have favoredtheir widespread distribution during the Quaternary ice ageIn particular H hoevenii appears to be the most recentlydispersed of the species under study with diversification anddispersal occurring approximately 03Ma ago (Figure 13)

4 Discussion

41 The Congruence of Morphological and Genetic AnalysesCombining genetic and morphological analyses to elucidatetaxonomy allowed us to better understand the diversificationand evolutionary relationships within this speciose group offishes Given the rapid and relatively constant rate of mtDNAevolution the deeper the branches within the phylogenetictree are the greater our ability to distinguish valid taxa

should be Conversely the lack of complete congruencebetween morphological- and genetic-based identificationsis expected to increase among the shallower branches of thephylogenetic tree resulting in the appearance of polyphyleticspecies Although 2 of the 8 species groups defined by Ngand Kottelat [7] were not sampled in this study the validityof the remaining 6 species groups and their componentspecies defined on the basis of morphological similaritywas largely confirmed by the analysis of the mitochondrialCyt 119887 bar code As expected congruence is strongest at thebase of the NJ and ML trees where the H guttatus speciesgroup formed a well-defined monophyletic group The Hmenoda and H wyckii species group were each representedby only one species but they were clearly distinct relativeto sequences characterizing the neighboring species groupsThe H planiceps H baramensis and H nemurus speciesgroups and their component species were also generallycongruent in morphological and genetic analyses Also asexpected most cases of discrepancy between morphological-based species identification and genetic-based speciesidentification occurred on relatively shallow branches of thephylogenetic tree These involve principally H spilopterusand the two mtDNA subclades composing H capitulumHowever a far more unexpected lack of congruence involvesthe definition of a deeply divergent unspecified genetic groupconfounding members of theH baramensis species group inthe morphological analysis of Ng and Kottelat [7]

Interpreting Polyphyly The apparent polyphyletic origin ofa species may be due to incomplete lineage sorting amongrecently diverged species the introgression of mtDNA dueto species hybridization or the erroneous identificationof species due to the lumping of morphologically crypticspecies In the absence of nuclear DNA analyses it isoften difficult to distinguish between introgression andincomplete lineage sorting For example several sequencescharacterizing the Indochinese clade of H capitulum(subclade E) are found in the Mekong and Chao PhrayaRivers well within the distributional area of H spilopterusH spilopterus and H capitulum first diverged at least 3MaOn the one hand this apparent case of polyphyly mayhave resulted from incomplete lineage sorting betweenrecently divergedH spilopterus andH capitulum We wouldhowever have expected similar cases of polyphyly amongpopulations of the Indochinese clade of H capitulum butthis was not detected On the other hand the sympatricoccurrence of H spilopterus subclade A and subcladeB sequences with H capitulum Indochinese subclade Esequences in the Mekong River may represent a migrationevent and local introgression of H capitulum with Hspilopterus A similar event may have occurred in theChao Phraya and Mae Khlong Rivers where H spilopterussequences cooccur withH capitulum sequences Computingthe divergence of H capitulum sequences (Indochinesesubclade E) cooccurring with H spilopterus and Hcapitulum sequences occurring in eastern PeninsularMalaysia (subclade A) reveals a mean sequence divergence119901 = 15 (04) Thus the H capitulum sequences occurringin sympatry with H spilopterus diverged at most 750 ka ago

Advances in Evolutionary Biology 17

H fortis

XXRAJ0604H furcatus

H sabanusH semotusH divaricatus

H planicepsGREND0206

VEBAH0907

H bonganPESAL0102

H wyckioidesH macropterus

H guttatusXXRAJ0301Leiocassis longirostris

Ma

4971

92

47

51

9999

99

9999

99

99

99

9999

99

99

99

99

65

96

96

4946

93

79

78

70

91

93

98

86

87

91

95

97

71

85

9999

05101520

005010015020 000

H capitulum Indochinese clades AndashE

H capitulum Sundaic clade

H hoevenii B

H hoevenii A

H spilopterus C

H spilopterus B

H spilopterus AH spilopterus A

H spp CH spp B

H spp A

Figure 13 Linearized tree with branch lengths estimated with the molecular clock using a mutation rate of 1 per Ma (=divergence rate of2 per Ma) The tree topology is identical to the ML reconstruction illustrated in Figure 3 (see the specificities of the tree reconstruction inthe caption of Figure 3) Boxes represent 95 confidence intervals of divergence time estimates for each node Vertical dashed lines redirectto dates indicated on the timescale (Ma) Numbers below timescale indicate the estimated number of substitution changes using Hasegawa-Kishino-Yanomodel (HKY)with gamma distribution (+119866 = 13290) after linearizing the treeH furcatus is synonymizedwithH baramensis

from eastern Peninsular Malaysia populations suggestinga recent introgression event Similarly H capitulum in theKapuas River is characterized by sequences of both theSundaic and the Indochinese clades As both sequences arebroadly sympatric it is not possible to distinguish betweenincomplete lineage sorting and introgression of these closelyrelated intraspecific lineages

A Possible Case of Morphological Stasis Clade 6 of thepresent analysis defines a well-supported monophyletic cladefound in Sabah Sarawak and the Endau River of easternpeninsula Malaysia that has no equivalent taxonomic statusin the revision of Ng and Kottelat [7] In Borneo they occursympatrically with the H baramensis species group and areconfounded as either H baramensis or H semotus These

two clades are genetically highly divergent having divergedbetween 8 and 12Ma ago It thus seems highly unlikely thatthey represent retained polymorphisms due to incompletelineage sorting Sequences previously identified as character-izing H chrysops [11] are part of clade 6 and extend to thesouth (Rajang and Sadong Rivers of Sarawak West Borneo)with one closely related genotype found in eastern peninsularMalaysia (Endau River) where it may well be confoundedwith H capitulum H chrysops was synonymized with Hcapitulum by Ng and Kottelat [7] based on their analysis ofspecimens ofH chrysops from the Rajang RiverThus despitea long and apparently independent phylogenetic historythe unspecified species group appears largely cryptic andconfounded principally with members of the H baramensisspecies group

18 Advances in Evolutionary Biology

We suggest that this situation is an example of mor-phological stasis involving the coexistence of a number ofgenetically divergent yet morphologically cryptic species thatare lumped together [24] A striking example of morpho-logical and ecological stasis among fishes is provided by thecoastal marine bonefishes (Albula spp) Distinct evolution-ary lineages (separated by sequence divergence between 5 and30 [25]) of bonefish coexist in several sample locations yetthey show little morphological or ecological differentiationin sympatry [25] Similarly a proportion of the grey mulletspecies (Mugilidae) also appears to consist of cryptic species[26] For exampleMugil cephalus in Taiwan is characterizedby three divergent lineages that have been shown to charac-terize 3 reproductively isolated but morphologically crypticspecies [26 27]

Here we have discovered 2 highly distinct and structuredclades (each composed of 3 mtDNA subclades) that aremorphologically confounded within different species Forexample H spp C of the unspecified species group isidentified as H baramensis in the Baram River and as Hsemotus in the Padas River We speculate that North Borneoharbors a morphologically cryptic species complex possiblycomposed of several species pairs inhabiting different riversystems The reconstruction of this phylogenetic history andthe taxonomic status of its components remains a majorchallenge to the revision of the Hemibagrus genus

42 Phylogeographic Hypotheses The phylogeography ofthe genus appears to have been principally influenced bytwo geological histories one encompassing the formationof North Borneo the second the emergence of the SundaIslands and their connectivity with the Indochinesemainlandassociated with recurrent sea-level changes North Borneoappears to have acted as a species sink promoting speciationand locally high biodiversity within the genus but with littleevidence of subsequent dispersal out of North Borneo Incontrast the geological events influencing the Indochinesemainland and the Sunda Islands have permitted bothspeciation and differential dispersal depending on thebiology of the species For example the H planiceps speciesgroup is found throughout the Greater Sunda Islands andtheThai-Malay Peninsula as is theH nemurus species groupWhereas the H nemurus species group generally occupiesthe low altitude lower reaches of large rivers the speciescomposing the H planiceps group are found in the upperreaches of large rivers The H nemurus species group iscomposed of a number of widely distributed closely relatedspecies whereas theH planiceps group is composed of a largenumber of species that are highly localized geographicallyIt thus appears that sea-level changes have had little impacton the dispersal history of the H planiceps species group buthave deeply influenced that of the H nemurus species group

The Case of North Borneo From 15Ma to 5Ma greater areasof Borneo became emergent and the central mountainson the Sarawak-Kalimantan border extending into Sabahbecame wider and higher which may have represented newbarriers to dispersal [28 29] Borneo gradually developedinto the present large island by emergence of land in the

north and east The H baramensis species group is restrictedto North Borneo as is the unspecified species group of theHnemurus species group H fortis is also distributed in NorthBorneo The isolation of the freshwater fish communities ofNorth Borneo (particularly in the Labuk-Segama watershed)has long been recognized based on the number of endemicspecies and the concentration of relict species [30] The latePliocene-early Pleistocene diversification of H fortis theunspecified species group and the H baramensis speciesgroup may thus be associated with their dispersal to andisolation within the watersheds of North Borneo Only onesequence of the unspecified species group is found outsideof North Borneo and H fortis appears to be distributedthroughout central and east Borneo [7]

ThePhylogeography of the H nemurus Species Group Periodicmarine transgressions on the Sundaland platform during theearly and middle Miocene [2 29] may have been sufficientto isolate populations of freshwater organisms and promotecladogenesis [31] This may have been responsible for theinitial mid to late Miocene divergence of the H nemurusspecies group The majority of sequences assigned to thisspecies group belong to 3 species (H spilopterus H capitulum(including H nemurus) and H hoevenii) all of which havebeen influenced by the more recent geological history of theSunda Islands and their connectivity with the Indochinesemainland H spilopterus is known from river drainages inIndochina including the Mekong Dong Nai Ban PakongChao Phraya and Mae Klong where it is found in a widevariety of lotic and lentic habitats [7] H spilopterus divergedin the late Pliocene-early Pleistocene possibly isolated tothe north of the Kra Peninsula during the late Pliocenemarine transgression that is purported to have created amajor biogeographic transition zone between the Sundaicand Indochinese biotas ([32] but see [33] for a critique ofthis biogeographic scenario) The existence of 2 subclades(A and B) in the Mekong River separated by 2 sequencedivergence is consistent with intraspecific divergence Thetwo subclades possibly represent 2 historical mtDNA lineagesthat are retained within the species reflecting a Pleistoceneisolation event followed by secondary contact within thecontemporary Mekong River Subclade C from the ChaoPhraya and Mae Khlong Rivers exhibits a level of geneticdivergence consistent with sibling species-level divergenceshowing a 5 sequence divergence relative to Mekong RiverH spilopterus

H capitulum may be considered as part of the Sundaicbiota located principally to the south of the Kra marinetransgression zone that may have isolated H spilopterus tothe north on the Indochinese mainlandThe biogeographicalevent leading to the subsequent formation of two intraspecificgroups within H capitulum is not at all clear The onlyarea where the two clades are clearly geographically separateis on the Thai-Malay Peninsula with the ldquoSundaicrdquo cladeon the west coast and the ldquoIndochineserdquo clade on theeast coast separated by the central Titiwangsa mountainrange The genetic distinctiveness of east and west coastpopulations of H capitulum has also been documented fornuclear DNA [34] A similar pattern of separation has been

Advances in Evolutionary Biology 19

reported for 5 additional freshwater fishes within this region[35] However as the divergence of the two H capitulumclades postdates the formation of the mountain range byapproximately 200Ma the geographic separation of the twoclades most likely represents differential dispersal rather thanancient vicariance The presence of the Sundaic clade on thewest coast of the Thai-Malay Peninsula as well as on theSundaic Islands reflects the historical connections betweeneast Sumatra and west Peninsular Malaysia across the Straitof Malacca This area encompasses the Northern CentralSumatra-Western Malaysia freshwater ecoregion [36] TheMuar River forms its southern border coincident with thesouthernmost distribution of the Sundaic clade on the Thai-Malay Peninsula It is important to note however that mostof Sumatra and Java emerged to achieve their present size andelevation only 5Ma ago and much of East Java continuedto be the site of marine deposition until late Pliocene-earlyPleistocene [29] We may speculate that the Sundaic clade ofH capitulum resulted from the relatively recent colonizationof these islands and Borneo The radiation within the cladeonly dates back to approximately 1Ma ago H nemurus fromWest Java may thus best be considered an isolated regionalpopulation of very recent origin

The diversification of the Indochinese clade dates fromat least 12Ma ago coincident with the mid-Pleistocenetransition at which time low-frequency high-amplitude andquasiperiodic (100 ka) glacial variability emerged It began125Ma ago and was complete by 07Ma ago [37] The tran-sition marked the beginning of major changes in sea levelsduring which the Sunda Shelf periodically emerged and wastraversed by enormous fluvial systems [3] The contributionof these events to the diversification of this clade cannotbe satisfactorily reconstructed One possible explanation isthat high sea levels produced barriers to gene flow and thepopulations of West Borneo (subclades B C and D) wereisolated from those of east Peninsular Malaysia This couldbe due to one or more dispersal events from Borneo via theGreat Sunda River (Figure 1) to east Peninsular Malaysia orlineage sorting whereby sequences of subclades A and E havebeen lost from West Borneo As in the case of the sympatricoccurrence of H spilopterus and H capitulum subclade E(see above) in the Mekong River we are unable to clearlydefine the relative roles of incomplete lineage sorting andintrogression followingmigration Given the recent origins ofthe Indochinese H capitulum sequences incomplete lineagesorting most probably blurs patterns of recent isolation

The last species of the H nemurus species group is Hhoevenii The species is widespread throughout PeninsularMalaysia and the Sundaic Islands It is found primarily inthe lower reaches of rivers and is apparently capable ofwithstanding low salt concentration [7] H hoevenii thusappears to be the most liable of all members of the Hnemurus species group to be impacted by Pleistocene sea-level changes This is clearly reflected in the widespreaddistribution of the sequences of this species Two lineages areevident having diverged no more than 03Ma ago suggest-ing a late Pleistocene isolation event followed by extensivedispersal and admixture in recent times This represents theclearest example of the dispersal opportunities afforded by

Pleistocene sea level changes on the Sunda Shelf In additionthe relatively low genetic diversity detected (only 6 sequencesin 42 fish) suggests a population bottleneck in the recent pastpossibly related to population declines in the lower reaches ofrivers flooded by increasing sea levels

43 Future Directions We have provided a global picture ofthe evolutionary taxonomy of the genus in Southeast AsiaClearly a more rigorous phylogenetic reconstruction of thegenus Hemibagrus requiring the construction of multiplegene trees based on the analysis of both mitochondrial andnuclear DNA markers from a wider taxonomic array ofhemibagrids will involve an even greater effort of samplingthroughout the region In particular we require samplingat smaller spatial and temporal scales For example smallerscale sampling in the Padas River and the Segama Riverof North Borneo using both mitochondrial and nucleargenetic markers would contribute to clarifying the natureof the cryptic species complex believed to inhabit theserivers The demonstration of reproductive isolation andormicroallopatric distribution among morphologically crypticspecies may result in the description of greater speciesrichness within the genus than presently described Thespecies status of H chrysops merits reconsideration givenits highly divergent evolutionary history Nuclear geneticmarkers in conjunction with mtDNA markers could revealcases of historical introgression particularly in the KapuasRiver and the Mekong River The hemibagrid catfishes con-tinue to provide multiple opportunities for the study of theevolutionary taxonomy and biogeography of a major faunalcomponent of the fluvial ecosystems of Southeast Asia

Disclosure

Ethanol-preserved tissue samples of many of the specimensidentified in Table S1 are freely available upon request fromJJD

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Goh Yan Yih for assistance in collectingspecimens and molecular analyses Mrs Francoise Colom-bani for molecular analyses and Dr Heok Hee Ng forspecies identification This work was funded by the NationalUniversity of Singapore grant to P K L Ng and a CanadianNatural Sciences and Engineering Research Council grant toJ J Dodson

References

[1] D J Lohman M de Bruyn T Page et al ldquoBiogeography ofthe Indo-Australian archipelagordquo Annual Review of EcologyEvolution and Systematics vol 42 pp 205ndash226 2011

20 Advances in Evolutionary Biology

[2] D S Woodruff ldquoNeogene marine transgressions palaeogeog-raphy and biogeographic transitions on the Thai-Malay Penin-sulardquo Journal of Biogeography vol 30 no 4 pp 551ndash567 2003

[3] H K Voris ldquoMaps of Pleistocene sea levels in SoutheastAsia shorelines river systems and time durationsrdquo Journal ofBiogeography vol 27 no 5 pp 1153ndash1167 2000

[4] S Y Yap ldquoOn the distributional patterns of Southeast-EastAsian freshwater fish and their historyrdquo Journal of Biogeographyvol 29 no 9 pp 1187ndash1199 2002

[5] S K J McConnell ldquoMapping aquatic faunal exchanges acrossthe Sunda shelf South-East Asia using distributional andgenetic data sets from the cyprinid fish Barbodes gonionotus(Bleeker 1850)rdquo Journal of Natural History vol 38 no 5 pp651ndash670 2004

[6] T Mo Anatomy Relationships and Systematics of the Bagridae(Teleostei Siluroidei) with a Hypothesis of Siluroid Phylogenyvol 17 ofTheses Zoologicae Koeltz Scientific Books 1991

[7] H H Ng and M Kottelat ldquoRevision of the Asian catfish genusHemibagrus bleeker 1862 (Teleostei Siluriformes Bagridae)rdquoRaffles Bulletin of Zoology vol 61 no 1 pp 205ndash291 2013

[8] P K L Ng and H H Ng ldquoHemibagrus gracilis a new speciesof large riverine catfish (Teleostei Bagridae) from PeninsularMalaysiardquo Raffles Bulletin of Zoology vol 43 pp 133ndash142 1995

[9] H H Tan and M Kottelat ldquoRedescription of Betta picta(Teleostei Osphronemidae) and description of Betta falx sp nfrom central Sumatrardquo Revue Suisse de Zoologie vol 105 no 3pp 557ndash568 1998

[10] J J Dodson F Colombani and P K Ng ldquoPhylogeographicstructure in mitochondrial DNA of a South-east Asian fresh-water fish Hemibagrus nemurus (Siluroidei Bagridae) andPleistocene sea-level changes on the Sunda shelfrdquo MolecularEcology vol 4 no 3 pp 331ndash346 1995

[11] H H Ng and J J Dodson ldquoMorphological and genetic descrip-tions of a new species of catfish Hemibagrus chrysops fromSarawak East Malaysia with an assessment of phylogeneticrelationships (Teleostei Bagridae)rdquo Raffles Bulletin of Zoologyvol 47 no 1 pp 45ndash57 1999

[12] P D N Hebert A Cywinska S L Ball and J R DeWaardldquoBiological identifications through DNA barcodesrdquo Proceedingsof the Royal Society B Biological Sciences vol 270 no 1512 pp313ndash321 2003

[13] J C Carolan T EMurray U Fitzpatrick et al ldquoColour patternsdo not diagnose species quantitative evaluation of a DNAbarcoded cryptic bumblebee complexrdquo PLoS ONE vol 7 no 1Article ID e29251 2012

[14] M Kekkonen and P D N Hebert ldquoDNA barcode-baseddelineation of putative species efficient start for taxonomicworkflowsrdquoMolecular Ecology Resources vol 14 no 4 pp 706ndash715 2014

[15] DM Hillis CMoritz and B KMarbleMolecular SystematicsSinauer Sunderland Mass USA 2nd edition 1996

[16] T D Kocher W K Thomas A Meyer et al ldquoDynamics ofmitochondrial DNA evolution in animals amplification andsequencingwith conserved primersrdquoProceedings of theNationalAcademy of Sciences of the United States of America vol 86 no16 pp 6196ndash6200 1989

[17] T A Hall ldquoBioEdit a user-friendly biological sequence align-ment editor and analysis program for Windows 9598NTrdquoNucleic Acids Symposium Series vol 41 pp 95ndash98 1999

[18] D A Benson I Karsch-Mizrachi K Clark D J Lipman JOstell and EW Sayers ldquoGenBankrdquoNucleic Acids Research vol40 no D1 pp D48ndashD53 2012

[19] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[20] X Ku Z Peng R Diogo and S He ldquoMtDNA phylogenyprovides evidence of generic polyphyleticism for East Asianbagrid catfishesrdquoHydrobiologia vol 579 no 1 pp 147ndash159 2007

[21] L Yang and S He ldquoPhylogeography of the freshwater catfishHemibagrus guttatus (Siluriformes Bagridae) implications forSouth China biogeography and influence of sea-level changesrdquoMolecular Phylogenetics and Evolution vol 49 no 1 pp 393ndash398 2008

[22] B W Bowen A L Bass L A Rocha W S Grant and DR Robertson ldquoPhylogeography of the trumpetfishes (Aulosto-mus) ring species complex on a global scalerdquo Evolution vol 55no 5 pp 1029ndash1039 2001

[23] Y P Kartavtsev ldquoDivergence at Cyt-b and Co-1 mtDNA geneson different taxonomic levels and genetics of speciation inanimalsrdquoMitochondrial DNA vol 22 no 3 pp 55ndash65 2011

[24] SWada Y Kameda and S Chiba ldquoLong-term stasis and short-term divergence in the phenotypes of microsnails on oceanicislandsrdquoMolecular Ecology vol 22 no 18 pp 4801ndash4810 2013

[25] J Colborn R E Crabtree J B Shaklee E Pfeiler and B WBowen ldquoThe evolutionary enigma of bonefishes (Albula spp)cryptic species and ancient separations in a globally distributedshorefishrdquo Evolution vol 55 no 4 pp 807ndash820 2001

[26] J-D Durand K-N Shen W-J Chen et al ldquoSystematics of thegrey mullets (Teleostei Mugiliformes Mugilidae) molecularphylogenetic evidence challenges two centuries ofmorphology-based taxonomyrdquo Molecular Phylogenetics and Evolution vol64 no 1 pp 73ndash92 2012

[27] K-N Shen B W Jamandre C-C Hsu W-N Tzeng and J-D Durand ldquoPlio-Pleistocene sea level and temperature fluc-tuations in the northwestern Pacific promoted speciation inthe globally-distributed flathead mullet Mugil cephalusrdquo BMCEvolutionary Biology vol 11 no 1 article 83 2011

[28] R Hall ldquoThe plate tectonics of Cenozoic SE Asia and thedistribution of land and seardquo in Biogeography and GeologicalEvolution of SE Asia R Hall and J D Holloway Eds pp 99ndash131Backhuys Publishers Leiden The Netherlands 1998

[29] R Hall ldquoSoutheast Asiarsquos changing palaeogeographyrdquo Blumeavol 54 no 1ndash3 pp 148ndash161 2009

[30] R F Inger and P K Chen ldquoThe freshwater fishes of NorthBorneordquo Fieldiana Zoology vol 45 pp 1ndash268 1962

[31] J Bohlen V Slechtova H H Tan and R Britz ldquoPhylogenyof the Southeast Asian freshwater fish genus Pangio (Cyprini-formes Cobitidae)rdquoMolecular Phylogenetics and Evolution vol61 no 3 pp 854ndash865 2011

[32] M de Bruyn E Nugroho M M Hossain J C Wilson andP B Mather ldquoPhylogeographic evidence for the existence ofan ancient biogeographic barrier the Isthmus of Kra SeawayrdquoHeredity vol 94 no 3 pp 370ndash378 2005

[33] J Parnell ldquoThe biogeography of the Isthmus of Kra region areviewrdquoNordic Journal of Botany vol 31 no 1 pp 001ndash015 2013

[34] S Usmani S G Tan S S Siraj and K Yusoff ldquoPopulationstructure of the Southeast Asian river catfishMystus nemurusrdquoAnimal Genetics vol 34 no 6 pp 462ndash464 2003

[35] M P Tan A F J Jamsari and M N Siti Azizah ldquoPhylo-geographic pattern of the striped snakehead Channa striatain Sundaland ancient river connectivity geographical and

Advances in Evolutionary Biology 21

anthropogenic singnaturesrdquo PLoS ONE vol 7 no 12 Article IDe52089 2012

[36] R Abell M L Thieme C Revenga et al ldquoFreshwater ecore-gions of the world a new map of biogeographic units forfreshwater biodiversity conservationrdquo BioScience vol 58 no 5pp 403ndash414 2008

[37] P U Clark D Archer D Pollard et al ldquoThemiddle Pleistocenetransition characteristics mechanisms and implications forlong-term changes in atmospheric pCO

2rdquo Quaternary Science

Reviews vol 25 no 23-24 pp 3150ndash3184 2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 8: ResearchArticle - Université Laval Julian... · ResearchArticle A DNA Barcode-Based Evaluation of the Southeast Asian Catfish Genus Hemibagrus Bleeker, 1862 (Teleostei: Siluriformes;

8 Advances in Evolutionary Biology

100∘

10∘

110∘

0∘

120∘

0

0 250

250

500

500

750(km)

(mi)

002

98

43

84

34

99

99

79

96

51

59

99

78

49

H peguensis

H guttatus

H macropterus

H wyckioides

PESAL0102

XXRAJ0301Leiocassis longirostris

MAPEA0201MAPEA0302MAPEA0101

GUPEA0501GURED0102

GUPEA0401GUPEA0602

WYCHP0401WYMEK1101

WYCHP0203

See Figures 7ndash12

See Figures 5 and 6

Shelf minus75mShelf minus120 m

Figure 4 Phylogenetic relationships of the individual sequences comprising four species H guttatus H macropterus (together forming theH guttatus species group) H wyckioides and H peguensisThe subtree is extracted from the taxon identification phenogram inferred usingthe Neighbor-Joining method (see Figure 2) The percentage of replicate trees in which the associated sequences clustered together in thebootstrap test (500 replicates) is shown next to the branches The distribution of these sequences is illustrated in the right panel

The group comprises three valid species H guttatus Hmacropterus andH vietnamicus (NampK) Specimens availablefor DNA analysis were obtained from the Pearl River andRed River (11 fish 7 sequences Table 1 and Table S1) Thetwo reciprocally monophyletic subclades comprising theH guttatus species group represent H guttatus and Hmacropterus (Figure 4) Although no ZRC specimens wereexamined by NampK species identification was confirmed(H H Ng personal communication) No H vietnamicusspecimens were available for DNA analysis The twospecies composing the group are closely related (119901 = 51(SE = 08))

mtDNA ldquoCladerdquo 3 The H menoda Species Group Thisisolated genotype (Figure 4) most likely identifies a cladeencompassing the H menoda species group although the

single sequence (Table S1) is shared by only 2 specimensof H peguensis from the Salween River Myanmar alsoincluded in the analysis of NampK (where its provenance wasidentified as the neighboring Irrawaddy River) The otherspecies comprising this group (H menoda H caveatusand H punctatus) were not sampled for DNA analysis Themonophyletic nature of this species group thus remains to bedemonstrated

31 The H planiceps + H baramensis Group The remainderof the gene tree is split into two large sister groups withmarginal bootstrap support (73 in ML and 43 in NJ)the H planiceps + H baramensis group and the H nemurusspecies group (Figures 2 and 3) The H planiceps + Hbaramensis group is strongly supported in both NJ and MLanalyses (84 and 90 bootstrap support respectively

Advances in Evolutionary Biology 9

110∘

47

53

69

81

69

5199

52

95

97

78

001

H bongan

H velox

H gracilis

H planiceps

H divaricatus

BOKAP1801

BOKAP1601BOKAP1701

BOKAP1402BOKAP1503

VEBAH0907GREND0206

PLCIM0114PLCIS0101

DIPER0401DIPER0301

DIPER0201

100∘

10∘

0∘

120∘

0

0 250 500

250 500

750(km)

(mi)

Shelf minus75mShelf minus120 m

Figure 5 Phylogenetic relationships of the individual sequences and species comprising theH planiceps species groupH bongan H veloxH gracilis H planiceps and H divaricatus The subtree is extracted from the taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicate trees in which the associated sequences clustered together in the bootstrap test(500 replicates) is shown next to the branches The distribution of these sequences is illustrated in the right panel

Figures 2 and 3) The two groups are genetically similarwith a genetic divergence estimate of 119901 = 121 (SE = 09)Although theH planiceps species group is strongly supportedin both trees (99) bootstrap support of the H baramensisspecies group is weak (Figure 2 32 Figure 3 34) Thegeographical distribution of the two groups differs Membersof the H planiceps species group are found throughout theGreater Sunda Islands and Peninsular Malaysia but they areisolated in the upper reaches of large rivers (Figure 5) Incontrast members of theH baramensis species group appearto be restricted to Sabah North Borneo (Figure 6)

mtDNA Clade 4 The H planiceps Species Group The Hplaniceps species group (Figure 5) is comprised of 5 closelyrelated but genetically distinct species (mean within-groupdivergence 119901 = 20 (SD = 033) H planiceps (Java)H gracilis (eastern Peninsular Malaysia) H divaricatus(western Peninsular Malaysia) H bongan (Borneo) andH velox (Sumatra)) H lacustrinus the sixth species of thisgroup (NampK) was not sampled in the present study WithintheH planiceps species groupH planiceps formed a distinctspecies (Figure 5) comprising two sequences One sequence(PLCIM0114 Table S1) characterized 14 specimens obtainedfrom Ci Manuk West Java 9 of which were identified as Hplaniceps by NampK (Table S1) A second sequence (PLCIS0101

Table S1) was obtained from one specimen sampled atCi Sadane West Java that we classified as H planicepsH bongan is represented by 5 sequences obtained from 8specimens sampled in the Kapuas River (Figure 5) Two ofthese specimens (ZRC 43013 and 43015) were identified asH bongan by NampK (Table S1) H divaricatus is composedof 3 sequences from three specimens obtained at Gerik onthe Perak River (western Peninsular Malaysia) One of thesespecimens (ZRC 41151) is the holotype of this new species(NampK) H gracilis is represented by only one sequence(GREND0206 Table S1) shared by 6 specimens sampledat Ulu Endau Jasin River eastern Peninsular Malaysiafour of which were identified by NampK (Table S1) ZRC21484 comprises the holotype and ZRC 21482 21486 and21487 comprise the paratypes (NampK) of this species (TableS1) Finally H velox is represented by only one sequence(VEBAH0907) from 7 specimens sampled at Kerinci BatangHari River Sumatra Three of these specimens (ZRC 41503Table S1) represent 3 paratypes of the species (NampK)

mtDNA Clade 5 The H baramensis Species Group The threespecies comprising the H baramensis species group (Figures2 and 3) form three monophyletic subclades (mean within-group divergence 119901 = 106 (08) Figure 6) H semotus iscomposed of two sequences (SEPAD0101 SEPAD0202) that

10 Advances in Evolutionary Biology

110∘

002

H sabanus

H semotus

H baramensis

SAKIN0204SASGM0706

BASGM0402BASGM0301

BASGM0201BASGM0104

SESGM0502SEUMA0101

SEPAD0202SEPAD0101

See Figure 5

100∘

10∘

0∘

120∘

0

0 250 500

250 500

750(km)

(mi)

99

57

32

83

99

72

99

56

66

54

99

Shelf minus75mShelf minus120 m

Figure 6 Phylogenetic relationships of the individual sequences and three species comprising the H baramensis species group H semotusH sabanus and H baramensis (furcatus) The subtree is extracted from the taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicate trees in which the associated sequences clustered together in the bootstrap test(500 replicates) is shown next to the branches The distribution of these sequences is illustrated in the right panel

characterize fish that are the holotype and paratypes of Hsemotus (ZRC 46122 Table S1) along with two somewhatmore divergent sequences (Figure 6) One of these sequences(SGM0502) was found in the Segama River (Danum Valley)and identified as H baramensis by NampK (ZRC 40525)(Table S1) The second sequence (UMA0101) was from afish sampled at Tawau Umas Umas River (ZRC 46123) alsoidentified as H baramensis by NampK These sequences mostlikely reflect distinct lineages within H semotus inhabitingdifferent rivers as the mean intraspecies sequence divergence(119901 = 16) indicates population-level divergence

H sabanus and H furcatus (synonymized with H bara-mensis by NampK) cluster together to form a distinct subcladebut clearly diverge at the species level (Figure 6) H sabanuscomprises 2 sequences that are shared respectively by 6specimens (ZRC 42741) from the Segama River Sabah and4 specimens (part of ZRC 42743) from the KinabatanganRiver Sabah (Table S1) All of these specimenswere identifiedas H sabanus by NampK (Table S1) Finally H baramensisforms a distant subclade (147 mean sequence divergencebetween H baramensis and H sabanus) composed of 4sequences (Figure 6) shared by 8 specimens all sampled inthe Segama River Danum Valley Conservation Area SabahSix of these specimens (ZRC 40523 (2 specimens) ZRC40527 (2 specimens) and ZRC 40526 (2 specimens) Table S1)comprise the paratypes of H baramensis (furcatus) (NampK)

32 The H nemurus Species Group In both NJ and ML trees(Figures 2 and 3) the H nemurus species group is strongly

supported (bootstrap support 98 in NJ and 99 in ML)indicating that it forms a distinct and widespread geneticentity within the genus Hemibagrus However relationshipswithin the group are not so clear In general the H nemurusspecies group is composed of 4 clades The first clade may beconsidered as a species group nested within the H nemurusspecies group (85 and 93 bootstrap support in NJ andML resp) encompassing three clades H hoevenii (clade9) the H capitulum Sundaic clade (clade 10 including Hnemurus) and the H capitulum Indochinese clade (clade 11)(Figures 2 and 3) An additional three clades (Figures 2 and3) are formed by an unidentified species group (clade 6) Hspilopterus (clade 7) andH fortis (clade 8)These three latterclades are distinct at the congeneric species level (unspecifiedversus H spilopterus 119901 = 77 (07) unspecified versus Hfortis 119901 = 85 (07) and H spilopterus versus H fortis119901 = 75 (08)) The distribution of these three clades issomewhat disparate H fortis and the unspecified speciesgroup are principally found in Borneo whereasH spilopterusis widely distributed on the Indochinese mainland

mtDNA Clade 6 Unspecified Species Group This geneticallydiverse group encompassing at least 3 subclades (H spp AB and C Figure 7 within-group mean sequence divergence119901 = 68(06)) is confoundedwith theH baramensis speciesgroup byNampK It is composed of sequences fromfish sampledin Sarawak Sabah and eastern PeninsularMalaysia Subclade119867 spp A (Figure 7) is particularly interesting as it contains2 sequences (CHSARA20 CHRAJ0401) that characterize

Advances in Evolutionary Biology 11

110∘

XXPAD0303XXPAD0601

XXPAD0401XXBEL0202

XXBRM0202XXRAJ0511

XXPAD0501

XXEND0410CHSARA20CHRAJ0401

XXBEL0101XXSEG0101

100∘

10∘

0∘

120∘

0

0 250 500

250 500

750(km)

(mi)

91

86

83

99

98

47

81

001

91

72

99

80

H spp C

H spp B

H spp A

Shelf minus75mShelf minus120 m

Figure 7 Phylogenetic relationships of the individual sequences comprising the unspecified species group containing three subcladesidentified as H spp A B and C The subtree is extracted from the taxon identification phenogram inferred using the Neighbor-Joiningmethod (see Figure 2) The percentage of replicate trees in which the associated sequences clustered together in the bootstrap test (500replicates) is shown next to the branches The distribution of these sequences is illustrated in the right panel

H chrysops a new species identified by Ng and Dodson [11]However specimens of H chrysops from the Rajang Riverwere subsequently synonymized by NampK with H capitulumof the H nemurus species group (ZRC 23008ndash23014 TableS1) However an additional 14 fish from the Sadong Riverat Serian bearing genotype SARA20 (Table S1) and thusgenetically identical to the Rajang H chrysops samples wereidentified by NampK as H spilopterus (ZRC 29583ndash96 TableS1) These specimens however were erroneously identifiedin NampK as originating in the Chao Phraya River (Thailand)Subclade A also contains one sequence obtained from 10 fishsampled on the Endau River (Mersing) in eastern PeninsularMalaysia These fish were not morphologically examinedFinally subclade A also contains one sequence from oneunidentified fish sampled in the Segaliud River Sabah andanother unidentified fish sampled in the Belait River BruneiSubclade H spp B encompasses 2 sequences one of whichcomes from a fish sampled in the Padas River (XXPAD0501)and part of the ZRC 46122 fish lot identified as H semotusby NampK (Table S1) The second sequence (XXRAJ0511) wasshared by 11 unidentified fish sampled at Katibas on theRajang River Sarawak (Table S1) Finally subclade 119867 sppC encompasses 5 sequences Three sequences (XXPAD0303XXPAD0401 and XXPAD0601) were obtained from thePadas River and identified byNampKasH semotus (ZRC46122Table S1) The fourth sequence (XXBRM0202) was obtainedfrom 2 fish sampled in the Baram River and was identified

as H baramensis by NampK (ZRC 42742) The 5th sequencecomposing this group was obtained from 2 unidentified fishsampled in the Belait River Brunei (Table S1)

mtDNA clade 6 is thus problematical within the contextof the revision of the genus Hemibagrus Two highlydivergent mtDNA clades (clade 5 and clade 6 Figures 2and 3) occur in sympatry principally in the Padas River ofSabah North Borneo (Figures 6 and 7) Specimens of theunspecified species group are morphologically similar tothe H baramensis species group where they are assigned inthe analysis of NampK However the mean genetic distancebetween the unspecified species group and theH baramensisspecies group is 119901 = 193 (28) close to genus leveldivergence Furthermore subclade 119867 spp A (Figure 7)contains not only H chrysops but also an unidentifiedpopulation from eastern Peninsular Malaysia (XXEND0410Table S1) and two additional Sabah sequences On theother hand the unspecified species group clusters with allother members of the H nemurus species group (meangenetic divergence of unspecified group versus H capitulum(Sundaic clade) 119901 = 76 (07) (Figure 2)) with strongbootstrap support Such genetic clustering may justify theinclusion of H chrysops and associated sequences in theH nemurus species group but its synonymization with Hcapitulum (NampK) is difficult to defend on genetic groundsWe tentatively conclude that H chrysops is a valid speciesbased on genetic identity [11] forming part of an unknown

12 Advances in Evolutionary Biology

110∘

SPCPCHMK12

SPMEK0202SPMEK0401

SPMEK0301SPMEK0103

SPMEK1401SPMEK0501

SPMEK1201SPMEK1301

SPMEK1601SPMEK1502

SPMEK0701SPMEK0801SPMEK1701

SPCHP0501SPCHA0201

SPCHA0304SPPAT0101

100∘

10∘

0∘

120∘

0

0 250 500

250 500

750(km)

(mi)

000499

99 98

60

47

48

54

96

7091

38

86

87

99

80

58

H spilopterus ldquoCrdquo

H spilopterus ldquoBrdquo

H spilopterus ldquoArdquo

Shelf minus75mShelf minus120 m

Figure 8 Phylogenetic relationships within H spilopterus comprised of 3 subclades identified as A B and C The subtree is extracted fromthe taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicate trees in whichthe associated sequences clustered together in the bootstrap test (500 replicates) is shown next to the branches The distribution of thesesequences is illustrated in the right panel

species group found throughout North Borneo (and toa lesser degree in eastern Peninsular Malaysia) that maybe considered as part of the H nemurus species groupHowever the morphological confusion of the unspecifiedspecies group with the sympatric highly divergent Hbaramensis species group suggests the existence of a crypticspecies assemblage (see Section 4)

mtDNA Clade 7 Clade 7 (Figures 2 and 3) is composed of atleast 3 distinct subclades that appear to reflect 3 geneticallydivergent populations ofH spilopterus Subclade A (Figure 8)encompasses 7 sequences obtained from 8 fish sampled inthe Mekong River Four fish bearing 3 of these sequenceswere identified as paratypes (ZRC 43324) of H spilopterusby NampK (Table S1) One additional fish was identified asH spilopterus by NampK (ZRC 46157 Table S1) Subclade Bencompasses 6 sequences obtained from 9 fish all sampledfrom the Mekong River (Table S1) The fish sampled at ThatPhanom (ZRC 46012) was identified as H spilopterus inthe analysis of HampK (Table S1) Subclade C encompasses 5sequences obtained from 19 fish sampled in the Chao PhrayaMae Khlong and Chanthaburi Rivers Thailand Onesequence (CPCHMK12) was found in all of these drainages(Table S1) A closely allied sequence was found in one fishsampled at Phatthalung just south of the Isthmus of Kra onthe Thai-Malay Peninsula None of these fish were examined

by NampK and thus we are unable to definitively identifythe species However given the widespread occurrence ofH spilopterus in the Chao Phraya and Mae Klong Riversreported by NampK we conclude that these fish are part of awidespread H spilopterus population complex The meanwithin-group (clade 7) genetic divergence (119901 = 29 (04))suggests intraspecific population-level divergence

mtDNA Clade 8 Clade 8 encompasses 3 sequencesobtained from 9 fish sampled in east Sabah specifically theKinabatangan Segama and Umas Umas Rivers (Figure 9)The Kinabatangan sequence is somewhat divergent withinthe clade and the specimen was not identified by NampK Onesequence (SGM0606) characterized 6 fish from the SegamaRiver 2 of which were identified as H fortis (ZEC 4052140522 Table S1) by NampK (Table S1) Finally one fish bearinga closely related genotype from the Umas Umas River wasidentified as H baramensis by NampK (Table S1)

mtDNA Clade 9 Clade 9 is a sister group to the remaining2 clades composing the H nemurus species group Thiswell-defined clade (Figure 10) clusters 6 sequences from42 fish that are clearly identified as H hoevenii (NampK)Some of these haplotypes are geographically widespread onesequence (JOMSKA11 Table S1) was found in the Johor River(East Peninsular Malaysia) the Musi River (Sumatra) and

Advances in Evolutionary Biology 13

100∘ 110∘

0∘

120∘

00 250

250500

500750

(km)

(mi)

FOKIN0102FOSGM0606

FOUMA0201H fortis

10∘

99

86

001

Shelf minus75mShelf minus120 m

Figure 9 Phylogenetic relationships within H fortis comprised of 3 sequences The subtree is extracted from the taxon identificationphenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicate trees in which the associated sequencesclustered together in the bootstrap test (500 replicates) is shown next to the branches The distribution of these sequences is illustrated in theright panel

the Kapuas River (West Borneo) and a second sequence(HMUBHKABM23 Table S1) was found inMuarRiver (WestPeninsular Malaysia) and the Batang Hari (Sumatra) theKapuas (Sarawak) and the Baram Rivers (Sarawak) Many ofthese fish were identified as H hoevenii by NampK (Table S1)The existence of twodistinct but very closely related subclades(Figure 10 119901 = 06 (03)) that are not geographicallysegregated suggests some recent isolation event followed bywidespread dispersal and secondary contact

33 The H capitulum Group The final two monophyleticgroups of the Hemibagrus gene tree are approximatelyequivalent to the remaining species composing the Hnemurus species group as defined by NampK Dodson etal [10] and Ng and Dodson [11] have previously referredto clade 10 as the Sundaic clade as most sequences wereobtained from fish sampled on the Greater Sunda Islandsand clade 11 as the Indochinese clade asmany sequences wereobtained from fish sampled on the Indochinese mainland(Figure 2 Table S1) Although most of these sequencesare assigned to H capitulum by NampK there are somesignificant phylogeographic differences between the 2 cladesIn particular there appears to be an east-west division in thedistribution of the two clades on the Thai-Malay Peninsulawith the Sundaic clade (clade 10) found on the west coast andthe Indochinese clade found on the east coast with sympatricoccurrence in the Muar River Nevertheless mean sequencedivergence between the two clades is 119901 = 29 (05) avalue that is indicative of population-level or subspecies

divergence The two clades are thus best considered ascomprising one species

mtDNA Clade 10 The Sundaic clade includes a number ofclosely related sequences that comprise part of H capitulumand H nemurus Very little genetic structure is apparentand bootstrap support for tree topology within this group isgenerally very weak (Figure 11) A widely spread genotype(NCLCTBA20) characterized 6 of 17 specimens from CiTarum at the Cirata reservoir (West Java) (Table S1) and5 were identified as H nemurus in the analysis of NampK(ZRC 42564) (Table S1) The remaining fish characterized bythis sequence were obtained from Ci Liwong at Sawangan(West Java) and one fish was obtained from the Barito Riverat Banjarmassin south Borneo This latter specimen (ZRC40553) was identified by NampK as H capitulum (Table S1)Another common genotype (NBHKPBR09) was identified aseither H capitulum H hoevenii or H nemurus dependingon the river of origin (Table S1) Many of the remainingsequences of the Sundaic clade appear to characterize fish thatNampK identified asH capitulum A geographically widespreadgenotype (CPEBEMUKA36) characterized 9 fish sampledon the Perak River at Gerik that were identified by NampKas H capitulum (ZRC 41150 Table S1) Another commongenotype within this clade (CABHMS20) included 2 fishfrom the Batang Hari at Jambi and identified by NampK asH capitulum (ZRC40534 Table S1) Another 2 sequences(CABAR0203 CABAR0305) characterized 8 fish sampledon the Barito River at Banjarmasin and were identified as

14 Advances in Evolutionary Biology

100∘

10∘

110∘

0∘

120∘

0

0 250

250

500

500

750(km)

(mi)

53

64

99

85

64

0002

HJOMSKA11

HOBAH0601

HOBHMS05

HMUBHKABM23

HOBAH0701

HOKAP0501

H hoevenii ldquoBrdquo

H hoevenii ldquoArdquo

Shelf minus75mShelf minus120 m

Figure 10 Phylogenetic relationships withinH hoevenii comprised of 2 subclades and 6 sequences The subtree is extracted from the taxonidentification phenogram inferred using theNeighbor-Joiningmethod (see Figure 2)The percentage of replicate trees in which the associatedsequences clustered together in the bootstrap test (500 replicates) is shown next to the branches The distribution of these sequences isillustrated in the right panel

110∘

XXMAH0110

CABAR0305

96 CAKPR0201

74CAKAP0401

5931 CAKAP0601

35 CPEBEMUKA36

646 CAKAP0903

33 CAKAP080114 CAKAP0701

CARAJ0210

4 CABAR0203CABRA0204

20

CBHKPBR0919

CABRA0301

15CABHMS20

24 NCLCTBA20

63 NECIT0201

H capitulumldquoSundaic claderdquo

H nemurus

0002 120∘

0 250 500 750

(km)

0 250 500

(mi)100

10∘

0∘

Shelf minus75mShelf minus120 m

Figure 11 Phylogenetic relationships within theH capitulum ldquoSundaicrdquo clade including 2 sequences associated withH nemurusThe subtreeis extracted from the taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicatetrees in which the associated sequences clustered together in the bootstrap test (500 replicates) is shown next to the branchesThe distributionof these sequences is illustrated in the right panel

Advances in Evolutionary Biology 15

H capitulum

0∘

10∘

100∘

110∘

120∘

66

63

88

59

38

39 68

71

6991

57

9754

88 4944

39

53

89

0002

ldquoIndochineserdquogroup ldquoErdquo

H capitulumldquoIndochineserdquo

group ldquoDrdquoH capitulumldquoIndochineserdquo

group ldquoCrdquo

H capitulumldquoIndochineserdquo

group ldquoBrdquo

H capitulumldquoIndochineserdquo

group ldquoArdquo0

0

250

250

500

500

750

(km)

(mi)

CATER0301CATERR0401

CKETEMU23

CAMEK0601CACHP0301

CAMKPA03

CAMEK0910CAMEK1003

XXMUBM02

CABMTU09

CABRM0601

CAKAP1201CAKAP1001

CAKAP1101CAKAP1301

CABRM0501

CASAD0102

CASAD0220

CAEND0301

CABAH0801

CAPABH05CATEEN03

CATER0701CATERR0801

CAKETE04CATER0501

CAPAH0303

Shelf minus75mShelf minus120 m

Figure 12 Phylogenetic relationships within theH capitulum ldquoIndochineserdquo clade encompassing 5 subclades identified as A B C D and EThe subtree is extracted from the taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2)The percentageof replicate trees in which the associated sequences clustered together in the bootstrap test (500 replicates) is shown next to the branchesThedistribution of these sequences is illustrated in the right panel

H capitulum by NampK (ZRC 40553 Table S1) Although notincluded in the analysis of NampK samples from the Sundaicclade were also found in the Kapuas River (West Borneo)Finally 10 fish sampled on the Mahakam River at Samarindaeast Borneo are all characterized by one relatively divergentgenotype (CAMAH0110) butwere considered asH capitulumin the analysis of NampK (ZRC 40533 Table S1)TheMahakampopulation sample is the most genetically distinct of theSundaic clade (119901 = 17 (04)) but nevertheless indicativeof intraspecific population-level divergence (Figure 12)

The topology of the Sundaic clade does not permit usto draw a clear distinction between H nemurus and theSundaic H capitulum The mean within-group divergenceof the Sundaic clade is only 119901 = 08 (02) well withinpopulation-level divergence values We postulate that Hnemurus may be one of several regional variants of awidespread Sundaic H capitulum clade that has radiatedrelatively recently This may explain the existence of manyshared sequences throughout the Sundaic islands assigned todifferent species in different regions

mtDNA Clade 11 Finally the Indochinese clade of Hcapitulum represents a geographically widespread andweakly diversified clade (within-group mean sequencedivergence 119901 = 11 (02) Figure 12) Although few fishfrom this genetic clade were included in the morphological

analysis of NampK most may be considered as H capitulumgiven their geographic distribution and close geneticrelationship to the Sundaic H capitulum Four sequencesin this clade were identified as H capitulum in the analysisof NampK 3 specimens characterized by one sequence(CABMTU09) found in the Tutong River of westernBorneo (ZRC 40482) 6 characterized by the same sequencefrom the Baram River (ZRC 40498) another 2 sequencesfrom the Baram River (ZRC 40498) and 20 specimenscharacterized by an abundant genotype (CASAD0220)found at 2 sites in the Sadong River also in western Borneo(ZRC 39526 5 specimens 39527 3 specimens and 38753 4specimens) (Table S1) Unfortunately none of the specimenscollected on the Indochinese mainland for genetic analysiswere examined by NampK Nevertheless the distribution ofspecimens analyzed for mtDNA resembles the distribution ofspecimens examined by NampK Although weakly structuredwe may recognize 5 subclades One genotype (CAPAH0303)is basal to the phenogram Subclade A encompasses 8sequences mainly found in eastern Peninsular Malaysia(Table S1 Figure 12) These rivers furnished many of thespecimens NampK examined to define H capitulum Howeversome sequences in this group occur in sympatry withsequences identified within the Sundaic H capitulum groupTwo sequences from subclade A occurred in the Batang HariSumatra (Table S1) Subclade E also encompasses sequences

16 Advances in Evolutionary Biology

found in Peninsular Malaysia as well as in the Mekong ChaoPhraya and Mea Khlong Rivers (Figure 12) In addition3 subclades encompass sequences found principally inwestern Borneo subclade B in the Baram and Sadong RiversSarawak subclade C in the Kapuas River West Kalimantanand subclade D in the Baram and Tutong Rivers of Sarawakand Brunei respectively (Figure 12) This latter subclade alsoincludes one genotype found in the Muar River of westernpeninsular Malaysia Finally subclade E encompasses 5sequences from the Mekong Chao Praya and Mae KhlongRivers all within the distribution area of H spilopterus

EvolutionaryHistoryThe evolutionary history of theHemiba-grus species groups analyzed here dates from at least theMiocene 15ndash20Ma (Figure 13) when considering the widelyused molecular clock of 2 sequence divergence per millionyears Bootstrap support for the sister group relationships ofH wyckioides theH guttatus species group andH peguensisare all weak (clearly due to undersampling) and little can besaid of their evolutionary history other than the fact that theyoccupy basal positions in the rooted trees The remainder ofboth the NJ and ML trees is composed of two major sistergroups the H planiceps + H baramensis group and the Hnemurus species group (Figures 2 3 and 13)TheH planiceps+ H baramensis group is strongly supported in both NJ andML analyses (84 and 90 bootstrap support resp Figures2 and 3) The H nemurus species group is also stronglysupported in both analyses (98 and 99 bootstrap supportFigures 2 and 3) These two major phylogenetic groups areestimated to have diverged at least 8 to 12Ma ago during themid to late Miocene (Figure 13)

Much of the evolutionary history of Hemibagrus hasoccurred during the Quaternary starting approximately26Ma ago This is particularly true of the H planicepsand H nemurus species groups The radiation of the 5species included in the H planiceps species group appearsto coincide with the middle Pleistocene transition period ofapproximately 1Ma ago Their high-elevation distributionhowever appears to have largely isolated them from theeffects of lowered sea levels during glacial maxima and theattendant opportunities for widespread dispersal (Figure 5)The evolutionary histories of theH baramensis species groupand the unspecified species group date back to the latePliocene In contrast the low-elevation distribution of theHnemurus species group in Sundaland appears to have favoredtheir widespread distribution during the Quaternary ice ageIn particular H hoevenii appears to be the most recentlydispersed of the species under study with diversification anddispersal occurring approximately 03Ma ago (Figure 13)

4 Discussion

41 The Congruence of Morphological and Genetic AnalysesCombining genetic and morphological analyses to elucidatetaxonomy allowed us to better understand the diversificationand evolutionary relationships within this speciose group offishes Given the rapid and relatively constant rate of mtDNAevolution the deeper the branches within the phylogenetictree are the greater our ability to distinguish valid taxa

should be Conversely the lack of complete congruencebetween morphological- and genetic-based identificationsis expected to increase among the shallower branches of thephylogenetic tree resulting in the appearance of polyphyleticspecies Although 2 of the 8 species groups defined by Ngand Kottelat [7] were not sampled in this study the validityof the remaining 6 species groups and their componentspecies defined on the basis of morphological similaritywas largely confirmed by the analysis of the mitochondrialCyt 119887 bar code As expected congruence is strongest at thebase of the NJ and ML trees where the H guttatus speciesgroup formed a well-defined monophyletic group The Hmenoda and H wyckii species group were each representedby only one species but they were clearly distinct relativeto sequences characterizing the neighboring species groupsThe H planiceps H baramensis and H nemurus speciesgroups and their component species were also generallycongruent in morphological and genetic analyses Also asexpected most cases of discrepancy between morphological-based species identification and genetic-based speciesidentification occurred on relatively shallow branches of thephylogenetic tree These involve principally H spilopterusand the two mtDNA subclades composing H capitulumHowever a far more unexpected lack of congruence involvesthe definition of a deeply divergent unspecified genetic groupconfounding members of theH baramensis species group inthe morphological analysis of Ng and Kottelat [7]

Interpreting Polyphyly The apparent polyphyletic origin ofa species may be due to incomplete lineage sorting amongrecently diverged species the introgression of mtDNA dueto species hybridization or the erroneous identificationof species due to the lumping of morphologically crypticspecies In the absence of nuclear DNA analyses it isoften difficult to distinguish between introgression andincomplete lineage sorting For example several sequencescharacterizing the Indochinese clade of H capitulum(subclade E) are found in the Mekong and Chao PhrayaRivers well within the distributional area of H spilopterusH spilopterus and H capitulum first diverged at least 3MaOn the one hand this apparent case of polyphyly mayhave resulted from incomplete lineage sorting betweenrecently divergedH spilopterus andH capitulum We wouldhowever have expected similar cases of polyphyly amongpopulations of the Indochinese clade of H capitulum butthis was not detected On the other hand the sympatricoccurrence of H spilopterus subclade A and subcladeB sequences with H capitulum Indochinese subclade Esequences in the Mekong River may represent a migrationevent and local introgression of H capitulum with Hspilopterus A similar event may have occurred in theChao Phraya and Mae Khlong Rivers where H spilopterussequences cooccur withH capitulum sequences Computingthe divergence of H capitulum sequences (Indochinesesubclade E) cooccurring with H spilopterus and Hcapitulum sequences occurring in eastern PeninsularMalaysia (subclade A) reveals a mean sequence divergence119901 = 15 (04) Thus the H capitulum sequences occurringin sympatry with H spilopterus diverged at most 750 ka ago

Advances in Evolutionary Biology 17

H fortis

XXRAJ0604H furcatus

H sabanusH semotusH divaricatus

H planicepsGREND0206

VEBAH0907

H bonganPESAL0102

H wyckioidesH macropterus

H guttatusXXRAJ0301Leiocassis longirostris

Ma

4971

92

47

51

9999

99

9999

99

99

99

9999

99

99

99

99

65

96

96

4946

93

79

78

70

91

93

98

86

87

91

95

97

71

85

9999

05101520

005010015020 000

H capitulum Indochinese clades AndashE

H capitulum Sundaic clade

H hoevenii B

H hoevenii A

H spilopterus C

H spilopterus B

H spilopterus AH spilopterus A

H spp CH spp B

H spp A

Figure 13 Linearized tree with branch lengths estimated with the molecular clock using a mutation rate of 1 per Ma (=divergence rate of2 per Ma) The tree topology is identical to the ML reconstruction illustrated in Figure 3 (see the specificities of the tree reconstruction inthe caption of Figure 3) Boxes represent 95 confidence intervals of divergence time estimates for each node Vertical dashed lines redirectto dates indicated on the timescale (Ma) Numbers below timescale indicate the estimated number of substitution changes using Hasegawa-Kishino-Yanomodel (HKY)with gamma distribution (+119866 = 13290) after linearizing the treeH furcatus is synonymizedwithH baramensis

from eastern Peninsular Malaysia populations suggestinga recent introgression event Similarly H capitulum in theKapuas River is characterized by sequences of both theSundaic and the Indochinese clades As both sequences arebroadly sympatric it is not possible to distinguish betweenincomplete lineage sorting and introgression of these closelyrelated intraspecific lineages

A Possible Case of Morphological Stasis Clade 6 of thepresent analysis defines a well-supported monophyletic cladefound in Sabah Sarawak and the Endau River of easternpeninsula Malaysia that has no equivalent taxonomic statusin the revision of Ng and Kottelat [7] In Borneo they occursympatrically with the H baramensis species group and areconfounded as either H baramensis or H semotus These

two clades are genetically highly divergent having divergedbetween 8 and 12Ma ago It thus seems highly unlikely thatthey represent retained polymorphisms due to incompletelineage sorting Sequences previously identified as character-izing H chrysops [11] are part of clade 6 and extend to thesouth (Rajang and Sadong Rivers of Sarawak West Borneo)with one closely related genotype found in eastern peninsularMalaysia (Endau River) where it may well be confoundedwith H capitulum H chrysops was synonymized with Hcapitulum by Ng and Kottelat [7] based on their analysis ofspecimens ofH chrysops from the Rajang RiverThus despitea long and apparently independent phylogenetic historythe unspecified species group appears largely cryptic andconfounded principally with members of the H baramensisspecies group

18 Advances in Evolutionary Biology

We suggest that this situation is an example of mor-phological stasis involving the coexistence of a number ofgenetically divergent yet morphologically cryptic species thatare lumped together [24] A striking example of morpho-logical and ecological stasis among fishes is provided by thecoastal marine bonefishes (Albula spp) Distinct evolution-ary lineages (separated by sequence divergence between 5 and30 [25]) of bonefish coexist in several sample locations yetthey show little morphological or ecological differentiationin sympatry [25] Similarly a proportion of the grey mulletspecies (Mugilidae) also appears to consist of cryptic species[26] For exampleMugil cephalus in Taiwan is characterizedby three divergent lineages that have been shown to charac-terize 3 reproductively isolated but morphologically crypticspecies [26 27]

Here we have discovered 2 highly distinct and structuredclades (each composed of 3 mtDNA subclades) that aremorphologically confounded within different species Forexample H spp C of the unspecified species group isidentified as H baramensis in the Baram River and as Hsemotus in the Padas River We speculate that North Borneoharbors a morphologically cryptic species complex possiblycomposed of several species pairs inhabiting different riversystems The reconstruction of this phylogenetic history andthe taxonomic status of its components remains a majorchallenge to the revision of the Hemibagrus genus

42 Phylogeographic Hypotheses The phylogeography ofthe genus appears to have been principally influenced bytwo geological histories one encompassing the formationof North Borneo the second the emergence of the SundaIslands and their connectivity with the Indochinesemainlandassociated with recurrent sea-level changes North Borneoappears to have acted as a species sink promoting speciationand locally high biodiversity within the genus but with littleevidence of subsequent dispersal out of North Borneo Incontrast the geological events influencing the Indochinesemainland and the Sunda Islands have permitted bothspeciation and differential dispersal depending on thebiology of the species For example the H planiceps speciesgroup is found throughout the Greater Sunda Islands andtheThai-Malay Peninsula as is theH nemurus species groupWhereas the H nemurus species group generally occupiesthe low altitude lower reaches of large rivers the speciescomposing the H planiceps group are found in the upperreaches of large rivers The H nemurus species group iscomposed of a number of widely distributed closely relatedspecies whereas theH planiceps group is composed of a largenumber of species that are highly localized geographicallyIt thus appears that sea-level changes have had little impacton the dispersal history of the H planiceps species group buthave deeply influenced that of the H nemurus species group

The Case of North Borneo From 15Ma to 5Ma greater areasof Borneo became emergent and the central mountainson the Sarawak-Kalimantan border extending into Sabahbecame wider and higher which may have represented newbarriers to dispersal [28 29] Borneo gradually developedinto the present large island by emergence of land in the

north and east The H baramensis species group is restrictedto North Borneo as is the unspecified species group of theHnemurus species group H fortis is also distributed in NorthBorneo The isolation of the freshwater fish communities ofNorth Borneo (particularly in the Labuk-Segama watershed)has long been recognized based on the number of endemicspecies and the concentration of relict species [30] The latePliocene-early Pleistocene diversification of H fortis theunspecified species group and the H baramensis speciesgroup may thus be associated with their dispersal to andisolation within the watersheds of North Borneo Only onesequence of the unspecified species group is found outsideof North Borneo and H fortis appears to be distributedthroughout central and east Borneo [7]

ThePhylogeography of the H nemurus Species Group Periodicmarine transgressions on the Sundaland platform during theearly and middle Miocene [2 29] may have been sufficientto isolate populations of freshwater organisms and promotecladogenesis [31] This may have been responsible for theinitial mid to late Miocene divergence of the H nemurusspecies group The majority of sequences assigned to thisspecies group belong to 3 species (H spilopterus H capitulum(including H nemurus) and H hoevenii) all of which havebeen influenced by the more recent geological history of theSunda Islands and their connectivity with the Indochinesemainland H spilopterus is known from river drainages inIndochina including the Mekong Dong Nai Ban PakongChao Phraya and Mae Klong where it is found in a widevariety of lotic and lentic habitats [7] H spilopterus divergedin the late Pliocene-early Pleistocene possibly isolated tothe north of the Kra Peninsula during the late Pliocenemarine transgression that is purported to have created amajor biogeographic transition zone between the Sundaicand Indochinese biotas ([32] but see [33] for a critique ofthis biogeographic scenario) The existence of 2 subclades(A and B) in the Mekong River separated by 2 sequencedivergence is consistent with intraspecific divergence Thetwo subclades possibly represent 2 historical mtDNA lineagesthat are retained within the species reflecting a Pleistoceneisolation event followed by secondary contact within thecontemporary Mekong River Subclade C from the ChaoPhraya and Mae Khlong Rivers exhibits a level of geneticdivergence consistent with sibling species-level divergenceshowing a 5 sequence divergence relative to Mekong RiverH spilopterus

H capitulum may be considered as part of the Sundaicbiota located principally to the south of the Kra marinetransgression zone that may have isolated H spilopterus tothe north on the Indochinese mainlandThe biogeographicalevent leading to the subsequent formation of two intraspecificgroups within H capitulum is not at all clear The onlyarea where the two clades are clearly geographically separateis on the Thai-Malay Peninsula with the ldquoSundaicrdquo cladeon the west coast and the ldquoIndochineserdquo clade on theeast coast separated by the central Titiwangsa mountainrange The genetic distinctiveness of east and west coastpopulations of H capitulum has also been documented fornuclear DNA [34] A similar pattern of separation has been

Advances in Evolutionary Biology 19

reported for 5 additional freshwater fishes within this region[35] However as the divergence of the two H capitulumclades postdates the formation of the mountain range byapproximately 200Ma the geographic separation of the twoclades most likely represents differential dispersal rather thanancient vicariance The presence of the Sundaic clade on thewest coast of the Thai-Malay Peninsula as well as on theSundaic Islands reflects the historical connections betweeneast Sumatra and west Peninsular Malaysia across the Straitof Malacca This area encompasses the Northern CentralSumatra-Western Malaysia freshwater ecoregion [36] TheMuar River forms its southern border coincident with thesouthernmost distribution of the Sundaic clade on the Thai-Malay Peninsula It is important to note however that mostof Sumatra and Java emerged to achieve their present size andelevation only 5Ma ago and much of East Java continuedto be the site of marine deposition until late Pliocene-earlyPleistocene [29] We may speculate that the Sundaic clade ofH capitulum resulted from the relatively recent colonizationof these islands and Borneo The radiation within the cladeonly dates back to approximately 1Ma ago H nemurus fromWest Java may thus best be considered an isolated regionalpopulation of very recent origin

The diversification of the Indochinese clade dates fromat least 12Ma ago coincident with the mid-Pleistocenetransition at which time low-frequency high-amplitude andquasiperiodic (100 ka) glacial variability emerged It began125Ma ago and was complete by 07Ma ago [37] The tran-sition marked the beginning of major changes in sea levelsduring which the Sunda Shelf periodically emerged and wastraversed by enormous fluvial systems [3] The contributionof these events to the diversification of this clade cannotbe satisfactorily reconstructed One possible explanation isthat high sea levels produced barriers to gene flow and thepopulations of West Borneo (subclades B C and D) wereisolated from those of east Peninsular Malaysia This couldbe due to one or more dispersal events from Borneo via theGreat Sunda River (Figure 1) to east Peninsular Malaysia orlineage sorting whereby sequences of subclades A and E havebeen lost from West Borneo As in the case of the sympatricoccurrence of H spilopterus and H capitulum subclade E(see above) in the Mekong River we are unable to clearlydefine the relative roles of incomplete lineage sorting andintrogression followingmigration Given the recent origins ofthe Indochinese H capitulum sequences incomplete lineagesorting most probably blurs patterns of recent isolation

The last species of the H nemurus species group is Hhoevenii The species is widespread throughout PeninsularMalaysia and the Sundaic Islands It is found primarily inthe lower reaches of rivers and is apparently capable ofwithstanding low salt concentration [7] H hoevenii thusappears to be the most liable of all members of the Hnemurus species group to be impacted by Pleistocene sea-level changes This is clearly reflected in the widespreaddistribution of the sequences of this species Two lineages areevident having diverged no more than 03Ma ago suggest-ing a late Pleistocene isolation event followed by extensivedispersal and admixture in recent times This represents theclearest example of the dispersal opportunities afforded by

Pleistocene sea level changes on the Sunda Shelf In additionthe relatively low genetic diversity detected (only 6 sequencesin 42 fish) suggests a population bottleneck in the recent pastpossibly related to population declines in the lower reaches ofrivers flooded by increasing sea levels

43 Future Directions We have provided a global picture ofthe evolutionary taxonomy of the genus in Southeast AsiaClearly a more rigorous phylogenetic reconstruction of thegenus Hemibagrus requiring the construction of multiplegene trees based on the analysis of both mitochondrial andnuclear DNA markers from a wider taxonomic array ofhemibagrids will involve an even greater effort of samplingthroughout the region In particular we require samplingat smaller spatial and temporal scales For example smallerscale sampling in the Padas River and the Segama Riverof North Borneo using both mitochondrial and nucleargenetic markers would contribute to clarifying the natureof the cryptic species complex believed to inhabit theserivers The demonstration of reproductive isolation andormicroallopatric distribution among morphologically crypticspecies may result in the description of greater speciesrichness within the genus than presently described Thespecies status of H chrysops merits reconsideration givenits highly divergent evolutionary history Nuclear geneticmarkers in conjunction with mtDNA markers could revealcases of historical introgression particularly in the KapuasRiver and the Mekong River The hemibagrid catfishes con-tinue to provide multiple opportunities for the study of theevolutionary taxonomy and biogeography of a major faunalcomponent of the fluvial ecosystems of Southeast Asia

Disclosure

Ethanol-preserved tissue samples of many of the specimensidentified in Table S1 are freely available upon request fromJJD

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Goh Yan Yih for assistance in collectingspecimens and molecular analyses Mrs Francoise Colom-bani for molecular analyses and Dr Heok Hee Ng forspecies identification This work was funded by the NationalUniversity of Singapore grant to P K L Ng and a CanadianNatural Sciences and Engineering Research Council grant toJ J Dodson

References

[1] D J Lohman M de Bruyn T Page et al ldquoBiogeography ofthe Indo-Australian archipelagordquo Annual Review of EcologyEvolution and Systematics vol 42 pp 205ndash226 2011

20 Advances in Evolutionary Biology

[2] D S Woodruff ldquoNeogene marine transgressions palaeogeog-raphy and biogeographic transitions on the Thai-Malay Penin-sulardquo Journal of Biogeography vol 30 no 4 pp 551ndash567 2003

[3] H K Voris ldquoMaps of Pleistocene sea levels in SoutheastAsia shorelines river systems and time durationsrdquo Journal ofBiogeography vol 27 no 5 pp 1153ndash1167 2000

[4] S Y Yap ldquoOn the distributional patterns of Southeast-EastAsian freshwater fish and their historyrdquo Journal of Biogeographyvol 29 no 9 pp 1187ndash1199 2002

[5] S K J McConnell ldquoMapping aquatic faunal exchanges acrossthe Sunda shelf South-East Asia using distributional andgenetic data sets from the cyprinid fish Barbodes gonionotus(Bleeker 1850)rdquo Journal of Natural History vol 38 no 5 pp651ndash670 2004

[6] T Mo Anatomy Relationships and Systematics of the Bagridae(Teleostei Siluroidei) with a Hypothesis of Siluroid Phylogenyvol 17 ofTheses Zoologicae Koeltz Scientific Books 1991

[7] H H Ng and M Kottelat ldquoRevision of the Asian catfish genusHemibagrus bleeker 1862 (Teleostei Siluriformes Bagridae)rdquoRaffles Bulletin of Zoology vol 61 no 1 pp 205ndash291 2013

[8] P K L Ng and H H Ng ldquoHemibagrus gracilis a new speciesof large riverine catfish (Teleostei Bagridae) from PeninsularMalaysiardquo Raffles Bulletin of Zoology vol 43 pp 133ndash142 1995

[9] H H Tan and M Kottelat ldquoRedescription of Betta picta(Teleostei Osphronemidae) and description of Betta falx sp nfrom central Sumatrardquo Revue Suisse de Zoologie vol 105 no 3pp 557ndash568 1998

[10] J J Dodson F Colombani and P K Ng ldquoPhylogeographicstructure in mitochondrial DNA of a South-east Asian fresh-water fish Hemibagrus nemurus (Siluroidei Bagridae) andPleistocene sea-level changes on the Sunda shelfrdquo MolecularEcology vol 4 no 3 pp 331ndash346 1995

[11] H H Ng and J J Dodson ldquoMorphological and genetic descrip-tions of a new species of catfish Hemibagrus chrysops fromSarawak East Malaysia with an assessment of phylogeneticrelationships (Teleostei Bagridae)rdquo Raffles Bulletin of Zoologyvol 47 no 1 pp 45ndash57 1999

[12] P D N Hebert A Cywinska S L Ball and J R DeWaardldquoBiological identifications through DNA barcodesrdquo Proceedingsof the Royal Society B Biological Sciences vol 270 no 1512 pp313ndash321 2003

[13] J C Carolan T EMurray U Fitzpatrick et al ldquoColour patternsdo not diagnose species quantitative evaluation of a DNAbarcoded cryptic bumblebee complexrdquo PLoS ONE vol 7 no 1Article ID e29251 2012

[14] M Kekkonen and P D N Hebert ldquoDNA barcode-baseddelineation of putative species efficient start for taxonomicworkflowsrdquoMolecular Ecology Resources vol 14 no 4 pp 706ndash715 2014

[15] DM Hillis CMoritz and B KMarbleMolecular SystematicsSinauer Sunderland Mass USA 2nd edition 1996

[16] T D Kocher W K Thomas A Meyer et al ldquoDynamics ofmitochondrial DNA evolution in animals amplification andsequencingwith conserved primersrdquoProceedings of theNationalAcademy of Sciences of the United States of America vol 86 no16 pp 6196ndash6200 1989

[17] T A Hall ldquoBioEdit a user-friendly biological sequence align-ment editor and analysis program for Windows 9598NTrdquoNucleic Acids Symposium Series vol 41 pp 95ndash98 1999

[18] D A Benson I Karsch-Mizrachi K Clark D J Lipman JOstell and EW Sayers ldquoGenBankrdquoNucleic Acids Research vol40 no D1 pp D48ndashD53 2012

[19] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[20] X Ku Z Peng R Diogo and S He ldquoMtDNA phylogenyprovides evidence of generic polyphyleticism for East Asianbagrid catfishesrdquoHydrobiologia vol 579 no 1 pp 147ndash159 2007

[21] L Yang and S He ldquoPhylogeography of the freshwater catfishHemibagrus guttatus (Siluriformes Bagridae) implications forSouth China biogeography and influence of sea-level changesrdquoMolecular Phylogenetics and Evolution vol 49 no 1 pp 393ndash398 2008

[22] B W Bowen A L Bass L A Rocha W S Grant and DR Robertson ldquoPhylogeography of the trumpetfishes (Aulosto-mus) ring species complex on a global scalerdquo Evolution vol 55no 5 pp 1029ndash1039 2001

[23] Y P Kartavtsev ldquoDivergence at Cyt-b and Co-1 mtDNA geneson different taxonomic levels and genetics of speciation inanimalsrdquoMitochondrial DNA vol 22 no 3 pp 55ndash65 2011

[24] SWada Y Kameda and S Chiba ldquoLong-term stasis and short-term divergence in the phenotypes of microsnails on oceanicislandsrdquoMolecular Ecology vol 22 no 18 pp 4801ndash4810 2013

[25] J Colborn R E Crabtree J B Shaklee E Pfeiler and B WBowen ldquoThe evolutionary enigma of bonefishes (Albula spp)cryptic species and ancient separations in a globally distributedshorefishrdquo Evolution vol 55 no 4 pp 807ndash820 2001

[26] J-D Durand K-N Shen W-J Chen et al ldquoSystematics of thegrey mullets (Teleostei Mugiliformes Mugilidae) molecularphylogenetic evidence challenges two centuries ofmorphology-based taxonomyrdquo Molecular Phylogenetics and Evolution vol64 no 1 pp 73ndash92 2012

[27] K-N Shen B W Jamandre C-C Hsu W-N Tzeng and J-D Durand ldquoPlio-Pleistocene sea level and temperature fluc-tuations in the northwestern Pacific promoted speciation inthe globally-distributed flathead mullet Mugil cephalusrdquo BMCEvolutionary Biology vol 11 no 1 article 83 2011

[28] R Hall ldquoThe plate tectonics of Cenozoic SE Asia and thedistribution of land and seardquo in Biogeography and GeologicalEvolution of SE Asia R Hall and J D Holloway Eds pp 99ndash131Backhuys Publishers Leiden The Netherlands 1998

[29] R Hall ldquoSoutheast Asiarsquos changing palaeogeographyrdquo Blumeavol 54 no 1ndash3 pp 148ndash161 2009

[30] R F Inger and P K Chen ldquoThe freshwater fishes of NorthBorneordquo Fieldiana Zoology vol 45 pp 1ndash268 1962

[31] J Bohlen V Slechtova H H Tan and R Britz ldquoPhylogenyof the Southeast Asian freshwater fish genus Pangio (Cyprini-formes Cobitidae)rdquoMolecular Phylogenetics and Evolution vol61 no 3 pp 854ndash865 2011

[32] M de Bruyn E Nugroho M M Hossain J C Wilson andP B Mather ldquoPhylogeographic evidence for the existence ofan ancient biogeographic barrier the Isthmus of Kra SeawayrdquoHeredity vol 94 no 3 pp 370ndash378 2005

[33] J Parnell ldquoThe biogeography of the Isthmus of Kra region areviewrdquoNordic Journal of Botany vol 31 no 1 pp 001ndash015 2013

[34] S Usmani S G Tan S S Siraj and K Yusoff ldquoPopulationstructure of the Southeast Asian river catfishMystus nemurusrdquoAnimal Genetics vol 34 no 6 pp 462ndash464 2003

[35] M P Tan A F J Jamsari and M N Siti Azizah ldquoPhylo-geographic pattern of the striped snakehead Channa striatain Sundaland ancient river connectivity geographical and

Advances in Evolutionary Biology 21

anthropogenic singnaturesrdquo PLoS ONE vol 7 no 12 Article IDe52089 2012

[36] R Abell M L Thieme C Revenga et al ldquoFreshwater ecore-gions of the world a new map of biogeographic units forfreshwater biodiversity conservationrdquo BioScience vol 58 no 5pp 403ndash414 2008

[37] P U Clark D Archer D Pollard et al ldquoThemiddle Pleistocenetransition characteristics mechanisms and implications forlong-term changes in atmospheric pCO

2rdquo Quaternary Science

Reviews vol 25 no 23-24 pp 3150ndash3184 2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 9: ResearchArticle - Université Laval Julian... · ResearchArticle A DNA Barcode-Based Evaluation of the Southeast Asian Catfish Genus Hemibagrus Bleeker, 1862 (Teleostei: Siluriformes;

Advances in Evolutionary Biology 9

110∘

47

53

69

81

69

5199

52

95

97

78

001

H bongan

H velox

H gracilis

H planiceps

H divaricatus

BOKAP1801

BOKAP1601BOKAP1701

BOKAP1402BOKAP1503

VEBAH0907GREND0206

PLCIM0114PLCIS0101

DIPER0401DIPER0301

DIPER0201

100∘

10∘

0∘

120∘

0

0 250 500

250 500

750(km)

(mi)

Shelf minus75mShelf minus120 m

Figure 5 Phylogenetic relationships of the individual sequences and species comprising theH planiceps species groupH bongan H veloxH gracilis H planiceps and H divaricatus The subtree is extracted from the taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicate trees in which the associated sequences clustered together in the bootstrap test(500 replicates) is shown next to the branches The distribution of these sequences is illustrated in the right panel

Figures 2 and 3) The two groups are genetically similarwith a genetic divergence estimate of 119901 = 121 (SE = 09)Although theH planiceps species group is strongly supportedin both trees (99) bootstrap support of the H baramensisspecies group is weak (Figure 2 32 Figure 3 34) Thegeographical distribution of the two groups differs Membersof the H planiceps species group are found throughout theGreater Sunda Islands and Peninsular Malaysia but they areisolated in the upper reaches of large rivers (Figure 5) Incontrast members of theH baramensis species group appearto be restricted to Sabah North Borneo (Figure 6)

mtDNA Clade 4 The H planiceps Species Group The Hplaniceps species group (Figure 5) is comprised of 5 closelyrelated but genetically distinct species (mean within-groupdivergence 119901 = 20 (SD = 033) H planiceps (Java)H gracilis (eastern Peninsular Malaysia) H divaricatus(western Peninsular Malaysia) H bongan (Borneo) andH velox (Sumatra)) H lacustrinus the sixth species of thisgroup (NampK) was not sampled in the present study WithintheH planiceps species groupH planiceps formed a distinctspecies (Figure 5) comprising two sequences One sequence(PLCIM0114 Table S1) characterized 14 specimens obtainedfrom Ci Manuk West Java 9 of which were identified as Hplaniceps by NampK (Table S1) A second sequence (PLCIS0101

Table S1) was obtained from one specimen sampled atCi Sadane West Java that we classified as H planicepsH bongan is represented by 5 sequences obtained from 8specimens sampled in the Kapuas River (Figure 5) Two ofthese specimens (ZRC 43013 and 43015) were identified asH bongan by NampK (Table S1) H divaricatus is composedof 3 sequences from three specimens obtained at Gerik onthe Perak River (western Peninsular Malaysia) One of thesespecimens (ZRC 41151) is the holotype of this new species(NampK) H gracilis is represented by only one sequence(GREND0206 Table S1) shared by 6 specimens sampledat Ulu Endau Jasin River eastern Peninsular Malaysiafour of which were identified by NampK (Table S1) ZRC21484 comprises the holotype and ZRC 21482 21486 and21487 comprise the paratypes (NampK) of this species (TableS1) Finally H velox is represented by only one sequence(VEBAH0907) from 7 specimens sampled at Kerinci BatangHari River Sumatra Three of these specimens (ZRC 41503Table S1) represent 3 paratypes of the species (NampK)

mtDNA Clade 5 The H baramensis Species Group The threespecies comprising the H baramensis species group (Figures2 and 3) form three monophyletic subclades (mean within-group divergence 119901 = 106 (08) Figure 6) H semotus iscomposed of two sequences (SEPAD0101 SEPAD0202) that

10 Advances in Evolutionary Biology

110∘

002

H sabanus

H semotus

H baramensis

SAKIN0204SASGM0706

BASGM0402BASGM0301

BASGM0201BASGM0104

SESGM0502SEUMA0101

SEPAD0202SEPAD0101

See Figure 5

100∘

10∘

0∘

120∘

0

0 250 500

250 500

750(km)

(mi)

99

57

32

83

99

72

99

56

66

54

99

Shelf minus75mShelf minus120 m

Figure 6 Phylogenetic relationships of the individual sequences and three species comprising the H baramensis species group H semotusH sabanus and H baramensis (furcatus) The subtree is extracted from the taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicate trees in which the associated sequences clustered together in the bootstrap test(500 replicates) is shown next to the branches The distribution of these sequences is illustrated in the right panel

characterize fish that are the holotype and paratypes of Hsemotus (ZRC 46122 Table S1) along with two somewhatmore divergent sequences (Figure 6) One of these sequences(SGM0502) was found in the Segama River (Danum Valley)and identified as H baramensis by NampK (ZRC 40525)(Table S1) The second sequence (UMA0101) was from afish sampled at Tawau Umas Umas River (ZRC 46123) alsoidentified as H baramensis by NampK These sequences mostlikely reflect distinct lineages within H semotus inhabitingdifferent rivers as the mean intraspecies sequence divergence(119901 = 16) indicates population-level divergence

H sabanus and H furcatus (synonymized with H bara-mensis by NampK) cluster together to form a distinct subcladebut clearly diverge at the species level (Figure 6) H sabanuscomprises 2 sequences that are shared respectively by 6specimens (ZRC 42741) from the Segama River Sabah and4 specimens (part of ZRC 42743) from the KinabatanganRiver Sabah (Table S1) All of these specimenswere identifiedas H sabanus by NampK (Table S1) Finally H baramensisforms a distant subclade (147 mean sequence divergencebetween H baramensis and H sabanus) composed of 4sequences (Figure 6) shared by 8 specimens all sampled inthe Segama River Danum Valley Conservation Area SabahSix of these specimens (ZRC 40523 (2 specimens) ZRC40527 (2 specimens) and ZRC 40526 (2 specimens) Table S1)comprise the paratypes of H baramensis (furcatus) (NampK)

32 The H nemurus Species Group In both NJ and ML trees(Figures 2 and 3) the H nemurus species group is strongly

supported (bootstrap support 98 in NJ and 99 in ML)indicating that it forms a distinct and widespread geneticentity within the genus Hemibagrus However relationshipswithin the group are not so clear In general the H nemurusspecies group is composed of 4 clades The first clade may beconsidered as a species group nested within the H nemurusspecies group (85 and 93 bootstrap support in NJ andML resp) encompassing three clades H hoevenii (clade9) the H capitulum Sundaic clade (clade 10 including Hnemurus) and the H capitulum Indochinese clade (clade 11)(Figures 2 and 3) An additional three clades (Figures 2 and3) are formed by an unidentified species group (clade 6) Hspilopterus (clade 7) andH fortis (clade 8)These three latterclades are distinct at the congeneric species level (unspecifiedversus H spilopterus 119901 = 77 (07) unspecified versus Hfortis 119901 = 85 (07) and H spilopterus versus H fortis119901 = 75 (08)) The distribution of these three clades issomewhat disparate H fortis and the unspecified speciesgroup are principally found in Borneo whereasH spilopterusis widely distributed on the Indochinese mainland

mtDNA Clade 6 Unspecified Species Group This geneticallydiverse group encompassing at least 3 subclades (H spp AB and C Figure 7 within-group mean sequence divergence119901 = 68(06)) is confoundedwith theH baramensis speciesgroup byNampK It is composed of sequences fromfish sampledin Sarawak Sabah and eastern PeninsularMalaysia Subclade119867 spp A (Figure 7) is particularly interesting as it contains2 sequences (CHSARA20 CHRAJ0401) that characterize

Advances in Evolutionary Biology 11

110∘

XXPAD0303XXPAD0601

XXPAD0401XXBEL0202

XXBRM0202XXRAJ0511

XXPAD0501

XXEND0410CHSARA20CHRAJ0401

XXBEL0101XXSEG0101

100∘

10∘

0∘

120∘

0

0 250 500

250 500

750(km)

(mi)

91

86

83

99

98

47

81

001

91

72

99

80

H spp C

H spp B

H spp A

Shelf minus75mShelf minus120 m

Figure 7 Phylogenetic relationships of the individual sequences comprising the unspecified species group containing three subcladesidentified as H spp A B and C The subtree is extracted from the taxon identification phenogram inferred using the Neighbor-Joiningmethod (see Figure 2) The percentage of replicate trees in which the associated sequences clustered together in the bootstrap test (500replicates) is shown next to the branches The distribution of these sequences is illustrated in the right panel

H chrysops a new species identified by Ng and Dodson [11]However specimens of H chrysops from the Rajang Riverwere subsequently synonymized by NampK with H capitulumof the H nemurus species group (ZRC 23008ndash23014 TableS1) However an additional 14 fish from the Sadong Riverat Serian bearing genotype SARA20 (Table S1) and thusgenetically identical to the Rajang H chrysops samples wereidentified by NampK as H spilopterus (ZRC 29583ndash96 TableS1) These specimens however were erroneously identifiedin NampK as originating in the Chao Phraya River (Thailand)Subclade A also contains one sequence obtained from 10 fishsampled on the Endau River (Mersing) in eastern PeninsularMalaysia These fish were not morphologically examinedFinally subclade A also contains one sequence from oneunidentified fish sampled in the Segaliud River Sabah andanother unidentified fish sampled in the Belait River BruneiSubclade H spp B encompasses 2 sequences one of whichcomes from a fish sampled in the Padas River (XXPAD0501)and part of the ZRC 46122 fish lot identified as H semotusby NampK (Table S1) The second sequence (XXRAJ0511) wasshared by 11 unidentified fish sampled at Katibas on theRajang River Sarawak (Table S1) Finally subclade 119867 sppC encompasses 5 sequences Three sequences (XXPAD0303XXPAD0401 and XXPAD0601) were obtained from thePadas River and identified byNampKasH semotus (ZRC46122Table S1) The fourth sequence (XXBRM0202) was obtainedfrom 2 fish sampled in the Baram River and was identified

as H baramensis by NampK (ZRC 42742) The 5th sequencecomposing this group was obtained from 2 unidentified fishsampled in the Belait River Brunei (Table S1)

mtDNA clade 6 is thus problematical within the contextof the revision of the genus Hemibagrus Two highlydivergent mtDNA clades (clade 5 and clade 6 Figures 2and 3) occur in sympatry principally in the Padas River ofSabah North Borneo (Figures 6 and 7) Specimens of theunspecified species group are morphologically similar tothe H baramensis species group where they are assigned inthe analysis of NampK However the mean genetic distancebetween the unspecified species group and theH baramensisspecies group is 119901 = 193 (28) close to genus leveldivergence Furthermore subclade 119867 spp A (Figure 7)contains not only H chrysops but also an unidentifiedpopulation from eastern Peninsular Malaysia (XXEND0410Table S1) and two additional Sabah sequences On theother hand the unspecified species group clusters with allother members of the H nemurus species group (meangenetic divergence of unspecified group versus H capitulum(Sundaic clade) 119901 = 76 (07) (Figure 2)) with strongbootstrap support Such genetic clustering may justify theinclusion of H chrysops and associated sequences in theH nemurus species group but its synonymization with Hcapitulum (NampK) is difficult to defend on genetic groundsWe tentatively conclude that H chrysops is a valid speciesbased on genetic identity [11] forming part of an unknown

12 Advances in Evolutionary Biology

110∘

SPCPCHMK12

SPMEK0202SPMEK0401

SPMEK0301SPMEK0103

SPMEK1401SPMEK0501

SPMEK1201SPMEK1301

SPMEK1601SPMEK1502

SPMEK0701SPMEK0801SPMEK1701

SPCHP0501SPCHA0201

SPCHA0304SPPAT0101

100∘

10∘

0∘

120∘

0

0 250 500

250 500

750(km)

(mi)

000499

99 98

60

47

48

54

96

7091

38

86

87

99

80

58

H spilopterus ldquoCrdquo

H spilopterus ldquoBrdquo

H spilopterus ldquoArdquo

Shelf minus75mShelf minus120 m

Figure 8 Phylogenetic relationships within H spilopterus comprised of 3 subclades identified as A B and C The subtree is extracted fromthe taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicate trees in whichthe associated sequences clustered together in the bootstrap test (500 replicates) is shown next to the branches The distribution of thesesequences is illustrated in the right panel

species group found throughout North Borneo (and toa lesser degree in eastern Peninsular Malaysia) that maybe considered as part of the H nemurus species groupHowever the morphological confusion of the unspecifiedspecies group with the sympatric highly divergent Hbaramensis species group suggests the existence of a crypticspecies assemblage (see Section 4)

mtDNA Clade 7 Clade 7 (Figures 2 and 3) is composed of atleast 3 distinct subclades that appear to reflect 3 geneticallydivergent populations ofH spilopterus Subclade A (Figure 8)encompasses 7 sequences obtained from 8 fish sampled inthe Mekong River Four fish bearing 3 of these sequenceswere identified as paratypes (ZRC 43324) of H spilopterusby NampK (Table S1) One additional fish was identified asH spilopterus by NampK (ZRC 46157 Table S1) Subclade Bencompasses 6 sequences obtained from 9 fish all sampledfrom the Mekong River (Table S1) The fish sampled at ThatPhanom (ZRC 46012) was identified as H spilopterus inthe analysis of HampK (Table S1) Subclade C encompasses 5sequences obtained from 19 fish sampled in the Chao PhrayaMae Khlong and Chanthaburi Rivers Thailand Onesequence (CPCHMK12) was found in all of these drainages(Table S1) A closely allied sequence was found in one fishsampled at Phatthalung just south of the Isthmus of Kra onthe Thai-Malay Peninsula None of these fish were examined

by NampK and thus we are unable to definitively identifythe species However given the widespread occurrence ofH spilopterus in the Chao Phraya and Mae Klong Riversreported by NampK we conclude that these fish are part of awidespread H spilopterus population complex The meanwithin-group (clade 7) genetic divergence (119901 = 29 (04))suggests intraspecific population-level divergence

mtDNA Clade 8 Clade 8 encompasses 3 sequencesobtained from 9 fish sampled in east Sabah specifically theKinabatangan Segama and Umas Umas Rivers (Figure 9)The Kinabatangan sequence is somewhat divergent withinthe clade and the specimen was not identified by NampK Onesequence (SGM0606) characterized 6 fish from the SegamaRiver 2 of which were identified as H fortis (ZEC 4052140522 Table S1) by NampK (Table S1) Finally one fish bearinga closely related genotype from the Umas Umas River wasidentified as H baramensis by NampK (Table S1)

mtDNA Clade 9 Clade 9 is a sister group to the remaining2 clades composing the H nemurus species group Thiswell-defined clade (Figure 10) clusters 6 sequences from42 fish that are clearly identified as H hoevenii (NampK)Some of these haplotypes are geographically widespread onesequence (JOMSKA11 Table S1) was found in the Johor River(East Peninsular Malaysia) the Musi River (Sumatra) and

Advances in Evolutionary Biology 13

100∘ 110∘

0∘

120∘

00 250

250500

500750

(km)

(mi)

FOKIN0102FOSGM0606

FOUMA0201H fortis

10∘

99

86

001

Shelf minus75mShelf minus120 m

Figure 9 Phylogenetic relationships within H fortis comprised of 3 sequences The subtree is extracted from the taxon identificationphenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicate trees in which the associated sequencesclustered together in the bootstrap test (500 replicates) is shown next to the branches The distribution of these sequences is illustrated in theright panel

the Kapuas River (West Borneo) and a second sequence(HMUBHKABM23 Table S1) was found inMuarRiver (WestPeninsular Malaysia) and the Batang Hari (Sumatra) theKapuas (Sarawak) and the Baram Rivers (Sarawak) Many ofthese fish were identified as H hoevenii by NampK (Table S1)The existence of twodistinct but very closely related subclades(Figure 10 119901 = 06 (03)) that are not geographicallysegregated suggests some recent isolation event followed bywidespread dispersal and secondary contact

33 The H capitulum Group The final two monophyleticgroups of the Hemibagrus gene tree are approximatelyequivalent to the remaining species composing the Hnemurus species group as defined by NampK Dodson etal [10] and Ng and Dodson [11] have previously referredto clade 10 as the Sundaic clade as most sequences wereobtained from fish sampled on the Greater Sunda Islandsand clade 11 as the Indochinese clade asmany sequences wereobtained from fish sampled on the Indochinese mainland(Figure 2 Table S1) Although most of these sequencesare assigned to H capitulum by NampK there are somesignificant phylogeographic differences between the 2 cladesIn particular there appears to be an east-west division in thedistribution of the two clades on the Thai-Malay Peninsulawith the Sundaic clade (clade 10) found on the west coast andthe Indochinese clade found on the east coast with sympatricoccurrence in the Muar River Nevertheless mean sequencedivergence between the two clades is 119901 = 29 (05) avalue that is indicative of population-level or subspecies

divergence The two clades are thus best considered ascomprising one species

mtDNA Clade 10 The Sundaic clade includes a number ofclosely related sequences that comprise part of H capitulumand H nemurus Very little genetic structure is apparentand bootstrap support for tree topology within this group isgenerally very weak (Figure 11) A widely spread genotype(NCLCTBA20) characterized 6 of 17 specimens from CiTarum at the Cirata reservoir (West Java) (Table S1) and5 were identified as H nemurus in the analysis of NampK(ZRC 42564) (Table S1) The remaining fish characterized bythis sequence were obtained from Ci Liwong at Sawangan(West Java) and one fish was obtained from the Barito Riverat Banjarmassin south Borneo This latter specimen (ZRC40553) was identified by NampK as H capitulum (Table S1)Another common genotype (NBHKPBR09) was identified aseither H capitulum H hoevenii or H nemurus dependingon the river of origin (Table S1) Many of the remainingsequences of the Sundaic clade appear to characterize fish thatNampK identified asH capitulum A geographically widespreadgenotype (CPEBEMUKA36) characterized 9 fish sampledon the Perak River at Gerik that were identified by NampKas H capitulum (ZRC 41150 Table S1) Another commongenotype within this clade (CABHMS20) included 2 fishfrom the Batang Hari at Jambi and identified by NampK asH capitulum (ZRC40534 Table S1) Another 2 sequences(CABAR0203 CABAR0305) characterized 8 fish sampledon the Barito River at Banjarmasin and were identified as

14 Advances in Evolutionary Biology

100∘

10∘

110∘

0∘

120∘

0

0 250

250

500

500

750(km)

(mi)

53

64

99

85

64

0002

HJOMSKA11

HOBAH0601

HOBHMS05

HMUBHKABM23

HOBAH0701

HOKAP0501

H hoevenii ldquoBrdquo

H hoevenii ldquoArdquo

Shelf minus75mShelf minus120 m

Figure 10 Phylogenetic relationships withinH hoevenii comprised of 2 subclades and 6 sequences The subtree is extracted from the taxonidentification phenogram inferred using theNeighbor-Joiningmethod (see Figure 2)The percentage of replicate trees in which the associatedsequences clustered together in the bootstrap test (500 replicates) is shown next to the branches The distribution of these sequences isillustrated in the right panel

110∘

XXMAH0110

CABAR0305

96 CAKPR0201

74CAKAP0401

5931 CAKAP0601

35 CPEBEMUKA36

646 CAKAP0903

33 CAKAP080114 CAKAP0701

CARAJ0210

4 CABAR0203CABRA0204

20

CBHKPBR0919

CABRA0301

15CABHMS20

24 NCLCTBA20

63 NECIT0201

H capitulumldquoSundaic claderdquo

H nemurus

0002 120∘

0 250 500 750

(km)

0 250 500

(mi)100

10∘

0∘

Shelf minus75mShelf minus120 m

Figure 11 Phylogenetic relationships within theH capitulum ldquoSundaicrdquo clade including 2 sequences associated withH nemurusThe subtreeis extracted from the taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicatetrees in which the associated sequences clustered together in the bootstrap test (500 replicates) is shown next to the branchesThe distributionof these sequences is illustrated in the right panel

Advances in Evolutionary Biology 15

H capitulum

0∘

10∘

100∘

110∘

120∘

66

63

88

59

38

39 68

71

6991

57

9754

88 4944

39

53

89

0002

ldquoIndochineserdquogroup ldquoErdquo

H capitulumldquoIndochineserdquo

group ldquoDrdquoH capitulumldquoIndochineserdquo

group ldquoCrdquo

H capitulumldquoIndochineserdquo

group ldquoBrdquo

H capitulumldquoIndochineserdquo

group ldquoArdquo0

0

250

250

500

500

750

(km)

(mi)

CATER0301CATERR0401

CKETEMU23

CAMEK0601CACHP0301

CAMKPA03

CAMEK0910CAMEK1003

XXMUBM02

CABMTU09

CABRM0601

CAKAP1201CAKAP1001

CAKAP1101CAKAP1301

CABRM0501

CASAD0102

CASAD0220

CAEND0301

CABAH0801

CAPABH05CATEEN03

CATER0701CATERR0801

CAKETE04CATER0501

CAPAH0303

Shelf minus75mShelf minus120 m

Figure 12 Phylogenetic relationships within theH capitulum ldquoIndochineserdquo clade encompassing 5 subclades identified as A B C D and EThe subtree is extracted from the taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2)The percentageof replicate trees in which the associated sequences clustered together in the bootstrap test (500 replicates) is shown next to the branchesThedistribution of these sequences is illustrated in the right panel

H capitulum by NampK (ZRC 40553 Table S1) Although notincluded in the analysis of NampK samples from the Sundaicclade were also found in the Kapuas River (West Borneo)Finally 10 fish sampled on the Mahakam River at Samarindaeast Borneo are all characterized by one relatively divergentgenotype (CAMAH0110) butwere considered asH capitulumin the analysis of NampK (ZRC 40533 Table S1)TheMahakampopulation sample is the most genetically distinct of theSundaic clade (119901 = 17 (04)) but nevertheless indicativeof intraspecific population-level divergence (Figure 12)

The topology of the Sundaic clade does not permit usto draw a clear distinction between H nemurus and theSundaic H capitulum The mean within-group divergenceof the Sundaic clade is only 119901 = 08 (02) well withinpopulation-level divergence values We postulate that Hnemurus may be one of several regional variants of awidespread Sundaic H capitulum clade that has radiatedrelatively recently This may explain the existence of manyshared sequences throughout the Sundaic islands assigned todifferent species in different regions

mtDNA Clade 11 Finally the Indochinese clade of Hcapitulum represents a geographically widespread andweakly diversified clade (within-group mean sequencedivergence 119901 = 11 (02) Figure 12) Although few fishfrom this genetic clade were included in the morphological

analysis of NampK most may be considered as H capitulumgiven their geographic distribution and close geneticrelationship to the Sundaic H capitulum Four sequencesin this clade were identified as H capitulum in the analysisof NampK 3 specimens characterized by one sequence(CABMTU09) found in the Tutong River of westernBorneo (ZRC 40482) 6 characterized by the same sequencefrom the Baram River (ZRC 40498) another 2 sequencesfrom the Baram River (ZRC 40498) and 20 specimenscharacterized by an abundant genotype (CASAD0220)found at 2 sites in the Sadong River also in western Borneo(ZRC 39526 5 specimens 39527 3 specimens and 38753 4specimens) (Table S1) Unfortunately none of the specimenscollected on the Indochinese mainland for genetic analysiswere examined by NampK Nevertheless the distribution ofspecimens analyzed for mtDNA resembles the distribution ofspecimens examined by NampK Although weakly structuredwe may recognize 5 subclades One genotype (CAPAH0303)is basal to the phenogram Subclade A encompasses 8sequences mainly found in eastern Peninsular Malaysia(Table S1 Figure 12) These rivers furnished many of thespecimens NampK examined to define H capitulum Howeversome sequences in this group occur in sympatry withsequences identified within the Sundaic H capitulum groupTwo sequences from subclade A occurred in the Batang HariSumatra (Table S1) Subclade E also encompasses sequences

16 Advances in Evolutionary Biology

found in Peninsular Malaysia as well as in the Mekong ChaoPhraya and Mea Khlong Rivers (Figure 12) In addition3 subclades encompass sequences found principally inwestern Borneo subclade B in the Baram and Sadong RiversSarawak subclade C in the Kapuas River West Kalimantanand subclade D in the Baram and Tutong Rivers of Sarawakand Brunei respectively (Figure 12) This latter subclade alsoincludes one genotype found in the Muar River of westernpeninsular Malaysia Finally subclade E encompasses 5sequences from the Mekong Chao Praya and Mae KhlongRivers all within the distribution area of H spilopterus

EvolutionaryHistoryThe evolutionary history of theHemiba-grus species groups analyzed here dates from at least theMiocene 15ndash20Ma (Figure 13) when considering the widelyused molecular clock of 2 sequence divergence per millionyears Bootstrap support for the sister group relationships ofH wyckioides theH guttatus species group andH peguensisare all weak (clearly due to undersampling) and little can besaid of their evolutionary history other than the fact that theyoccupy basal positions in the rooted trees The remainder ofboth the NJ and ML trees is composed of two major sistergroups the H planiceps + H baramensis group and the Hnemurus species group (Figures 2 3 and 13)TheH planiceps+ H baramensis group is strongly supported in both NJ andML analyses (84 and 90 bootstrap support resp Figures2 and 3) The H nemurus species group is also stronglysupported in both analyses (98 and 99 bootstrap supportFigures 2 and 3) These two major phylogenetic groups areestimated to have diverged at least 8 to 12Ma ago during themid to late Miocene (Figure 13)

Much of the evolutionary history of Hemibagrus hasoccurred during the Quaternary starting approximately26Ma ago This is particularly true of the H planicepsand H nemurus species groups The radiation of the 5species included in the H planiceps species group appearsto coincide with the middle Pleistocene transition period ofapproximately 1Ma ago Their high-elevation distributionhowever appears to have largely isolated them from theeffects of lowered sea levels during glacial maxima and theattendant opportunities for widespread dispersal (Figure 5)The evolutionary histories of theH baramensis species groupand the unspecified species group date back to the latePliocene In contrast the low-elevation distribution of theHnemurus species group in Sundaland appears to have favoredtheir widespread distribution during the Quaternary ice ageIn particular H hoevenii appears to be the most recentlydispersed of the species under study with diversification anddispersal occurring approximately 03Ma ago (Figure 13)

4 Discussion

41 The Congruence of Morphological and Genetic AnalysesCombining genetic and morphological analyses to elucidatetaxonomy allowed us to better understand the diversificationand evolutionary relationships within this speciose group offishes Given the rapid and relatively constant rate of mtDNAevolution the deeper the branches within the phylogenetictree are the greater our ability to distinguish valid taxa

should be Conversely the lack of complete congruencebetween morphological- and genetic-based identificationsis expected to increase among the shallower branches of thephylogenetic tree resulting in the appearance of polyphyleticspecies Although 2 of the 8 species groups defined by Ngand Kottelat [7] were not sampled in this study the validityof the remaining 6 species groups and their componentspecies defined on the basis of morphological similaritywas largely confirmed by the analysis of the mitochondrialCyt 119887 bar code As expected congruence is strongest at thebase of the NJ and ML trees where the H guttatus speciesgroup formed a well-defined monophyletic group The Hmenoda and H wyckii species group were each representedby only one species but they were clearly distinct relativeto sequences characterizing the neighboring species groupsThe H planiceps H baramensis and H nemurus speciesgroups and their component species were also generallycongruent in morphological and genetic analyses Also asexpected most cases of discrepancy between morphological-based species identification and genetic-based speciesidentification occurred on relatively shallow branches of thephylogenetic tree These involve principally H spilopterusand the two mtDNA subclades composing H capitulumHowever a far more unexpected lack of congruence involvesthe definition of a deeply divergent unspecified genetic groupconfounding members of theH baramensis species group inthe morphological analysis of Ng and Kottelat [7]

Interpreting Polyphyly The apparent polyphyletic origin ofa species may be due to incomplete lineage sorting amongrecently diverged species the introgression of mtDNA dueto species hybridization or the erroneous identificationof species due to the lumping of morphologically crypticspecies In the absence of nuclear DNA analyses it isoften difficult to distinguish between introgression andincomplete lineage sorting For example several sequencescharacterizing the Indochinese clade of H capitulum(subclade E) are found in the Mekong and Chao PhrayaRivers well within the distributional area of H spilopterusH spilopterus and H capitulum first diverged at least 3MaOn the one hand this apparent case of polyphyly mayhave resulted from incomplete lineage sorting betweenrecently divergedH spilopterus andH capitulum We wouldhowever have expected similar cases of polyphyly amongpopulations of the Indochinese clade of H capitulum butthis was not detected On the other hand the sympatricoccurrence of H spilopterus subclade A and subcladeB sequences with H capitulum Indochinese subclade Esequences in the Mekong River may represent a migrationevent and local introgression of H capitulum with Hspilopterus A similar event may have occurred in theChao Phraya and Mae Khlong Rivers where H spilopterussequences cooccur withH capitulum sequences Computingthe divergence of H capitulum sequences (Indochinesesubclade E) cooccurring with H spilopterus and Hcapitulum sequences occurring in eastern PeninsularMalaysia (subclade A) reveals a mean sequence divergence119901 = 15 (04) Thus the H capitulum sequences occurringin sympatry with H spilopterus diverged at most 750 ka ago

Advances in Evolutionary Biology 17

H fortis

XXRAJ0604H furcatus

H sabanusH semotusH divaricatus

H planicepsGREND0206

VEBAH0907

H bonganPESAL0102

H wyckioidesH macropterus

H guttatusXXRAJ0301Leiocassis longirostris

Ma

4971

92

47

51

9999

99

9999

99

99

99

9999

99

99

99

99

65

96

96

4946

93

79

78

70

91

93

98

86

87

91

95

97

71

85

9999

05101520

005010015020 000

H capitulum Indochinese clades AndashE

H capitulum Sundaic clade

H hoevenii B

H hoevenii A

H spilopterus C

H spilopterus B

H spilopterus AH spilopterus A

H spp CH spp B

H spp A

Figure 13 Linearized tree with branch lengths estimated with the molecular clock using a mutation rate of 1 per Ma (=divergence rate of2 per Ma) The tree topology is identical to the ML reconstruction illustrated in Figure 3 (see the specificities of the tree reconstruction inthe caption of Figure 3) Boxes represent 95 confidence intervals of divergence time estimates for each node Vertical dashed lines redirectto dates indicated on the timescale (Ma) Numbers below timescale indicate the estimated number of substitution changes using Hasegawa-Kishino-Yanomodel (HKY)with gamma distribution (+119866 = 13290) after linearizing the treeH furcatus is synonymizedwithH baramensis

from eastern Peninsular Malaysia populations suggestinga recent introgression event Similarly H capitulum in theKapuas River is characterized by sequences of both theSundaic and the Indochinese clades As both sequences arebroadly sympatric it is not possible to distinguish betweenincomplete lineage sorting and introgression of these closelyrelated intraspecific lineages

A Possible Case of Morphological Stasis Clade 6 of thepresent analysis defines a well-supported monophyletic cladefound in Sabah Sarawak and the Endau River of easternpeninsula Malaysia that has no equivalent taxonomic statusin the revision of Ng and Kottelat [7] In Borneo they occursympatrically with the H baramensis species group and areconfounded as either H baramensis or H semotus These

two clades are genetically highly divergent having divergedbetween 8 and 12Ma ago It thus seems highly unlikely thatthey represent retained polymorphisms due to incompletelineage sorting Sequences previously identified as character-izing H chrysops [11] are part of clade 6 and extend to thesouth (Rajang and Sadong Rivers of Sarawak West Borneo)with one closely related genotype found in eastern peninsularMalaysia (Endau River) where it may well be confoundedwith H capitulum H chrysops was synonymized with Hcapitulum by Ng and Kottelat [7] based on their analysis ofspecimens ofH chrysops from the Rajang RiverThus despitea long and apparently independent phylogenetic historythe unspecified species group appears largely cryptic andconfounded principally with members of the H baramensisspecies group

18 Advances in Evolutionary Biology

We suggest that this situation is an example of mor-phological stasis involving the coexistence of a number ofgenetically divergent yet morphologically cryptic species thatare lumped together [24] A striking example of morpho-logical and ecological stasis among fishes is provided by thecoastal marine bonefishes (Albula spp) Distinct evolution-ary lineages (separated by sequence divergence between 5 and30 [25]) of bonefish coexist in several sample locations yetthey show little morphological or ecological differentiationin sympatry [25] Similarly a proportion of the grey mulletspecies (Mugilidae) also appears to consist of cryptic species[26] For exampleMugil cephalus in Taiwan is characterizedby three divergent lineages that have been shown to charac-terize 3 reproductively isolated but morphologically crypticspecies [26 27]

Here we have discovered 2 highly distinct and structuredclades (each composed of 3 mtDNA subclades) that aremorphologically confounded within different species Forexample H spp C of the unspecified species group isidentified as H baramensis in the Baram River and as Hsemotus in the Padas River We speculate that North Borneoharbors a morphologically cryptic species complex possiblycomposed of several species pairs inhabiting different riversystems The reconstruction of this phylogenetic history andthe taxonomic status of its components remains a majorchallenge to the revision of the Hemibagrus genus

42 Phylogeographic Hypotheses The phylogeography ofthe genus appears to have been principally influenced bytwo geological histories one encompassing the formationof North Borneo the second the emergence of the SundaIslands and their connectivity with the Indochinesemainlandassociated with recurrent sea-level changes North Borneoappears to have acted as a species sink promoting speciationand locally high biodiversity within the genus but with littleevidence of subsequent dispersal out of North Borneo Incontrast the geological events influencing the Indochinesemainland and the Sunda Islands have permitted bothspeciation and differential dispersal depending on thebiology of the species For example the H planiceps speciesgroup is found throughout the Greater Sunda Islands andtheThai-Malay Peninsula as is theH nemurus species groupWhereas the H nemurus species group generally occupiesthe low altitude lower reaches of large rivers the speciescomposing the H planiceps group are found in the upperreaches of large rivers The H nemurus species group iscomposed of a number of widely distributed closely relatedspecies whereas theH planiceps group is composed of a largenumber of species that are highly localized geographicallyIt thus appears that sea-level changes have had little impacton the dispersal history of the H planiceps species group buthave deeply influenced that of the H nemurus species group

The Case of North Borneo From 15Ma to 5Ma greater areasof Borneo became emergent and the central mountainson the Sarawak-Kalimantan border extending into Sabahbecame wider and higher which may have represented newbarriers to dispersal [28 29] Borneo gradually developedinto the present large island by emergence of land in the

north and east The H baramensis species group is restrictedto North Borneo as is the unspecified species group of theHnemurus species group H fortis is also distributed in NorthBorneo The isolation of the freshwater fish communities ofNorth Borneo (particularly in the Labuk-Segama watershed)has long been recognized based on the number of endemicspecies and the concentration of relict species [30] The latePliocene-early Pleistocene diversification of H fortis theunspecified species group and the H baramensis speciesgroup may thus be associated with their dispersal to andisolation within the watersheds of North Borneo Only onesequence of the unspecified species group is found outsideof North Borneo and H fortis appears to be distributedthroughout central and east Borneo [7]

ThePhylogeography of the H nemurus Species Group Periodicmarine transgressions on the Sundaland platform during theearly and middle Miocene [2 29] may have been sufficientto isolate populations of freshwater organisms and promotecladogenesis [31] This may have been responsible for theinitial mid to late Miocene divergence of the H nemurusspecies group The majority of sequences assigned to thisspecies group belong to 3 species (H spilopterus H capitulum(including H nemurus) and H hoevenii) all of which havebeen influenced by the more recent geological history of theSunda Islands and their connectivity with the Indochinesemainland H spilopterus is known from river drainages inIndochina including the Mekong Dong Nai Ban PakongChao Phraya and Mae Klong where it is found in a widevariety of lotic and lentic habitats [7] H spilopterus divergedin the late Pliocene-early Pleistocene possibly isolated tothe north of the Kra Peninsula during the late Pliocenemarine transgression that is purported to have created amajor biogeographic transition zone between the Sundaicand Indochinese biotas ([32] but see [33] for a critique ofthis biogeographic scenario) The existence of 2 subclades(A and B) in the Mekong River separated by 2 sequencedivergence is consistent with intraspecific divergence Thetwo subclades possibly represent 2 historical mtDNA lineagesthat are retained within the species reflecting a Pleistoceneisolation event followed by secondary contact within thecontemporary Mekong River Subclade C from the ChaoPhraya and Mae Khlong Rivers exhibits a level of geneticdivergence consistent with sibling species-level divergenceshowing a 5 sequence divergence relative to Mekong RiverH spilopterus

H capitulum may be considered as part of the Sundaicbiota located principally to the south of the Kra marinetransgression zone that may have isolated H spilopterus tothe north on the Indochinese mainlandThe biogeographicalevent leading to the subsequent formation of two intraspecificgroups within H capitulum is not at all clear The onlyarea where the two clades are clearly geographically separateis on the Thai-Malay Peninsula with the ldquoSundaicrdquo cladeon the west coast and the ldquoIndochineserdquo clade on theeast coast separated by the central Titiwangsa mountainrange The genetic distinctiveness of east and west coastpopulations of H capitulum has also been documented fornuclear DNA [34] A similar pattern of separation has been

Advances in Evolutionary Biology 19

reported for 5 additional freshwater fishes within this region[35] However as the divergence of the two H capitulumclades postdates the formation of the mountain range byapproximately 200Ma the geographic separation of the twoclades most likely represents differential dispersal rather thanancient vicariance The presence of the Sundaic clade on thewest coast of the Thai-Malay Peninsula as well as on theSundaic Islands reflects the historical connections betweeneast Sumatra and west Peninsular Malaysia across the Straitof Malacca This area encompasses the Northern CentralSumatra-Western Malaysia freshwater ecoregion [36] TheMuar River forms its southern border coincident with thesouthernmost distribution of the Sundaic clade on the Thai-Malay Peninsula It is important to note however that mostof Sumatra and Java emerged to achieve their present size andelevation only 5Ma ago and much of East Java continuedto be the site of marine deposition until late Pliocene-earlyPleistocene [29] We may speculate that the Sundaic clade ofH capitulum resulted from the relatively recent colonizationof these islands and Borneo The radiation within the cladeonly dates back to approximately 1Ma ago H nemurus fromWest Java may thus best be considered an isolated regionalpopulation of very recent origin

The diversification of the Indochinese clade dates fromat least 12Ma ago coincident with the mid-Pleistocenetransition at which time low-frequency high-amplitude andquasiperiodic (100 ka) glacial variability emerged It began125Ma ago and was complete by 07Ma ago [37] The tran-sition marked the beginning of major changes in sea levelsduring which the Sunda Shelf periodically emerged and wastraversed by enormous fluvial systems [3] The contributionof these events to the diversification of this clade cannotbe satisfactorily reconstructed One possible explanation isthat high sea levels produced barriers to gene flow and thepopulations of West Borneo (subclades B C and D) wereisolated from those of east Peninsular Malaysia This couldbe due to one or more dispersal events from Borneo via theGreat Sunda River (Figure 1) to east Peninsular Malaysia orlineage sorting whereby sequences of subclades A and E havebeen lost from West Borneo As in the case of the sympatricoccurrence of H spilopterus and H capitulum subclade E(see above) in the Mekong River we are unable to clearlydefine the relative roles of incomplete lineage sorting andintrogression followingmigration Given the recent origins ofthe Indochinese H capitulum sequences incomplete lineagesorting most probably blurs patterns of recent isolation

The last species of the H nemurus species group is Hhoevenii The species is widespread throughout PeninsularMalaysia and the Sundaic Islands It is found primarily inthe lower reaches of rivers and is apparently capable ofwithstanding low salt concentration [7] H hoevenii thusappears to be the most liable of all members of the Hnemurus species group to be impacted by Pleistocene sea-level changes This is clearly reflected in the widespreaddistribution of the sequences of this species Two lineages areevident having diverged no more than 03Ma ago suggest-ing a late Pleistocene isolation event followed by extensivedispersal and admixture in recent times This represents theclearest example of the dispersal opportunities afforded by

Pleistocene sea level changes on the Sunda Shelf In additionthe relatively low genetic diversity detected (only 6 sequencesin 42 fish) suggests a population bottleneck in the recent pastpossibly related to population declines in the lower reaches ofrivers flooded by increasing sea levels

43 Future Directions We have provided a global picture ofthe evolutionary taxonomy of the genus in Southeast AsiaClearly a more rigorous phylogenetic reconstruction of thegenus Hemibagrus requiring the construction of multiplegene trees based on the analysis of both mitochondrial andnuclear DNA markers from a wider taxonomic array ofhemibagrids will involve an even greater effort of samplingthroughout the region In particular we require samplingat smaller spatial and temporal scales For example smallerscale sampling in the Padas River and the Segama Riverof North Borneo using both mitochondrial and nucleargenetic markers would contribute to clarifying the natureof the cryptic species complex believed to inhabit theserivers The demonstration of reproductive isolation andormicroallopatric distribution among morphologically crypticspecies may result in the description of greater speciesrichness within the genus than presently described Thespecies status of H chrysops merits reconsideration givenits highly divergent evolutionary history Nuclear geneticmarkers in conjunction with mtDNA markers could revealcases of historical introgression particularly in the KapuasRiver and the Mekong River The hemibagrid catfishes con-tinue to provide multiple opportunities for the study of theevolutionary taxonomy and biogeography of a major faunalcomponent of the fluvial ecosystems of Southeast Asia

Disclosure

Ethanol-preserved tissue samples of many of the specimensidentified in Table S1 are freely available upon request fromJJD

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Goh Yan Yih for assistance in collectingspecimens and molecular analyses Mrs Francoise Colom-bani for molecular analyses and Dr Heok Hee Ng forspecies identification This work was funded by the NationalUniversity of Singapore grant to P K L Ng and a CanadianNatural Sciences and Engineering Research Council grant toJ J Dodson

References

[1] D J Lohman M de Bruyn T Page et al ldquoBiogeography ofthe Indo-Australian archipelagordquo Annual Review of EcologyEvolution and Systematics vol 42 pp 205ndash226 2011

20 Advances in Evolutionary Biology

[2] D S Woodruff ldquoNeogene marine transgressions palaeogeog-raphy and biogeographic transitions on the Thai-Malay Penin-sulardquo Journal of Biogeography vol 30 no 4 pp 551ndash567 2003

[3] H K Voris ldquoMaps of Pleistocene sea levels in SoutheastAsia shorelines river systems and time durationsrdquo Journal ofBiogeography vol 27 no 5 pp 1153ndash1167 2000

[4] S Y Yap ldquoOn the distributional patterns of Southeast-EastAsian freshwater fish and their historyrdquo Journal of Biogeographyvol 29 no 9 pp 1187ndash1199 2002

[5] S K J McConnell ldquoMapping aquatic faunal exchanges acrossthe Sunda shelf South-East Asia using distributional andgenetic data sets from the cyprinid fish Barbodes gonionotus(Bleeker 1850)rdquo Journal of Natural History vol 38 no 5 pp651ndash670 2004

[6] T Mo Anatomy Relationships and Systematics of the Bagridae(Teleostei Siluroidei) with a Hypothesis of Siluroid Phylogenyvol 17 ofTheses Zoologicae Koeltz Scientific Books 1991

[7] H H Ng and M Kottelat ldquoRevision of the Asian catfish genusHemibagrus bleeker 1862 (Teleostei Siluriformes Bagridae)rdquoRaffles Bulletin of Zoology vol 61 no 1 pp 205ndash291 2013

[8] P K L Ng and H H Ng ldquoHemibagrus gracilis a new speciesof large riverine catfish (Teleostei Bagridae) from PeninsularMalaysiardquo Raffles Bulletin of Zoology vol 43 pp 133ndash142 1995

[9] H H Tan and M Kottelat ldquoRedescription of Betta picta(Teleostei Osphronemidae) and description of Betta falx sp nfrom central Sumatrardquo Revue Suisse de Zoologie vol 105 no 3pp 557ndash568 1998

[10] J J Dodson F Colombani and P K Ng ldquoPhylogeographicstructure in mitochondrial DNA of a South-east Asian fresh-water fish Hemibagrus nemurus (Siluroidei Bagridae) andPleistocene sea-level changes on the Sunda shelfrdquo MolecularEcology vol 4 no 3 pp 331ndash346 1995

[11] H H Ng and J J Dodson ldquoMorphological and genetic descrip-tions of a new species of catfish Hemibagrus chrysops fromSarawak East Malaysia with an assessment of phylogeneticrelationships (Teleostei Bagridae)rdquo Raffles Bulletin of Zoologyvol 47 no 1 pp 45ndash57 1999

[12] P D N Hebert A Cywinska S L Ball and J R DeWaardldquoBiological identifications through DNA barcodesrdquo Proceedingsof the Royal Society B Biological Sciences vol 270 no 1512 pp313ndash321 2003

[13] J C Carolan T EMurray U Fitzpatrick et al ldquoColour patternsdo not diagnose species quantitative evaluation of a DNAbarcoded cryptic bumblebee complexrdquo PLoS ONE vol 7 no 1Article ID e29251 2012

[14] M Kekkonen and P D N Hebert ldquoDNA barcode-baseddelineation of putative species efficient start for taxonomicworkflowsrdquoMolecular Ecology Resources vol 14 no 4 pp 706ndash715 2014

[15] DM Hillis CMoritz and B KMarbleMolecular SystematicsSinauer Sunderland Mass USA 2nd edition 1996

[16] T D Kocher W K Thomas A Meyer et al ldquoDynamics ofmitochondrial DNA evolution in animals amplification andsequencingwith conserved primersrdquoProceedings of theNationalAcademy of Sciences of the United States of America vol 86 no16 pp 6196ndash6200 1989

[17] T A Hall ldquoBioEdit a user-friendly biological sequence align-ment editor and analysis program for Windows 9598NTrdquoNucleic Acids Symposium Series vol 41 pp 95ndash98 1999

[18] D A Benson I Karsch-Mizrachi K Clark D J Lipman JOstell and EW Sayers ldquoGenBankrdquoNucleic Acids Research vol40 no D1 pp D48ndashD53 2012

[19] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[20] X Ku Z Peng R Diogo and S He ldquoMtDNA phylogenyprovides evidence of generic polyphyleticism for East Asianbagrid catfishesrdquoHydrobiologia vol 579 no 1 pp 147ndash159 2007

[21] L Yang and S He ldquoPhylogeography of the freshwater catfishHemibagrus guttatus (Siluriformes Bagridae) implications forSouth China biogeography and influence of sea-level changesrdquoMolecular Phylogenetics and Evolution vol 49 no 1 pp 393ndash398 2008

[22] B W Bowen A L Bass L A Rocha W S Grant and DR Robertson ldquoPhylogeography of the trumpetfishes (Aulosto-mus) ring species complex on a global scalerdquo Evolution vol 55no 5 pp 1029ndash1039 2001

[23] Y P Kartavtsev ldquoDivergence at Cyt-b and Co-1 mtDNA geneson different taxonomic levels and genetics of speciation inanimalsrdquoMitochondrial DNA vol 22 no 3 pp 55ndash65 2011

[24] SWada Y Kameda and S Chiba ldquoLong-term stasis and short-term divergence in the phenotypes of microsnails on oceanicislandsrdquoMolecular Ecology vol 22 no 18 pp 4801ndash4810 2013

[25] J Colborn R E Crabtree J B Shaklee E Pfeiler and B WBowen ldquoThe evolutionary enigma of bonefishes (Albula spp)cryptic species and ancient separations in a globally distributedshorefishrdquo Evolution vol 55 no 4 pp 807ndash820 2001

[26] J-D Durand K-N Shen W-J Chen et al ldquoSystematics of thegrey mullets (Teleostei Mugiliformes Mugilidae) molecularphylogenetic evidence challenges two centuries ofmorphology-based taxonomyrdquo Molecular Phylogenetics and Evolution vol64 no 1 pp 73ndash92 2012

[27] K-N Shen B W Jamandre C-C Hsu W-N Tzeng and J-D Durand ldquoPlio-Pleistocene sea level and temperature fluc-tuations in the northwestern Pacific promoted speciation inthe globally-distributed flathead mullet Mugil cephalusrdquo BMCEvolutionary Biology vol 11 no 1 article 83 2011

[28] R Hall ldquoThe plate tectonics of Cenozoic SE Asia and thedistribution of land and seardquo in Biogeography and GeologicalEvolution of SE Asia R Hall and J D Holloway Eds pp 99ndash131Backhuys Publishers Leiden The Netherlands 1998

[29] R Hall ldquoSoutheast Asiarsquos changing palaeogeographyrdquo Blumeavol 54 no 1ndash3 pp 148ndash161 2009

[30] R F Inger and P K Chen ldquoThe freshwater fishes of NorthBorneordquo Fieldiana Zoology vol 45 pp 1ndash268 1962

[31] J Bohlen V Slechtova H H Tan and R Britz ldquoPhylogenyof the Southeast Asian freshwater fish genus Pangio (Cyprini-formes Cobitidae)rdquoMolecular Phylogenetics and Evolution vol61 no 3 pp 854ndash865 2011

[32] M de Bruyn E Nugroho M M Hossain J C Wilson andP B Mather ldquoPhylogeographic evidence for the existence ofan ancient biogeographic barrier the Isthmus of Kra SeawayrdquoHeredity vol 94 no 3 pp 370ndash378 2005

[33] J Parnell ldquoThe biogeography of the Isthmus of Kra region areviewrdquoNordic Journal of Botany vol 31 no 1 pp 001ndash015 2013

[34] S Usmani S G Tan S S Siraj and K Yusoff ldquoPopulationstructure of the Southeast Asian river catfishMystus nemurusrdquoAnimal Genetics vol 34 no 6 pp 462ndash464 2003

[35] M P Tan A F J Jamsari and M N Siti Azizah ldquoPhylo-geographic pattern of the striped snakehead Channa striatain Sundaland ancient river connectivity geographical and

Advances in Evolutionary Biology 21

anthropogenic singnaturesrdquo PLoS ONE vol 7 no 12 Article IDe52089 2012

[36] R Abell M L Thieme C Revenga et al ldquoFreshwater ecore-gions of the world a new map of biogeographic units forfreshwater biodiversity conservationrdquo BioScience vol 58 no 5pp 403ndash414 2008

[37] P U Clark D Archer D Pollard et al ldquoThemiddle Pleistocenetransition characteristics mechanisms and implications forlong-term changes in atmospheric pCO

2rdquo Quaternary Science

Reviews vol 25 no 23-24 pp 3150ndash3184 2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 10: ResearchArticle - Université Laval Julian... · ResearchArticle A DNA Barcode-Based Evaluation of the Southeast Asian Catfish Genus Hemibagrus Bleeker, 1862 (Teleostei: Siluriformes;

10 Advances in Evolutionary Biology

110∘

002

H sabanus

H semotus

H baramensis

SAKIN0204SASGM0706

BASGM0402BASGM0301

BASGM0201BASGM0104

SESGM0502SEUMA0101

SEPAD0202SEPAD0101

See Figure 5

100∘

10∘

0∘

120∘

0

0 250 500

250 500

750(km)

(mi)

99

57

32

83

99

72

99

56

66

54

99

Shelf minus75mShelf minus120 m

Figure 6 Phylogenetic relationships of the individual sequences and three species comprising the H baramensis species group H semotusH sabanus and H baramensis (furcatus) The subtree is extracted from the taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicate trees in which the associated sequences clustered together in the bootstrap test(500 replicates) is shown next to the branches The distribution of these sequences is illustrated in the right panel

characterize fish that are the holotype and paratypes of Hsemotus (ZRC 46122 Table S1) along with two somewhatmore divergent sequences (Figure 6) One of these sequences(SGM0502) was found in the Segama River (Danum Valley)and identified as H baramensis by NampK (ZRC 40525)(Table S1) The second sequence (UMA0101) was from afish sampled at Tawau Umas Umas River (ZRC 46123) alsoidentified as H baramensis by NampK These sequences mostlikely reflect distinct lineages within H semotus inhabitingdifferent rivers as the mean intraspecies sequence divergence(119901 = 16) indicates population-level divergence

H sabanus and H furcatus (synonymized with H bara-mensis by NampK) cluster together to form a distinct subcladebut clearly diverge at the species level (Figure 6) H sabanuscomprises 2 sequences that are shared respectively by 6specimens (ZRC 42741) from the Segama River Sabah and4 specimens (part of ZRC 42743) from the KinabatanganRiver Sabah (Table S1) All of these specimenswere identifiedas H sabanus by NampK (Table S1) Finally H baramensisforms a distant subclade (147 mean sequence divergencebetween H baramensis and H sabanus) composed of 4sequences (Figure 6) shared by 8 specimens all sampled inthe Segama River Danum Valley Conservation Area SabahSix of these specimens (ZRC 40523 (2 specimens) ZRC40527 (2 specimens) and ZRC 40526 (2 specimens) Table S1)comprise the paratypes of H baramensis (furcatus) (NampK)

32 The H nemurus Species Group In both NJ and ML trees(Figures 2 and 3) the H nemurus species group is strongly

supported (bootstrap support 98 in NJ and 99 in ML)indicating that it forms a distinct and widespread geneticentity within the genus Hemibagrus However relationshipswithin the group are not so clear In general the H nemurusspecies group is composed of 4 clades The first clade may beconsidered as a species group nested within the H nemurusspecies group (85 and 93 bootstrap support in NJ andML resp) encompassing three clades H hoevenii (clade9) the H capitulum Sundaic clade (clade 10 including Hnemurus) and the H capitulum Indochinese clade (clade 11)(Figures 2 and 3) An additional three clades (Figures 2 and3) are formed by an unidentified species group (clade 6) Hspilopterus (clade 7) andH fortis (clade 8)These three latterclades are distinct at the congeneric species level (unspecifiedversus H spilopterus 119901 = 77 (07) unspecified versus Hfortis 119901 = 85 (07) and H spilopterus versus H fortis119901 = 75 (08)) The distribution of these three clades issomewhat disparate H fortis and the unspecified speciesgroup are principally found in Borneo whereasH spilopterusis widely distributed on the Indochinese mainland

mtDNA Clade 6 Unspecified Species Group This geneticallydiverse group encompassing at least 3 subclades (H spp AB and C Figure 7 within-group mean sequence divergence119901 = 68(06)) is confoundedwith theH baramensis speciesgroup byNampK It is composed of sequences fromfish sampledin Sarawak Sabah and eastern PeninsularMalaysia Subclade119867 spp A (Figure 7) is particularly interesting as it contains2 sequences (CHSARA20 CHRAJ0401) that characterize

Advances in Evolutionary Biology 11

110∘

XXPAD0303XXPAD0601

XXPAD0401XXBEL0202

XXBRM0202XXRAJ0511

XXPAD0501

XXEND0410CHSARA20CHRAJ0401

XXBEL0101XXSEG0101

100∘

10∘

0∘

120∘

0

0 250 500

250 500

750(km)

(mi)

91

86

83

99

98

47

81

001

91

72

99

80

H spp C

H spp B

H spp A

Shelf minus75mShelf minus120 m

Figure 7 Phylogenetic relationships of the individual sequences comprising the unspecified species group containing three subcladesidentified as H spp A B and C The subtree is extracted from the taxon identification phenogram inferred using the Neighbor-Joiningmethod (see Figure 2) The percentage of replicate trees in which the associated sequences clustered together in the bootstrap test (500replicates) is shown next to the branches The distribution of these sequences is illustrated in the right panel

H chrysops a new species identified by Ng and Dodson [11]However specimens of H chrysops from the Rajang Riverwere subsequently synonymized by NampK with H capitulumof the H nemurus species group (ZRC 23008ndash23014 TableS1) However an additional 14 fish from the Sadong Riverat Serian bearing genotype SARA20 (Table S1) and thusgenetically identical to the Rajang H chrysops samples wereidentified by NampK as H spilopterus (ZRC 29583ndash96 TableS1) These specimens however were erroneously identifiedin NampK as originating in the Chao Phraya River (Thailand)Subclade A also contains one sequence obtained from 10 fishsampled on the Endau River (Mersing) in eastern PeninsularMalaysia These fish were not morphologically examinedFinally subclade A also contains one sequence from oneunidentified fish sampled in the Segaliud River Sabah andanother unidentified fish sampled in the Belait River BruneiSubclade H spp B encompasses 2 sequences one of whichcomes from a fish sampled in the Padas River (XXPAD0501)and part of the ZRC 46122 fish lot identified as H semotusby NampK (Table S1) The second sequence (XXRAJ0511) wasshared by 11 unidentified fish sampled at Katibas on theRajang River Sarawak (Table S1) Finally subclade 119867 sppC encompasses 5 sequences Three sequences (XXPAD0303XXPAD0401 and XXPAD0601) were obtained from thePadas River and identified byNampKasH semotus (ZRC46122Table S1) The fourth sequence (XXBRM0202) was obtainedfrom 2 fish sampled in the Baram River and was identified

as H baramensis by NampK (ZRC 42742) The 5th sequencecomposing this group was obtained from 2 unidentified fishsampled in the Belait River Brunei (Table S1)

mtDNA clade 6 is thus problematical within the contextof the revision of the genus Hemibagrus Two highlydivergent mtDNA clades (clade 5 and clade 6 Figures 2and 3) occur in sympatry principally in the Padas River ofSabah North Borneo (Figures 6 and 7) Specimens of theunspecified species group are morphologically similar tothe H baramensis species group where they are assigned inthe analysis of NampK However the mean genetic distancebetween the unspecified species group and theH baramensisspecies group is 119901 = 193 (28) close to genus leveldivergence Furthermore subclade 119867 spp A (Figure 7)contains not only H chrysops but also an unidentifiedpopulation from eastern Peninsular Malaysia (XXEND0410Table S1) and two additional Sabah sequences On theother hand the unspecified species group clusters with allother members of the H nemurus species group (meangenetic divergence of unspecified group versus H capitulum(Sundaic clade) 119901 = 76 (07) (Figure 2)) with strongbootstrap support Such genetic clustering may justify theinclusion of H chrysops and associated sequences in theH nemurus species group but its synonymization with Hcapitulum (NampK) is difficult to defend on genetic groundsWe tentatively conclude that H chrysops is a valid speciesbased on genetic identity [11] forming part of an unknown

12 Advances in Evolutionary Biology

110∘

SPCPCHMK12

SPMEK0202SPMEK0401

SPMEK0301SPMEK0103

SPMEK1401SPMEK0501

SPMEK1201SPMEK1301

SPMEK1601SPMEK1502

SPMEK0701SPMEK0801SPMEK1701

SPCHP0501SPCHA0201

SPCHA0304SPPAT0101

100∘

10∘

0∘

120∘

0

0 250 500

250 500

750(km)

(mi)

000499

99 98

60

47

48

54

96

7091

38

86

87

99

80

58

H spilopterus ldquoCrdquo

H spilopterus ldquoBrdquo

H spilopterus ldquoArdquo

Shelf minus75mShelf minus120 m

Figure 8 Phylogenetic relationships within H spilopterus comprised of 3 subclades identified as A B and C The subtree is extracted fromthe taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicate trees in whichthe associated sequences clustered together in the bootstrap test (500 replicates) is shown next to the branches The distribution of thesesequences is illustrated in the right panel

species group found throughout North Borneo (and toa lesser degree in eastern Peninsular Malaysia) that maybe considered as part of the H nemurus species groupHowever the morphological confusion of the unspecifiedspecies group with the sympatric highly divergent Hbaramensis species group suggests the existence of a crypticspecies assemblage (see Section 4)

mtDNA Clade 7 Clade 7 (Figures 2 and 3) is composed of atleast 3 distinct subclades that appear to reflect 3 geneticallydivergent populations ofH spilopterus Subclade A (Figure 8)encompasses 7 sequences obtained from 8 fish sampled inthe Mekong River Four fish bearing 3 of these sequenceswere identified as paratypes (ZRC 43324) of H spilopterusby NampK (Table S1) One additional fish was identified asH spilopterus by NampK (ZRC 46157 Table S1) Subclade Bencompasses 6 sequences obtained from 9 fish all sampledfrom the Mekong River (Table S1) The fish sampled at ThatPhanom (ZRC 46012) was identified as H spilopterus inthe analysis of HampK (Table S1) Subclade C encompasses 5sequences obtained from 19 fish sampled in the Chao PhrayaMae Khlong and Chanthaburi Rivers Thailand Onesequence (CPCHMK12) was found in all of these drainages(Table S1) A closely allied sequence was found in one fishsampled at Phatthalung just south of the Isthmus of Kra onthe Thai-Malay Peninsula None of these fish were examined

by NampK and thus we are unable to definitively identifythe species However given the widespread occurrence ofH spilopterus in the Chao Phraya and Mae Klong Riversreported by NampK we conclude that these fish are part of awidespread H spilopterus population complex The meanwithin-group (clade 7) genetic divergence (119901 = 29 (04))suggests intraspecific population-level divergence

mtDNA Clade 8 Clade 8 encompasses 3 sequencesobtained from 9 fish sampled in east Sabah specifically theKinabatangan Segama and Umas Umas Rivers (Figure 9)The Kinabatangan sequence is somewhat divergent withinthe clade and the specimen was not identified by NampK Onesequence (SGM0606) characterized 6 fish from the SegamaRiver 2 of which were identified as H fortis (ZEC 4052140522 Table S1) by NampK (Table S1) Finally one fish bearinga closely related genotype from the Umas Umas River wasidentified as H baramensis by NampK (Table S1)

mtDNA Clade 9 Clade 9 is a sister group to the remaining2 clades composing the H nemurus species group Thiswell-defined clade (Figure 10) clusters 6 sequences from42 fish that are clearly identified as H hoevenii (NampK)Some of these haplotypes are geographically widespread onesequence (JOMSKA11 Table S1) was found in the Johor River(East Peninsular Malaysia) the Musi River (Sumatra) and

Advances in Evolutionary Biology 13

100∘ 110∘

0∘

120∘

00 250

250500

500750

(km)

(mi)

FOKIN0102FOSGM0606

FOUMA0201H fortis

10∘

99

86

001

Shelf minus75mShelf minus120 m

Figure 9 Phylogenetic relationships within H fortis comprised of 3 sequences The subtree is extracted from the taxon identificationphenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicate trees in which the associated sequencesclustered together in the bootstrap test (500 replicates) is shown next to the branches The distribution of these sequences is illustrated in theright panel

the Kapuas River (West Borneo) and a second sequence(HMUBHKABM23 Table S1) was found inMuarRiver (WestPeninsular Malaysia) and the Batang Hari (Sumatra) theKapuas (Sarawak) and the Baram Rivers (Sarawak) Many ofthese fish were identified as H hoevenii by NampK (Table S1)The existence of twodistinct but very closely related subclades(Figure 10 119901 = 06 (03)) that are not geographicallysegregated suggests some recent isolation event followed bywidespread dispersal and secondary contact

33 The H capitulum Group The final two monophyleticgroups of the Hemibagrus gene tree are approximatelyequivalent to the remaining species composing the Hnemurus species group as defined by NampK Dodson etal [10] and Ng and Dodson [11] have previously referredto clade 10 as the Sundaic clade as most sequences wereobtained from fish sampled on the Greater Sunda Islandsand clade 11 as the Indochinese clade asmany sequences wereobtained from fish sampled on the Indochinese mainland(Figure 2 Table S1) Although most of these sequencesare assigned to H capitulum by NampK there are somesignificant phylogeographic differences between the 2 cladesIn particular there appears to be an east-west division in thedistribution of the two clades on the Thai-Malay Peninsulawith the Sundaic clade (clade 10) found on the west coast andthe Indochinese clade found on the east coast with sympatricoccurrence in the Muar River Nevertheless mean sequencedivergence between the two clades is 119901 = 29 (05) avalue that is indicative of population-level or subspecies

divergence The two clades are thus best considered ascomprising one species

mtDNA Clade 10 The Sundaic clade includes a number ofclosely related sequences that comprise part of H capitulumand H nemurus Very little genetic structure is apparentand bootstrap support for tree topology within this group isgenerally very weak (Figure 11) A widely spread genotype(NCLCTBA20) characterized 6 of 17 specimens from CiTarum at the Cirata reservoir (West Java) (Table S1) and5 were identified as H nemurus in the analysis of NampK(ZRC 42564) (Table S1) The remaining fish characterized bythis sequence were obtained from Ci Liwong at Sawangan(West Java) and one fish was obtained from the Barito Riverat Banjarmassin south Borneo This latter specimen (ZRC40553) was identified by NampK as H capitulum (Table S1)Another common genotype (NBHKPBR09) was identified aseither H capitulum H hoevenii or H nemurus dependingon the river of origin (Table S1) Many of the remainingsequences of the Sundaic clade appear to characterize fish thatNampK identified asH capitulum A geographically widespreadgenotype (CPEBEMUKA36) characterized 9 fish sampledon the Perak River at Gerik that were identified by NampKas H capitulum (ZRC 41150 Table S1) Another commongenotype within this clade (CABHMS20) included 2 fishfrom the Batang Hari at Jambi and identified by NampK asH capitulum (ZRC40534 Table S1) Another 2 sequences(CABAR0203 CABAR0305) characterized 8 fish sampledon the Barito River at Banjarmasin and were identified as

14 Advances in Evolutionary Biology

100∘

10∘

110∘

0∘

120∘

0

0 250

250

500

500

750(km)

(mi)

53

64

99

85

64

0002

HJOMSKA11

HOBAH0601

HOBHMS05

HMUBHKABM23

HOBAH0701

HOKAP0501

H hoevenii ldquoBrdquo

H hoevenii ldquoArdquo

Shelf minus75mShelf minus120 m

Figure 10 Phylogenetic relationships withinH hoevenii comprised of 2 subclades and 6 sequences The subtree is extracted from the taxonidentification phenogram inferred using theNeighbor-Joiningmethod (see Figure 2)The percentage of replicate trees in which the associatedsequences clustered together in the bootstrap test (500 replicates) is shown next to the branches The distribution of these sequences isillustrated in the right panel

110∘

XXMAH0110

CABAR0305

96 CAKPR0201

74CAKAP0401

5931 CAKAP0601

35 CPEBEMUKA36

646 CAKAP0903

33 CAKAP080114 CAKAP0701

CARAJ0210

4 CABAR0203CABRA0204

20

CBHKPBR0919

CABRA0301

15CABHMS20

24 NCLCTBA20

63 NECIT0201

H capitulumldquoSundaic claderdquo

H nemurus

0002 120∘

0 250 500 750

(km)

0 250 500

(mi)100

10∘

0∘

Shelf minus75mShelf minus120 m

Figure 11 Phylogenetic relationships within theH capitulum ldquoSundaicrdquo clade including 2 sequences associated withH nemurusThe subtreeis extracted from the taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicatetrees in which the associated sequences clustered together in the bootstrap test (500 replicates) is shown next to the branchesThe distributionof these sequences is illustrated in the right panel

Advances in Evolutionary Biology 15

H capitulum

0∘

10∘

100∘

110∘

120∘

66

63

88

59

38

39 68

71

6991

57

9754

88 4944

39

53

89

0002

ldquoIndochineserdquogroup ldquoErdquo

H capitulumldquoIndochineserdquo

group ldquoDrdquoH capitulumldquoIndochineserdquo

group ldquoCrdquo

H capitulumldquoIndochineserdquo

group ldquoBrdquo

H capitulumldquoIndochineserdquo

group ldquoArdquo0

0

250

250

500

500

750

(km)

(mi)

CATER0301CATERR0401

CKETEMU23

CAMEK0601CACHP0301

CAMKPA03

CAMEK0910CAMEK1003

XXMUBM02

CABMTU09

CABRM0601

CAKAP1201CAKAP1001

CAKAP1101CAKAP1301

CABRM0501

CASAD0102

CASAD0220

CAEND0301

CABAH0801

CAPABH05CATEEN03

CATER0701CATERR0801

CAKETE04CATER0501

CAPAH0303

Shelf minus75mShelf minus120 m

Figure 12 Phylogenetic relationships within theH capitulum ldquoIndochineserdquo clade encompassing 5 subclades identified as A B C D and EThe subtree is extracted from the taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2)The percentageof replicate trees in which the associated sequences clustered together in the bootstrap test (500 replicates) is shown next to the branchesThedistribution of these sequences is illustrated in the right panel

H capitulum by NampK (ZRC 40553 Table S1) Although notincluded in the analysis of NampK samples from the Sundaicclade were also found in the Kapuas River (West Borneo)Finally 10 fish sampled on the Mahakam River at Samarindaeast Borneo are all characterized by one relatively divergentgenotype (CAMAH0110) butwere considered asH capitulumin the analysis of NampK (ZRC 40533 Table S1)TheMahakampopulation sample is the most genetically distinct of theSundaic clade (119901 = 17 (04)) but nevertheless indicativeof intraspecific population-level divergence (Figure 12)

The topology of the Sundaic clade does not permit usto draw a clear distinction between H nemurus and theSundaic H capitulum The mean within-group divergenceof the Sundaic clade is only 119901 = 08 (02) well withinpopulation-level divergence values We postulate that Hnemurus may be one of several regional variants of awidespread Sundaic H capitulum clade that has radiatedrelatively recently This may explain the existence of manyshared sequences throughout the Sundaic islands assigned todifferent species in different regions

mtDNA Clade 11 Finally the Indochinese clade of Hcapitulum represents a geographically widespread andweakly diversified clade (within-group mean sequencedivergence 119901 = 11 (02) Figure 12) Although few fishfrom this genetic clade were included in the morphological

analysis of NampK most may be considered as H capitulumgiven their geographic distribution and close geneticrelationship to the Sundaic H capitulum Four sequencesin this clade were identified as H capitulum in the analysisof NampK 3 specimens characterized by one sequence(CABMTU09) found in the Tutong River of westernBorneo (ZRC 40482) 6 characterized by the same sequencefrom the Baram River (ZRC 40498) another 2 sequencesfrom the Baram River (ZRC 40498) and 20 specimenscharacterized by an abundant genotype (CASAD0220)found at 2 sites in the Sadong River also in western Borneo(ZRC 39526 5 specimens 39527 3 specimens and 38753 4specimens) (Table S1) Unfortunately none of the specimenscollected on the Indochinese mainland for genetic analysiswere examined by NampK Nevertheless the distribution ofspecimens analyzed for mtDNA resembles the distribution ofspecimens examined by NampK Although weakly structuredwe may recognize 5 subclades One genotype (CAPAH0303)is basal to the phenogram Subclade A encompasses 8sequences mainly found in eastern Peninsular Malaysia(Table S1 Figure 12) These rivers furnished many of thespecimens NampK examined to define H capitulum Howeversome sequences in this group occur in sympatry withsequences identified within the Sundaic H capitulum groupTwo sequences from subclade A occurred in the Batang HariSumatra (Table S1) Subclade E also encompasses sequences

16 Advances in Evolutionary Biology

found in Peninsular Malaysia as well as in the Mekong ChaoPhraya and Mea Khlong Rivers (Figure 12) In addition3 subclades encompass sequences found principally inwestern Borneo subclade B in the Baram and Sadong RiversSarawak subclade C in the Kapuas River West Kalimantanand subclade D in the Baram and Tutong Rivers of Sarawakand Brunei respectively (Figure 12) This latter subclade alsoincludes one genotype found in the Muar River of westernpeninsular Malaysia Finally subclade E encompasses 5sequences from the Mekong Chao Praya and Mae KhlongRivers all within the distribution area of H spilopterus

EvolutionaryHistoryThe evolutionary history of theHemiba-grus species groups analyzed here dates from at least theMiocene 15ndash20Ma (Figure 13) when considering the widelyused molecular clock of 2 sequence divergence per millionyears Bootstrap support for the sister group relationships ofH wyckioides theH guttatus species group andH peguensisare all weak (clearly due to undersampling) and little can besaid of their evolutionary history other than the fact that theyoccupy basal positions in the rooted trees The remainder ofboth the NJ and ML trees is composed of two major sistergroups the H planiceps + H baramensis group and the Hnemurus species group (Figures 2 3 and 13)TheH planiceps+ H baramensis group is strongly supported in both NJ andML analyses (84 and 90 bootstrap support resp Figures2 and 3) The H nemurus species group is also stronglysupported in both analyses (98 and 99 bootstrap supportFigures 2 and 3) These two major phylogenetic groups areestimated to have diverged at least 8 to 12Ma ago during themid to late Miocene (Figure 13)

Much of the evolutionary history of Hemibagrus hasoccurred during the Quaternary starting approximately26Ma ago This is particularly true of the H planicepsand H nemurus species groups The radiation of the 5species included in the H planiceps species group appearsto coincide with the middle Pleistocene transition period ofapproximately 1Ma ago Their high-elevation distributionhowever appears to have largely isolated them from theeffects of lowered sea levels during glacial maxima and theattendant opportunities for widespread dispersal (Figure 5)The evolutionary histories of theH baramensis species groupand the unspecified species group date back to the latePliocene In contrast the low-elevation distribution of theHnemurus species group in Sundaland appears to have favoredtheir widespread distribution during the Quaternary ice ageIn particular H hoevenii appears to be the most recentlydispersed of the species under study with diversification anddispersal occurring approximately 03Ma ago (Figure 13)

4 Discussion

41 The Congruence of Morphological and Genetic AnalysesCombining genetic and morphological analyses to elucidatetaxonomy allowed us to better understand the diversificationand evolutionary relationships within this speciose group offishes Given the rapid and relatively constant rate of mtDNAevolution the deeper the branches within the phylogenetictree are the greater our ability to distinguish valid taxa

should be Conversely the lack of complete congruencebetween morphological- and genetic-based identificationsis expected to increase among the shallower branches of thephylogenetic tree resulting in the appearance of polyphyleticspecies Although 2 of the 8 species groups defined by Ngand Kottelat [7] were not sampled in this study the validityof the remaining 6 species groups and their componentspecies defined on the basis of morphological similaritywas largely confirmed by the analysis of the mitochondrialCyt 119887 bar code As expected congruence is strongest at thebase of the NJ and ML trees where the H guttatus speciesgroup formed a well-defined monophyletic group The Hmenoda and H wyckii species group were each representedby only one species but they were clearly distinct relativeto sequences characterizing the neighboring species groupsThe H planiceps H baramensis and H nemurus speciesgroups and their component species were also generallycongruent in morphological and genetic analyses Also asexpected most cases of discrepancy between morphological-based species identification and genetic-based speciesidentification occurred on relatively shallow branches of thephylogenetic tree These involve principally H spilopterusand the two mtDNA subclades composing H capitulumHowever a far more unexpected lack of congruence involvesthe definition of a deeply divergent unspecified genetic groupconfounding members of theH baramensis species group inthe morphological analysis of Ng and Kottelat [7]

Interpreting Polyphyly The apparent polyphyletic origin ofa species may be due to incomplete lineage sorting amongrecently diverged species the introgression of mtDNA dueto species hybridization or the erroneous identificationof species due to the lumping of morphologically crypticspecies In the absence of nuclear DNA analyses it isoften difficult to distinguish between introgression andincomplete lineage sorting For example several sequencescharacterizing the Indochinese clade of H capitulum(subclade E) are found in the Mekong and Chao PhrayaRivers well within the distributional area of H spilopterusH spilopterus and H capitulum first diverged at least 3MaOn the one hand this apparent case of polyphyly mayhave resulted from incomplete lineage sorting betweenrecently divergedH spilopterus andH capitulum We wouldhowever have expected similar cases of polyphyly amongpopulations of the Indochinese clade of H capitulum butthis was not detected On the other hand the sympatricoccurrence of H spilopterus subclade A and subcladeB sequences with H capitulum Indochinese subclade Esequences in the Mekong River may represent a migrationevent and local introgression of H capitulum with Hspilopterus A similar event may have occurred in theChao Phraya and Mae Khlong Rivers where H spilopterussequences cooccur withH capitulum sequences Computingthe divergence of H capitulum sequences (Indochinesesubclade E) cooccurring with H spilopterus and Hcapitulum sequences occurring in eastern PeninsularMalaysia (subclade A) reveals a mean sequence divergence119901 = 15 (04) Thus the H capitulum sequences occurringin sympatry with H spilopterus diverged at most 750 ka ago

Advances in Evolutionary Biology 17

H fortis

XXRAJ0604H furcatus

H sabanusH semotusH divaricatus

H planicepsGREND0206

VEBAH0907

H bonganPESAL0102

H wyckioidesH macropterus

H guttatusXXRAJ0301Leiocassis longirostris

Ma

4971

92

47

51

9999

99

9999

99

99

99

9999

99

99

99

99

65

96

96

4946

93

79

78

70

91

93

98

86

87

91

95

97

71

85

9999

05101520

005010015020 000

H capitulum Indochinese clades AndashE

H capitulum Sundaic clade

H hoevenii B

H hoevenii A

H spilopterus C

H spilopterus B

H spilopterus AH spilopterus A

H spp CH spp B

H spp A

Figure 13 Linearized tree with branch lengths estimated with the molecular clock using a mutation rate of 1 per Ma (=divergence rate of2 per Ma) The tree topology is identical to the ML reconstruction illustrated in Figure 3 (see the specificities of the tree reconstruction inthe caption of Figure 3) Boxes represent 95 confidence intervals of divergence time estimates for each node Vertical dashed lines redirectto dates indicated on the timescale (Ma) Numbers below timescale indicate the estimated number of substitution changes using Hasegawa-Kishino-Yanomodel (HKY)with gamma distribution (+119866 = 13290) after linearizing the treeH furcatus is synonymizedwithH baramensis

from eastern Peninsular Malaysia populations suggestinga recent introgression event Similarly H capitulum in theKapuas River is characterized by sequences of both theSundaic and the Indochinese clades As both sequences arebroadly sympatric it is not possible to distinguish betweenincomplete lineage sorting and introgression of these closelyrelated intraspecific lineages

A Possible Case of Morphological Stasis Clade 6 of thepresent analysis defines a well-supported monophyletic cladefound in Sabah Sarawak and the Endau River of easternpeninsula Malaysia that has no equivalent taxonomic statusin the revision of Ng and Kottelat [7] In Borneo they occursympatrically with the H baramensis species group and areconfounded as either H baramensis or H semotus These

two clades are genetically highly divergent having divergedbetween 8 and 12Ma ago It thus seems highly unlikely thatthey represent retained polymorphisms due to incompletelineage sorting Sequences previously identified as character-izing H chrysops [11] are part of clade 6 and extend to thesouth (Rajang and Sadong Rivers of Sarawak West Borneo)with one closely related genotype found in eastern peninsularMalaysia (Endau River) where it may well be confoundedwith H capitulum H chrysops was synonymized with Hcapitulum by Ng and Kottelat [7] based on their analysis ofspecimens ofH chrysops from the Rajang RiverThus despitea long and apparently independent phylogenetic historythe unspecified species group appears largely cryptic andconfounded principally with members of the H baramensisspecies group

18 Advances in Evolutionary Biology

We suggest that this situation is an example of mor-phological stasis involving the coexistence of a number ofgenetically divergent yet morphologically cryptic species thatare lumped together [24] A striking example of morpho-logical and ecological stasis among fishes is provided by thecoastal marine bonefishes (Albula spp) Distinct evolution-ary lineages (separated by sequence divergence between 5 and30 [25]) of bonefish coexist in several sample locations yetthey show little morphological or ecological differentiationin sympatry [25] Similarly a proportion of the grey mulletspecies (Mugilidae) also appears to consist of cryptic species[26] For exampleMugil cephalus in Taiwan is characterizedby three divergent lineages that have been shown to charac-terize 3 reproductively isolated but morphologically crypticspecies [26 27]

Here we have discovered 2 highly distinct and structuredclades (each composed of 3 mtDNA subclades) that aremorphologically confounded within different species Forexample H spp C of the unspecified species group isidentified as H baramensis in the Baram River and as Hsemotus in the Padas River We speculate that North Borneoharbors a morphologically cryptic species complex possiblycomposed of several species pairs inhabiting different riversystems The reconstruction of this phylogenetic history andthe taxonomic status of its components remains a majorchallenge to the revision of the Hemibagrus genus

42 Phylogeographic Hypotheses The phylogeography ofthe genus appears to have been principally influenced bytwo geological histories one encompassing the formationof North Borneo the second the emergence of the SundaIslands and their connectivity with the Indochinesemainlandassociated with recurrent sea-level changes North Borneoappears to have acted as a species sink promoting speciationand locally high biodiversity within the genus but with littleevidence of subsequent dispersal out of North Borneo Incontrast the geological events influencing the Indochinesemainland and the Sunda Islands have permitted bothspeciation and differential dispersal depending on thebiology of the species For example the H planiceps speciesgroup is found throughout the Greater Sunda Islands andtheThai-Malay Peninsula as is theH nemurus species groupWhereas the H nemurus species group generally occupiesthe low altitude lower reaches of large rivers the speciescomposing the H planiceps group are found in the upperreaches of large rivers The H nemurus species group iscomposed of a number of widely distributed closely relatedspecies whereas theH planiceps group is composed of a largenumber of species that are highly localized geographicallyIt thus appears that sea-level changes have had little impacton the dispersal history of the H planiceps species group buthave deeply influenced that of the H nemurus species group

The Case of North Borneo From 15Ma to 5Ma greater areasof Borneo became emergent and the central mountainson the Sarawak-Kalimantan border extending into Sabahbecame wider and higher which may have represented newbarriers to dispersal [28 29] Borneo gradually developedinto the present large island by emergence of land in the

north and east The H baramensis species group is restrictedto North Borneo as is the unspecified species group of theHnemurus species group H fortis is also distributed in NorthBorneo The isolation of the freshwater fish communities ofNorth Borneo (particularly in the Labuk-Segama watershed)has long been recognized based on the number of endemicspecies and the concentration of relict species [30] The latePliocene-early Pleistocene diversification of H fortis theunspecified species group and the H baramensis speciesgroup may thus be associated with their dispersal to andisolation within the watersheds of North Borneo Only onesequence of the unspecified species group is found outsideof North Borneo and H fortis appears to be distributedthroughout central and east Borneo [7]

ThePhylogeography of the H nemurus Species Group Periodicmarine transgressions on the Sundaland platform during theearly and middle Miocene [2 29] may have been sufficientto isolate populations of freshwater organisms and promotecladogenesis [31] This may have been responsible for theinitial mid to late Miocene divergence of the H nemurusspecies group The majority of sequences assigned to thisspecies group belong to 3 species (H spilopterus H capitulum(including H nemurus) and H hoevenii) all of which havebeen influenced by the more recent geological history of theSunda Islands and their connectivity with the Indochinesemainland H spilopterus is known from river drainages inIndochina including the Mekong Dong Nai Ban PakongChao Phraya and Mae Klong where it is found in a widevariety of lotic and lentic habitats [7] H spilopterus divergedin the late Pliocene-early Pleistocene possibly isolated tothe north of the Kra Peninsula during the late Pliocenemarine transgression that is purported to have created amajor biogeographic transition zone between the Sundaicand Indochinese biotas ([32] but see [33] for a critique ofthis biogeographic scenario) The existence of 2 subclades(A and B) in the Mekong River separated by 2 sequencedivergence is consistent with intraspecific divergence Thetwo subclades possibly represent 2 historical mtDNA lineagesthat are retained within the species reflecting a Pleistoceneisolation event followed by secondary contact within thecontemporary Mekong River Subclade C from the ChaoPhraya and Mae Khlong Rivers exhibits a level of geneticdivergence consistent with sibling species-level divergenceshowing a 5 sequence divergence relative to Mekong RiverH spilopterus

H capitulum may be considered as part of the Sundaicbiota located principally to the south of the Kra marinetransgression zone that may have isolated H spilopterus tothe north on the Indochinese mainlandThe biogeographicalevent leading to the subsequent formation of two intraspecificgroups within H capitulum is not at all clear The onlyarea where the two clades are clearly geographically separateis on the Thai-Malay Peninsula with the ldquoSundaicrdquo cladeon the west coast and the ldquoIndochineserdquo clade on theeast coast separated by the central Titiwangsa mountainrange The genetic distinctiveness of east and west coastpopulations of H capitulum has also been documented fornuclear DNA [34] A similar pattern of separation has been

Advances in Evolutionary Biology 19

reported for 5 additional freshwater fishes within this region[35] However as the divergence of the two H capitulumclades postdates the formation of the mountain range byapproximately 200Ma the geographic separation of the twoclades most likely represents differential dispersal rather thanancient vicariance The presence of the Sundaic clade on thewest coast of the Thai-Malay Peninsula as well as on theSundaic Islands reflects the historical connections betweeneast Sumatra and west Peninsular Malaysia across the Straitof Malacca This area encompasses the Northern CentralSumatra-Western Malaysia freshwater ecoregion [36] TheMuar River forms its southern border coincident with thesouthernmost distribution of the Sundaic clade on the Thai-Malay Peninsula It is important to note however that mostof Sumatra and Java emerged to achieve their present size andelevation only 5Ma ago and much of East Java continuedto be the site of marine deposition until late Pliocene-earlyPleistocene [29] We may speculate that the Sundaic clade ofH capitulum resulted from the relatively recent colonizationof these islands and Borneo The radiation within the cladeonly dates back to approximately 1Ma ago H nemurus fromWest Java may thus best be considered an isolated regionalpopulation of very recent origin

The diversification of the Indochinese clade dates fromat least 12Ma ago coincident with the mid-Pleistocenetransition at which time low-frequency high-amplitude andquasiperiodic (100 ka) glacial variability emerged It began125Ma ago and was complete by 07Ma ago [37] The tran-sition marked the beginning of major changes in sea levelsduring which the Sunda Shelf periodically emerged and wastraversed by enormous fluvial systems [3] The contributionof these events to the diversification of this clade cannotbe satisfactorily reconstructed One possible explanation isthat high sea levels produced barriers to gene flow and thepopulations of West Borneo (subclades B C and D) wereisolated from those of east Peninsular Malaysia This couldbe due to one or more dispersal events from Borneo via theGreat Sunda River (Figure 1) to east Peninsular Malaysia orlineage sorting whereby sequences of subclades A and E havebeen lost from West Borneo As in the case of the sympatricoccurrence of H spilopterus and H capitulum subclade E(see above) in the Mekong River we are unable to clearlydefine the relative roles of incomplete lineage sorting andintrogression followingmigration Given the recent origins ofthe Indochinese H capitulum sequences incomplete lineagesorting most probably blurs patterns of recent isolation

The last species of the H nemurus species group is Hhoevenii The species is widespread throughout PeninsularMalaysia and the Sundaic Islands It is found primarily inthe lower reaches of rivers and is apparently capable ofwithstanding low salt concentration [7] H hoevenii thusappears to be the most liable of all members of the Hnemurus species group to be impacted by Pleistocene sea-level changes This is clearly reflected in the widespreaddistribution of the sequences of this species Two lineages areevident having diverged no more than 03Ma ago suggest-ing a late Pleistocene isolation event followed by extensivedispersal and admixture in recent times This represents theclearest example of the dispersal opportunities afforded by

Pleistocene sea level changes on the Sunda Shelf In additionthe relatively low genetic diversity detected (only 6 sequencesin 42 fish) suggests a population bottleneck in the recent pastpossibly related to population declines in the lower reaches ofrivers flooded by increasing sea levels

43 Future Directions We have provided a global picture ofthe evolutionary taxonomy of the genus in Southeast AsiaClearly a more rigorous phylogenetic reconstruction of thegenus Hemibagrus requiring the construction of multiplegene trees based on the analysis of both mitochondrial andnuclear DNA markers from a wider taxonomic array ofhemibagrids will involve an even greater effort of samplingthroughout the region In particular we require samplingat smaller spatial and temporal scales For example smallerscale sampling in the Padas River and the Segama Riverof North Borneo using both mitochondrial and nucleargenetic markers would contribute to clarifying the natureof the cryptic species complex believed to inhabit theserivers The demonstration of reproductive isolation andormicroallopatric distribution among morphologically crypticspecies may result in the description of greater speciesrichness within the genus than presently described Thespecies status of H chrysops merits reconsideration givenits highly divergent evolutionary history Nuclear geneticmarkers in conjunction with mtDNA markers could revealcases of historical introgression particularly in the KapuasRiver and the Mekong River The hemibagrid catfishes con-tinue to provide multiple opportunities for the study of theevolutionary taxonomy and biogeography of a major faunalcomponent of the fluvial ecosystems of Southeast Asia

Disclosure

Ethanol-preserved tissue samples of many of the specimensidentified in Table S1 are freely available upon request fromJJD

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Goh Yan Yih for assistance in collectingspecimens and molecular analyses Mrs Francoise Colom-bani for molecular analyses and Dr Heok Hee Ng forspecies identification This work was funded by the NationalUniversity of Singapore grant to P K L Ng and a CanadianNatural Sciences and Engineering Research Council grant toJ J Dodson

References

[1] D J Lohman M de Bruyn T Page et al ldquoBiogeography ofthe Indo-Australian archipelagordquo Annual Review of EcologyEvolution and Systematics vol 42 pp 205ndash226 2011

20 Advances in Evolutionary Biology

[2] D S Woodruff ldquoNeogene marine transgressions palaeogeog-raphy and biogeographic transitions on the Thai-Malay Penin-sulardquo Journal of Biogeography vol 30 no 4 pp 551ndash567 2003

[3] H K Voris ldquoMaps of Pleistocene sea levels in SoutheastAsia shorelines river systems and time durationsrdquo Journal ofBiogeography vol 27 no 5 pp 1153ndash1167 2000

[4] S Y Yap ldquoOn the distributional patterns of Southeast-EastAsian freshwater fish and their historyrdquo Journal of Biogeographyvol 29 no 9 pp 1187ndash1199 2002

[5] S K J McConnell ldquoMapping aquatic faunal exchanges acrossthe Sunda shelf South-East Asia using distributional andgenetic data sets from the cyprinid fish Barbodes gonionotus(Bleeker 1850)rdquo Journal of Natural History vol 38 no 5 pp651ndash670 2004

[6] T Mo Anatomy Relationships and Systematics of the Bagridae(Teleostei Siluroidei) with a Hypothesis of Siluroid Phylogenyvol 17 ofTheses Zoologicae Koeltz Scientific Books 1991

[7] H H Ng and M Kottelat ldquoRevision of the Asian catfish genusHemibagrus bleeker 1862 (Teleostei Siluriformes Bagridae)rdquoRaffles Bulletin of Zoology vol 61 no 1 pp 205ndash291 2013

[8] P K L Ng and H H Ng ldquoHemibagrus gracilis a new speciesof large riverine catfish (Teleostei Bagridae) from PeninsularMalaysiardquo Raffles Bulletin of Zoology vol 43 pp 133ndash142 1995

[9] H H Tan and M Kottelat ldquoRedescription of Betta picta(Teleostei Osphronemidae) and description of Betta falx sp nfrom central Sumatrardquo Revue Suisse de Zoologie vol 105 no 3pp 557ndash568 1998

[10] J J Dodson F Colombani and P K Ng ldquoPhylogeographicstructure in mitochondrial DNA of a South-east Asian fresh-water fish Hemibagrus nemurus (Siluroidei Bagridae) andPleistocene sea-level changes on the Sunda shelfrdquo MolecularEcology vol 4 no 3 pp 331ndash346 1995

[11] H H Ng and J J Dodson ldquoMorphological and genetic descrip-tions of a new species of catfish Hemibagrus chrysops fromSarawak East Malaysia with an assessment of phylogeneticrelationships (Teleostei Bagridae)rdquo Raffles Bulletin of Zoologyvol 47 no 1 pp 45ndash57 1999

[12] P D N Hebert A Cywinska S L Ball and J R DeWaardldquoBiological identifications through DNA barcodesrdquo Proceedingsof the Royal Society B Biological Sciences vol 270 no 1512 pp313ndash321 2003

[13] J C Carolan T EMurray U Fitzpatrick et al ldquoColour patternsdo not diagnose species quantitative evaluation of a DNAbarcoded cryptic bumblebee complexrdquo PLoS ONE vol 7 no 1Article ID e29251 2012

[14] M Kekkonen and P D N Hebert ldquoDNA barcode-baseddelineation of putative species efficient start for taxonomicworkflowsrdquoMolecular Ecology Resources vol 14 no 4 pp 706ndash715 2014

[15] DM Hillis CMoritz and B KMarbleMolecular SystematicsSinauer Sunderland Mass USA 2nd edition 1996

[16] T D Kocher W K Thomas A Meyer et al ldquoDynamics ofmitochondrial DNA evolution in animals amplification andsequencingwith conserved primersrdquoProceedings of theNationalAcademy of Sciences of the United States of America vol 86 no16 pp 6196ndash6200 1989

[17] T A Hall ldquoBioEdit a user-friendly biological sequence align-ment editor and analysis program for Windows 9598NTrdquoNucleic Acids Symposium Series vol 41 pp 95ndash98 1999

[18] D A Benson I Karsch-Mizrachi K Clark D J Lipman JOstell and EW Sayers ldquoGenBankrdquoNucleic Acids Research vol40 no D1 pp D48ndashD53 2012

[19] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[20] X Ku Z Peng R Diogo and S He ldquoMtDNA phylogenyprovides evidence of generic polyphyleticism for East Asianbagrid catfishesrdquoHydrobiologia vol 579 no 1 pp 147ndash159 2007

[21] L Yang and S He ldquoPhylogeography of the freshwater catfishHemibagrus guttatus (Siluriformes Bagridae) implications forSouth China biogeography and influence of sea-level changesrdquoMolecular Phylogenetics and Evolution vol 49 no 1 pp 393ndash398 2008

[22] B W Bowen A L Bass L A Rocha W S Grant and DR Robertson ldquoPhylogeography of the trumpetfishes (Aulosto-mus) ring species complex on a global scalerdquo Evolution vol 55no 5 pp 1029ndash1039 2001

[23] Y P Kartavtsev ldquoDivergence at Cyt-b and Co-1 mtDNA geneson different taxonomic levels and genetics of speciation inanimalsrdquoMitochondrial DNA vol 22 no 3 pp 55ndash65 2011

[24] SWada Y Kameda and S Chiba ldquoLong-term stasis and short-term divergence in the phenotypes of microsnails on oceanicislandsrdquoMolecular Ecology vol 22 no 18 pp 4801ndash4810 2013

[25] J Colborn R E Crabtree J B Shaklee E Pfeiler and B WBowen ldquoThe evolutionary enigma of bonefishes (Albula spp)cryptic species and ancient separations in a globally distributedshorefishrdquo Evolution vol 55 no 4 pp 807ndash820 2001

[26] J-D Durand K-N Shen W-J Chen et al ldquoSystematics of thegrey mullets (Teleostei Mugiliformes Mugilidae) molecularphylogenetic evidence challenges two centuries ofmorphology-based taxonomyrdquo Molecular Phylogenetics and Evolution vol64 no 1 pp 73ndash92 2012

[27] K-N Shen B W Jamandre C-C Hsu W-N Tzeng and J-D Durand ldquoPlio-Pleistocene sea level and temperature fluc-tuations in the northwestern Pacific promoted speciation inthe globally-distributed flathead mullet Mugil cephalusrdquo BMCEvolutionary Biology vol 11 no 1 article 83 2011

[28] R Hall ldquoThe plate tectonics of Cenozoic SE Asia and thedistribution of land and seardquo in Biogeography and GeologicalEvolution of SE Asia R Hall and J D Holloway Eds pp 99ndash131Backhuys Publishers Leiden The Netherlands 1998

[29] R Hall ldquoSoutheast Asiarsquos changing palaeogeographyrdquo Blumeavol 54 no 1ndash3 pp 148ndash161 2009

[30] R F Inger and P K Chen ldquoThe freshwater fishes of NorthBorneordquo Fieldiana Zoology vol 45 pp 1ndash268 1962

[31] J Bohlen V Slechtova H H Tan and R Britz ldquoPhylogenyof the Southeast Asian freshwater fish genus Pangio (Cyprini-formes Cobitidae)rdquoMolecular Phylogenetics and Evolution vol61 no 3 pp 854ndash865 2011

[32] M de Bruyn E Nugroho M M Hossain J C Wilson andP B Mather ldquoPhylogeographic evidence for the existence ofan ancient biogeographic barrier the Isthmus of Kra SeawayrdquoHeredity vol 94 no 3 pp 370ndash378 2005

[33] J Parnell ldquoThe biogeography of the Isthmus of Kra region areviewrdquoNordic Journal of Botany vol 31 no 1 pp 001ndash015 2013

[34] S Usmani S G Tan S S Siraj and K Yusoff ldquoPopulationstructure of the Southeast Asian river catfishMystus nemurusrdquoAnimal Genetics vol 34 no 6 pp 462ndash464 2003

[35] M P Tan A F J Jamsari and M N Siti Azizah ldquoPhylo-geographic pattern of the striped snakehead Channa striatain Sundaland ancient river connectivity geographical and

Advances in Evolutionary Biology 21

anthropogenic singnaturesrdquo PLoS ONE vol 7 no 12 Article IDe52089 2012

[36] R Abell M L Thieme C Revenga et al ldquoFreshwater ecore-gions of the world a new map of biogeographic units forfreshwater biodiversity conservationrdquo BioScience vol 58 no 5pp 403ndash414 2008

[37] P U Clark D Archer D Pollard et al ldquoThemiddle Pleistocenetransition characteristics mechanisms and implications forlong-term changes in atmospheric pCO

2rdquo Quaternary Science

Reviews vol 25 no 23-24 pp 3150ndash3184 2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

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Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Advances in

Virolog y

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Nucleic AcidsJournal of

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Stem CellsInternational

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Enzyme Research

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International Journal of

Microbiology

Page 11: ResearchArticle - Université Laval Julian... · ResearchArticle A DNA Barcode-Based Evaluation of the Southeast Asian Catfish Genus Hemibagrus Bleeker, 1862 (Teleostei: Siluriformes;

Advances in Evolutionary Biology 11

110∘

XXPAD0303XXPAD0601

XXPAD0401XXBEL0202

XXBRM0202XXRAJ0511

XXPAD0501

XXEND0410CHSARA20CHRAJ0401

XXBEL0101XXSEG0101

100∘

10∘

0∘

120∘

0

0 250 500

250 500

750(km)

(mi)

91

86

83

99

98

47

81

001

91

72

99

80

H spp C

H spp B

H spp A

Shelf minus75mShelf minus120 m

Figure 7 Phylogenetic relationships of the individual sequences comprising the unspecified species group containing three subcladesidentified as H spp A B and C The subtree is extracted from the taxon identification phenogram inferred using the Neighbor-Joiningmethod (see Figure 2) The percentage of replicate trees in which the associated sequences clustered together in the bootstrap test (500replicates) is shown next to the branches The distribution of these sequences is illustrated in the right panel

H chrysops a new species identified by Ng and Dodson [11]However specimens of H chrysops from the Rajang Riverwere subsequently synonymized by NampK with H capitulumof the H nemurus species group (ZRC 23008ndash23014 TableS1) However an additional 14 fish from the Sadong Riverat Serian bearing genotype SARA20 (Table S1) and thusgenetically identical to the Rajang H chrysops samples wereidentified by NampK as H spilopterus (ZRC 29583ndash96 TableS1) These specimens however were erroneously identifiedin NampK as originating in the Chao Phraya River (Thailand)Subclade A also contains one sequence obtained from 10 fishsampled on the Endau River (Mersing) in eastern PeninsularMalaysia These fish were not morphologically examinedFinally subclade A also contains one sequence from oneunidentified fish sampled in the Segaliud River Sabah andanother unidentified fish sampled in the Belait River BruneiSubclade H spp B encompasses 2 sequences one of whichcomes from a fish sampled in the Padas River (XXPAD0501)and part of the ZRC 46122 fish lot identified as H semotusby NampK (Table S1) The second sequence (XXRAJ0511) wasshared by 11 unidentified fish sampled at Katibas on theRajang River Sarawak (Table S1) Finally subclade 119867 sppC encompasses 5 sequences Three sequences (XXPAD0303XXPAD0401 and XXPAD0601) were obtained from thePadas River and identified byNampKasH semotus (ZRC46122Table S1) The fourth sequence (XXBRM0202) was obtainedfrom 2 fish sampled in the Baram River and was identified

as H baramensis by NampK (ZRC 42742) The 5th sequencecomposing this group was obtained from 2 unidentified fishsampled in the Belait River Brunei (Table S1)

mtDNA clade 6 is thus problematical within the contextof the revision of the genus Hemibagrus Two highlydivergent mtDNA clades (clade 5 and clade 6 Figures 2and 3) occur in sympatry principally in the Padas River ofSabah North Borneo (Figures 6 and 7) Specimens of theunspecified species group are morphologically similar tothe H baramensis species group where they are assigned inthe analysis of NampK However the mean genetic distancebetween the unspecified species group and theH baramensisspecies group is 119901 = 193 (28) close to genus leveldivergence Furthermore subclade 119867 spp A (Figure 7)contains not only H chrysops but also an unidentifiedpopulation from eastern Peninsular Malaysia (XXEND0410Table S1) and two additional Sabah sequences On theother hand the unspecified species group clusters with allother members of the H nemurus species group (meangenetic divergence of unspecified group versus H capitulum(Sundaic clade) 119901 = 76 (07) (Figure 2)) with strongbootstrap support Such genetic clustering may justify theinclusion of H chrysops and associated sequences in theH nemurus species group but its synonymization with Hcapitulum (NampK) is difficult to defend on genetic groundsWe tentatively conclude that H chrysops is a valid speciesbased on genetic identity [11] forming part of an unknown

12 Advances in Evolutionary Biology

110∘

SPCPCHMK12

SPMEK0202SPMEK0401

SPMEK0301SPMEK0103

SPMEK1401SPMEK0501

SPMEK1201SPMEK1301

SPMEK1601SPMEK1502

SPMEK0701SPMEK0801SPMEK1701

SPCHP0501SPCHA0201

SPCHA0304SPPAT0101

100∘

10∘

0∘

120∘

0

0 250 500

250 500

750(km)

(mi)

000499

99 98

60

47

48

54

96

7091

38

86

87

99

80

58

H spilopterus ldquoCrdquo

H spilopterus ldquoBrdquo

H spilopterus ldquoArdquo

Shelf minus75mShelf minus120 m

Figure 8 Phylogenetic relationships within H spilopterus comprised of 3 subclades identified as A B and C The subtree is extracted fromthe taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicate trees in whichthe associated sequences clustered together in the bootstrap test (500 replicates) is shown next to the branches The distribution of thesesequences is illustrated in the right panel

species group found throughout North Borneo (and toa lesser degree in eastern Peninsular Malaysia) that maybe considered as part of the H nemurus species groupHowever the morphological confusion of the unspecifiedspecies group with the sympatric highly divergent Hbaramensis species group suggests the existence of a crypticspecies assemblage (see Section 4)

mtDNA Clade 7 Clade 7 (Figures 2 and 3) is composed of atleast 3 distinct subclades that appear to reflect 3 geneticallydivergent populations ofH spilopterus Subclade A (Figure 8)encompasses 7 sequences obtained from 8 fish sampled inthe Mekong River Four fish bearing 3 of these sequenceswere identified as paratypes (ZRC 43324) of H spilopterusby NampK (Table S1) One additional fish was identified asH spilopterus by NampK (ZRC 46157 Table S1) Subclade Bencompasses 6 sequences obtained from 9 fish all sampledfrom the Mekong River (Table S1) The fish sampled at ThatPhanom (ZRC 46012) was identified as H spilopterus inthe analysis of HampK (Table S1) Subclade C encompasses 5sequences obtained from 19 fish sampled in the Chao PhrayaMae Khlong and Chanthaburi Rivers Thailand Onesequence (CPCHMK12) was found in all of these drainages(Table S1) A closely allied sequence was found in one fishsampled at Phatthalung just south of the Isthmus of Kra onthe Thai-Malay Peninsula None of these fish were examined

by NampK and thus we are unable to definitively identifythe species However given the widespread occurrence ofH spilopterus in the Chao Phraya and Mae Klong Riversreported by NampK we conclude that these fish are part of awidespread H spilopterus population complex The meanwithin-group (clade 7) genetic divergence (119901 = 29 (04))suggests intraspecific population-level divergence

mtDNA Clade 8 Clade 8 encompasses 3 sequencesobtained from 9 fish sampled in east Sabah specifically theKinabatangan Segama and Umas Umas Rivers (Figure 9)The Kinabatangan sequence is somewhat divergent withinthe clade and the specimen was not identified by NampK Onesequence (SGM0606) characterized 6 fish from the SegamaRiver 2 of which were identified as H fortis (ZEC 4052140522 Table S1) by NampK (Table S1) Finally one fish bearinga closely related genotype from the Umas Umas River wasidentified as H baramensis by NampK (Table S1)

mtDNA Clade 9 Clade 9 is a sister group to the remaining2 clades composing the H nemurus species group Thiswell-defined clade (Figure 10) clusters 6 sequences from42 fish that are clearly identified as H hoevenii (NampK)Some of these haplotypes are geographically widespread onesequence (JOMSKA11 Table S1) was found in the Johor River(East Peninsular Malaysia) the Musi River (Sumatra) and

Advances in Evolutionary Biology 13

100∘ 110∘

0∘

120∘

00 250

250500

500750

(km)

(mi)

FOKIN0102FOSGM0606

FOUMA0201H fortis

10∘

99

86

001

Shelf minus75mShelf minus120 m

Figure 9 Phylogenetic relationships within H fortis comprised of 3 sequences The subtree is extracted from the taxon identificationphenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicate trees in which the associated sequencesclustered together in the bootstrap test (500 replicates) is shown next to the branches The distribution of these sequences is illustrated in theright panel

the Kapuas River (West Borneo) and a second sequence(HMUBHKABM23 Table S1) was found inMuarRiver (WestPeninsular Malaysia) and the Batang Hari (Sumatra) theKapuas (Sarawak) and the Baram Rivers (Sarawak) Many ofthese fish were identified as H hoevenii by NampK (Table S1)The existence of twodistinct but very closely related subclades(Figure 10 119901 = 06 (03)) that are not geographicallysegregated suggests some recent isolation event followed bywidespread dispersal and secondary contact

33 The H capitulum Group The final two monophyleticgroups of the Hemibagrus gene tree are approximatelyequivalent to the remaining species composing the Hnemurus species group as defined by NampK Dodson etal [10] and Ng and Dodson [11] have previously referredto clade 10 as the Sundaic clade as most sequences wereobtained from fish sampled on the Greater Sunda Islandsand clade 11 as the Indochinese clade asmany sequences wereobtained from fish sampled on the Indochinese mainland(Figure 2 Table S1) Although most of these sequencesare assigned to H capitulum by NampK there are somesignificant phylogeographic differences between the 2 cladesIn particular there appears to be an east-west division in thedistribution of the two clades on the Thai-Malay Peninsulawith the Sundaic clade (clade 10) found on the west coast andthe Indochinese clade found on the east coast with sympatricoccurrence in the Muar River Nevertheless mean sequencedivergence between the two clades is 119901 = 29 (05) avalue that is indicative of population-level or subspecies

divergence The two clades are thus best considered ascomprising one species

mtDNA Clade 10 The Sundaic clade includes a number ofclosely related sequences that comprise part of H capitulumand H nemurus Very little genetic structure is apparentand bootstrap support for tree topology within this group isgenerally very weak (Figure 11) A widely spread genotype(NCLCTBA20) characterized 6 of 17 specimens from CiTarum at the Cirata reservoir (West Java) (Table S1) and5 were identified as H nemurus in the analysis of NampK(ZRC 42564) (Table S1) The remaining fish characterized bythis sequence were obtained from Ci Liwong at Sawangan(West Java) and one fish was obtained from the Barito Riverat Banjarmassin south Borneo This latter specimen (ZRC40553) was identified by NampK as H capitulum (Table S1)Another common genotype (NBHKPBR09) was identified aseither H capitulum H hoevenii or H nemurus dependingon the river of origin (Table S1) Many of the remainingsequences of the Sundaic clade appear to characterize fish thatNampK identified asH capitulum A geographically widespreadgenotype (CPEBEMUKA36) characterized 9 fish sampledon the Perak River at Gerik that were identified by NampKas H capitulum (ZRC 41150 Table S1) Another commongenotype within this clade (CABHMS20) included 2 fishfrom the Batang Hari at Jambi and identified by NampK asH capitulum (ZRC40534 Table S1) Another 2 sequences(CABAR0203 CABAR0305) characterized 8 fish sampledon the Barito River at Banjarmasin and were identified as

14 Advances in Evolutionary Biology

100∘

10∘

110∘

0∘

120∘

0

0 250

250

500

500

750(km)

(mi)

53

64

99

85

64

0002

HJOMSKA11

HOBAH0601

HOBHMS05

HMUBHKABM23

HOBAH0701

HOKAP0501

H hoevenii ldquoBrdquo

H hoevenii ldquoArdquo

Shelf minus75mShelf minus120 m

Figure 10 Phylogenetic relationships withinH hoevenii comprised of 2 subclades and 6 sequences The subtree is extracted from the taxonidentification phenogram inferred using theNeighbor-Joiningmethod (see Figure 2)The percentage of replicate trees in which the associatedsequences clustered together in the bootstrap test (500 replicates) is shown next to the branches The distribution of these sequences isillustrated in the right panel

110∘

XXMAH0110

CABAR0305

96 CAKPR0201

74CAKAP0401

5931 CAKAP0601

35 CPEBEMUKA36

646 CAKAP0903

33 CAKAP080114 CAKAP0701

CARAJ0210

4 CABAR0203CABRA0204

20

CBHKPBR0919

CABRA0301

15CABHMS20

24 NCLCTBA20

63 NECIT0201

H capitulumldquoSundaic claderdquo

H nemurus

0002 120∘

0 250 500 750

(km)

0 250 500

(mi)100

10∘

0∘

Shelf minus75mShelf minus120 m

Figure 11 Phylogenetic relationships within theH capitulum ldquoSundaicrdquo clade including 2 sequences associated withH nemurusThe subtreeis extracted from the taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicatetrees in which the associated sequences clustered together in the bootstrap test (500 replicates) is shown next to the branchesThe distributionof these sequences is illustrated in the right panel

Advances in Evolutionary Biology 15

H capitulum

0∘

10∘

100∘

110∘

120∘

66

63

88

59

38

39 68

71

6991

57

9754

88 4944

39

53

89

0002

ldquoIndochineserdquogroup ldquoErdquo

H capitulumldquoIndochineserdquo

group ldquoDrdquoH capitulumldquoIndochineserdquo

group ldquoCrdquo

H capitulumldquoIndochineserdquo

group ldquoBrdquo

H capitulumldquoIndochineserdquo

group ldquoArdquo0

0

250

250

500

500

750

(km)

(mi)

CATER0301CATERR0401

CKETEMU23

CAMEK0601CACHP0301

CAMKPA03

CAMEK0910CAMEK1003

XXMUBM02

CABMTU09

CABRM0601

CAKAP1201CAKAP1001

CAKAP1101CAKAP1301

CABRM0501

CASAD0102

CASAD0220

CAEND0301

CABAH0801

CAPABH05CATEEN03

CATER0701CATERR0801

CAKETE04CATER0501

CAPAH0303

Shelf minus75mShelf minus120 m

Figure 12 Phylogenetic relationships within theH capitulum ldquoIndochineserdquo clade encompassing 5 subclades identified as A B C D and EThe subtree is extracted from the taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2)The percentageof replicate trees in which the associated sequences clustered together in the bootstrap test (500 replicates) is shown next to the branchesThedistribution of these sequences is illustrated in the right panel

H capitulum by NampK (ZRC 40553 Table S1) Although notincluded in the analysis of NampK samples from the Sundaicclade were also found in the Kapuas River (West Borneo)Finally 10 fish sampled on the Mahakam River at Samarindaeast Borneo are all characterized by one relatively divergentgenotype (CAMAH0110) butwere considered asH capitulumin the analysis of NampK (ZRC 40533 Table S1)TheMahakampopulation sample is the most genetically distinct of theSundaic clade (119901 = 17 (04)) but nevertheless indicativeof intraspecific population-level divergence (Figure 12)

The topology of the Sundaic clade does not permit usto draw a clear distinction between H nemurus and theSundaic H capitulum The mean within-group divergenceof the Sundaic clade is only 119901 = 08 (02) well withinpopulation-level divergence values We postulate that Hnemurus may be one of several regional variants of awidespread Sundaic H capitulum clade that has radiatedrelatively recently This may explain the existence of manyshared sequences throughout the Sundaic islands assigned todifferent species in different regions

mtDNA Clade 11 Finally the Indochinese clade of Hcapitulum represents a geographically widespread andweakly diversified clade (within-group mean sequencedivergence 119901 = 11 (02) Figure 12) Although few fishfrom this genetic clade were included in the morphological

analysis of NampK most may be considered as H capitulumgiven their geographic distribution and close geneticrelationship to the Sundaic H capitulum Four sequencesin this clade were identified as H capitulum in the analysisof NampK 3 specimens characterized by one sequence(CABMTU09) found in the Tutong River of westernBorneo (ZRC 40482) 6 characterized by the same sequencefrom the Baram River (ZRC 40498) another 2 sequencesfrom the Baram River (ZRC 40498) and 20 specimenscharacterized by an abundant genotype (CASAD0220)found at 2 sites in the Sadong River also in western Borneo(ZRC 39526 5 specimens 39527 3 specimens and 38753 4specimens) (Table S1) Unfortunately none of the specimenscollected on the Indochinese mainland for genetic analysiswere examined by NampK Nevertheless the distribution ofspecimens analyzed for mtDNA resembles the distribution ofspecimens examined by NampK Although weakly structuredwe may recognize 5 subclades One genotype (CAPAH0303)is basal to the phenogram Subclade A encompasses 8sequences mainly found in eastern Peninsular Malaysia(Table S1 Figure 12) These rivers furnished many of thespecimens NampK examined to define H capitulum Howeversome sequences in this group occur in sympatry withsequences identified within the Sundaic H capitulum groupTwo sequences from subclade A occurred in the Batang HariSumatra (Table S1) Subclade E also encompasses sequences

16 Advances in Evolutionary Biology

found in Peninsular Malaysia as well as in the Mekong ChaoPhraya and Mea Khlong Rivers (Figure 12) In addition3 subclades encompass sequences found principally inwestern Borneo subclade B in the Baram and Sadong RiversSarawak subclade C in the Kapuas River West Kalimantanand subclade D in the Baram and Tutong Rivers of Sarawakand Brunei respectively (Figure 12) This latter subclade alsoincludes one genotype found in the Muar River of westernpeninsular Malaysia Finally subclade E encompasses 5sequences from the Mekong Chao Praya and Mae KhlongRivers all within the distribution area of H spilopterus

EvolutionaryHistoryThe evolutionary history of theHemiba-grus species groups analyzed here dates from at least theMiocene 15ndash20Ma (Figure 13) when considering the widelyused molecular clock of 2 sequence divergence per millionyears Bootstrap support for the sister group relationships ofH wyckioides theH guttatus species group andH peguensisare all weak (clearly due to undersampling) and little can besaid of their evolutionary history other than the fact that theyoccupy basal positions in the rooted trees The remainder ofboth the NJ and ML trees is composed of two major sistergroups the H planiceps + H baramensis group and the Hnemurus species group (Figures 2 3 and 13)TheH planiceps+ H baramensis group is strongly supported in both NJ andML analyses (84 and 90 bootstrap support resp Figures2 and 3) The H nemurus species group is also stronglysupported in both analyses (98 and 99 bootstrap supportFigures 2 and 3) These two major phylogenetic groups areestimated to have diverged at least 8 to 12Ma ago during themid to late Miocene (Figure 13)

Much of the evolutionary history of Hemibagrus hasoccurred during the Quaternary starting approximately26Ma ago This is particularly true of the H planicepsand H nemurus species groups The radiation of the 5species included in the H planiceps species group appearsto coincide with the middle Pleistocene transition period ofapproximately 1Ma ago Their high-elevation distributionhowever appears to have largely isolated them from theeffects of lowered sea levels during glacial maxima and theattendant opportunities for widespread dispersal (Figure 5)The evolutionary histories of theH baramensis species groupand the unspecified species group date back to the latePliocene In contrast the low-elevation distribution of theHnemurus species group in Sundaland appears to have favoredtheir widespread distribution during the Quaternary ice ageIn particular H hoevenii appears to be the most recentlydispersed of the species under study with diversification anddispersal occurring approximately 03Ma ago (Figure 13)

4 Discussion

41 The Congruence of Morphological and Genetic AnalysesCombining genetic and morphological analyses to elucidatetaxonomy allowed us to better understand the diversificationand evolutionary relationships within this speciose group offishes Given the rapid and relatively constant rate of mtDNAevolution the deeper the branches within the phylogenetictree are the greater our ability to distinguish valid taxa

should be Conversely the lack of complete congruencebetween morphological- and genetic-based identificationsis expected to increase among the shallower branches of thephylogenetic tree resulting in the appearance of polyphyleticspecies Although 2 of the 8 species groups defined by Ngand Kottelat [7] were not sampled in this study the validityof the remaining 6 species groups and their componentspecies defined on the basis of morphological similaritywas largely confirmed by the analysis of the mitochondrialCyt 119887 bar code As expected congruence is strongest at thebase of the NJ and ML trees where the H guttatus speciesgroup formed a well-defined monophyletic group The Hmenoda and H wyckii species group were each representedby only one species but they were clearly distinct relativeto sequences characterizing the neighboring species groupsThe H planiceps H baramensis and H nemurus speciesgroups and their component species were also generallycongruent in morphological and genetic analyses Also asexpected most cases of discrepancy between morphological-based species identification and genetic-based speciesidentification occurred on relatively shallow branches of thephylogenetic tree These involve principally H spilopterusand the two mtDNA subclades composing H capitulumHowever a far more unexpected lack of congruence involvesthe definition of a deeply divergent unspecified genetic groupconfounding members of theH baramensis species group inthe morphological analysis of Ng and Kottelat [7]

Interpreting Polyphyly The apparent polyphyletic origin ofa species may be due to incomplete lineage sorting amongrecently diverged species the introgression of mtDNA dueto species hybridization or the erroneous identificationof species due to the lumping of morphologically crypticspecies In the absence of nuclear DNA analyses it isoften difficult to distinguish between introgression andincomplete lineage sorting For example several sequencescharacterizing the Indochinese clade of H capitulum(subclade E) are found in the Mekong and Chao PhrayaRivers well within the distributional area of H spilopterusH spilopterus and H capitulum first diverged at least 3MaOn the one hand this apparent case of polyphyly mayhave resulted from incomplete lineage sorting betweenrecently divergedH spilopterus andH capitulum We wouldhowever have expected similar cases of polyphyly amongpopulations of the Indochinese clade of H capitulum butthis was not detected On the other hand the sympatricoccurrence of H spilopterus subclade A and subcladeB sequences with H capitulum Indochinese subclade Esequences in the Mekong River may represent a migrationevent and local introgression of H capitulum with Hspilopterus A similar event may have occurred in theChao Phraya and Mae Khlong Rivers where H spilopterussequences cooccur withH capitulum sequences Computingthe divergence of H capitulum sequences (Indochinesesubclade E) cooccurring with H spilopterus and Hcapitulum sequences occurring in eastern PeninsularMalaysia (subclade A) reveals a mean sequence divergence119901 = 15 (04) Thus the H capitulum sequences occurringin sympatry with H spilopterus diverged at most 750 ka ago

Advances in Evolutionary Biology 17

H fortis

XXRAJ0604H furcatus

H sabanusH semotusH divaricatus

H planicepsGREND0206

VEBAH0907

H bonganPESAL0102

H wyckioidesH macropterus

H guttatusXXRAJ0301Leiocassis longirostris

Ma

4971

92

47

51

9999

99

9999

99

99

99

9999

99

99

99

99

65

96

96

4946

93

79

78

70

91

93

98

86

87

91

95

97

71

85

9999

05101520

005010015020 000

H capitulum Indochinese clades AndashE

H capitulum Sundaic clade

H hoevenii B

H hoevenii A

H spilopterus C

H spilopterus B

H spilopterus AH spilopterus A

H spp CH spp B

H spp A

Figure 13 Linearized tree with branch lengths estimated with the molecular clock using a mutation rate of 1 per Ma (=divergence rate of2 per Ma) The tree topology is identical to the ML reconstruction illustrated in Figure 3 (see the specificities of the tree reconstruction inthe caption of Figure 3) Boxes represent 95 confidence intervals of divergence time estimates for each node Vertical dashed lines redirectto dates indicated on the timescale (Ma) Numbers below timescale indicate the estimated number of substitution changes using Hasegawa-Kishino-Yanomodel (HKY)with gamma distribution (+119866 = 13290) after linearizing the treeH furcatus is synonymizedwithH baramensis

from eastern Peninsular Malaysia populations suggestinga recent introgression event Similarly H capitulum in theKapuas River is characterized by sequences of both theSundaic and the Indochinese clades As both sequences arebroadly sympatric it is not possible to distinguish betweenincomplete lineage sorting and introgression of these closelyrelated intraspecific lineages

A Possible Case of Morphological Stasis Clade 6 of thepresent analysis defines a well-supported monophyletic cladefound in Sabah Sarawak and the Endau River of easternpeninsula Malaysia that has no equivalent taxonomic statusin the revision of Ng and Kottelat [7] In Borneo they occursympatrically with the H baramensis species group and areconfounded as either H baramensis or H semotus These

two clades are genetically highly divergent having divergedbetween 8 and 12Ma ago It thus seems highly unlikely thatthey represent retained polymorphisms due to incompletelineage sorting Sequences previously identified as character-izing H chrysops [11] are part of clade 6 and extend to thesouth (Rajang and Sadong Rivers of Sarawak West Borneo)with one closely related genotype found in eastern peninsularMalaysia (Endau River) where it may well be confoundedwith H capitulum H chrysops was synonymized with Hcapitulum by Ng and Kottelat [7] based on their analysis ofspecimens ofH chrysops from the Rajang RiverThus despitea long and apparently independent phylogenetic historythe unspecified species group appears largely cryptic andconfounded principally with members of the H baramensisspecies group

18 Advances in Evolutionary Biology

We suggest that this situation is an example of mor-phological stasis involving the coexistence of a number ofgenetically divergent yet morphologically cryptic species thatare lumped together [24] A striking example of morpho-logical and ecological stasis among fishes is provided by thecoastal marine bonefishes (Albula spp) Distinct evolution-ary lineages (separated by sequence divergence between 5 and30 [25]) of bonefish coexist in several sample locations yetthey show little morphological or ecological differentiationin sympatry [25] Similarly a proportion of the grey mulletspecies (Mugilidae) also appears to consist of cryptic species[26] For exampleMugil cephalus in Taiwan is characterizedby three divergent lineages that have been shown to charac-terize 3 reproductively isolated but morphologically crypticspecies [26 27]

Here we have discovered 2 highly distinct and structuredclades (each composed of 3 mtDNA subclades) that aremorphologically confounded within different species Forexample H spp C of the unspecified species group isidentified as H baramensis in the Baram River and as Hsemotus in the Padas River We speculate that North Borneoharbors a morphologically cryptic species complex possiblycomposed of several species pairs inhabiting different riversystems The reconstruction of this phylogenetic history andthe taxonomic status of its components remains a majorchallenge to the revision of the Hemibagrus genus

42 Phylogeographic Hypotheses The phylogeography ofthe genus appears to have been principally influenced bytwo geological histories one encompassing the formationof North Borneo the second the emergence of the SundaIslands and their connectivity with the Indochinesemainlandassociated with recurrent sea-level changes North Borneoappears to have acted as a species sink promoting speciationand locally high biodiversity within the genus but with littleevidence of subsequent dispersal out of North Borneo Incontrast the geological events influencing the Indochinesemainland and the Sunda Islands have permitted bothspeciation and differential dispersal depending on thebiology of the species For example the H planiceps speciesgroup is found throughout the Greater Sunda Islands andtheThai-Malay Peninsula as is theH nemurus species groupWhereas the H nemurus species group generally occupiesthe low altitude lower reaches of large rivers the speciescomposing the H planiceps group are found in the upperreaches of large rivers The H nemurus species group iscomposed of a number of widely distributed closely relatedspecies whereas theH planiceps group is composed of a largenumber of species that are highly localized geographicallyIt thus appears that sea-level changes have had little impacton the dispersal history of the H planiceps species group buthave deeply influenced that of the H nemurus species group

The Case of North Borneo From 15Ma to 5Ma greater areasof Borneo became emergent and the central mountainson the Sarawak-Kalimantan border extending into Sabahbecame wider and higher which may have represented newbarriers to dispersal [28 29] Borneo gradually developedinto the present large island by emergence of land in the

north and east The H baramensis species group is restrictedto North Borneo as is the unspecified species group of theHnemurus species group H fortis is also distributed in NorthBorneo The isolation of the freshwater fish communities ofNorth Borneo (particularly in the Labuk-Segama watershed)has long been recognized based on the number of endemicspecies and the concentration of relict species [30] The latePliocene-early Pleistocene diversification of H fortis theunspecified species group and the H baramensis speciesgroup may thus be associated with their dispersal to andisolation within the watersheds of North Borneo Only onesequence of the unspecified species group is found outsideof North Borneo and H fortis appears to be distributedthroughout central and east Borneo [7]

ThePhylogeography of the H nemurus Species Group Periodicmarine transgressions on the Sundaland platform during theearly and middle Miocene [2 29] may have been sufficientto isolate populations of freshwater organisms and promotecladogenesis [31] This may have been responsible for theinitial mid to late Miocene divergence of the H nemurusspecies group The majority of sequences assigned to thisspecies group belong to 3 species (H spilopterus H capitulum(including H nemurus) and H hoevenii) all of which havebeen influenced by the more recent geological history of theSunda Islands and their connectivity with the Indochinesemainland H spilopterus is known from river drainages inIndochina including the Mekong Dong Nai Ban PakongChao Phraya and Mae Klong where it is found in a widevariety of lotic and lentic habitats [7] H spilopterus divergedin the late Pliocene-early Pleistocene possibly isolated tothe north of the Kra Peninsula during the late Pliocenemarine transgression that is purported to have created amajor biogeographic transition zone between the Sundaicand Indochinese biotas ([32] but see [33] for a critique ofthis biogeographic scenario) The existence of 2 subclades(A and B) in the Mekong River separated by 2 sequencedivergence is consistent with intraspecific divergence Thetwo subclades possibly represent 2 historical mtDNA lineagesthat are retained within the species reflecting a Pleistoceneisolation event followed by secondary contact within thecontemporary Mekong River Subclade C from the ChaoPhraya and Mae Khlong Rivers exhibits a level of geneticdivergence consistent with sibling species-level divergenceshowing a 5 sequence divergence relative to Mekong RiverH spilopterus

H capitulum may be considered as part of the Sundaicbiota located principally to the south of the Kra marinetransgression zone that may have isolated H spilopterus tothe north on the Indochinese mainlandThe biogeographicalevent leading to the subsequent formation of two intraspecificgroups within H capitulum is not at all clear The onlyarea where the two clades are clearly geographically separateis on the Thai-Malay Peninsula with the ldquoSundaicrdquo cladeon the west coast and the ldquoIndochineserdquo clade on theeast coast separated by the central Titiwangsa mountainrange The genetic distinctiveness of east and west coastpopulations of H capitulum has also been documented fornuclear DNA [34] A similar pattern of separation has been

Advances in Evolutionary Biology 19

reported for 5 additional freshwater fishes within this region[35] However as the divergence of the two H capitulumclades postdates the formation of the mountain range byapproximately 200Ma the geographic separation of the twoclades most likely represents differential dispersal rather thanancient vicariance The presence of the Sundaic clade on thewest coast of the Thai-Malay Peninsula as well as on theSundaic Islands reflects the historical connections betweeneast Sumatra and west Peninsular Malaysia across the Straitof Malacca This area encompasses the Northern CentralSumatra-Western Malaysia freshwater ecoregion [36] TheMuar River forms its southern border coincident with thesouthernmost distribution of the Sundaic clade on the Thai-Malay Peninsula It is important to note however that mostof Sumatra and Java emerged to achieve their present size andelevation only 5Ma ago and much of East Java continuedto be the site of marine deposition until late Pliocene-earlyPleistocene [29] We may speculate that the Sundaic clade ofH capitulum resulted from the relatively recent colonizationof these islands and Borneo The radiation within the cladeonly dates back to approximately 1Ma ago H nemurus fromWest Java may thus best be considered an isolated regionalpopulation of very recent origin

The diversification of the Indochinese clade dates fromat least 12Ma ago coincident with the mid-Pleistocenetransition at which time low-frequency high-amplitude andquasiperiodic (100 ka) glacial variability emerged It began125Ma ago and was complete by 07Ma ago [37] The tran-sition marked the beginning of major changes in sea levelsduring which the Sunda Shelf periodically emerged and wastraversed by enormous fluvial systems [3] The contributionof these events to the diversification of this clade cannotbe satisfactorily reconstructed One possible explanation isthat high sea levels produced barriers to gene flow and thepopulations of West Borneo (subclades B C and D) wereisolated from those of east Peninsular Malaysia This couldbe due to one or more dispersal events from Borneo via theGreat Sunda River (Figure 1) to east Peninsular Malaysia orlineage sorting whereby sequences of subclades A and E havebeen lost from West Borneo As in the case of the sympatricoccurrence of H spilopterus and H capitulum subclade E(see above) in the Mekong River we are unable to clearlydefine the relative roles of incomplete lineage sorting andintrogression followingmigration Given the recent origins ofthe Indochinese H capitulum sequences incomplete lineagesorting most probably blurs patterns of recent isolation

The last species of the H nemurus species group is Hhoevenii The species is widespread throughout PeninsularMalaysia and the Sundaic Islands It is found primarily inthe lower reaches of rivers and is apparently capable ofwithstanding low salt concentration [7] H hoevenii thusappears to be the most liable of all members of the Hnemurus species group to be impacted by Pleistocene sea-level changes This is clearly reflected in the widespreaddistribution of the sequences of this species Two lineages areevident having diverged no more than 03Ma ago suggest-ing a late Pleistocene isolation event followed by extensivedispersal and admixture in recent times This represents theclearest example of the dispersal opportunities afforded by

Pleistocene sea level changes on the Sunda Shelf In additionthe relatively low genetic diversity detected (only 6 sequencesin 42 fish) suggests a population bottleneck in the recent pastpossibly related to population declines in the lower reaches ofrivers flooded by increasing sea levels

43 Future Directions We have provided a global picture ofthe evolutionary taxonomy of the genus in Southeast AsiaClearly a more rigorous phylogenetic reconstruction of thegenus Hemibagrus requiring the construction of multiplegene trees based on the analysis of both mitochondrial andnuclear DNA markers from a wider taxonomic array ofhemibagrids will involve an even greater effort of samplingthroughout the region In particular we require samplingat smaller spatial and temporal scales For example smallerscale sampling in the Padas River and the Segama Riverof North Borneo using both mitochondrial and nucleargenetic markers would contribute to clarifying the natureof the cryptic species complex believed to inhabit theserivers The demonstration of reproductive isolation andormicroallopatric distribution among morphologically crypticspecies may result in the description of greater speciesrichness within the genus than presently described Thespecies status of H chrysops merits reconsideration givenits highly divergent evolutionary history Nuclear geneticmarkers in conjunction with mtDNA markers could revealcases of historical introgression particularly in the KapuasRiver and the Mekong River The hemibagrid catfishes con-tinue to provide multiple opportunities for the study of theevolutionary taxonomy and biogeography of a major faunalcomponent of the fluvial ecosystems of Southeast Asia

Disclosure

Ethanol-preserved tissue samples of many of the specimensidentified in Table S1 are freely available upon request fromJJD

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Goh Yan Yih for assistance in collectingspecimens and molecular analyses Mrs Francoise Colom-bani for molecular analyses and Dr Heok Hee Ng forspecies identification This work was funded by the NationalUniversity of Singapore grant to P K L Ng and a CanadianNatural Sciences and Engineering Research Council grant toJ J Dodson

References

[1] D J Lohman M de Bruyn T Page et al ldquoBiogeography ofthe Indo-Australian archipelagordquo Annual Review of EcologyEvolution and Systematics vol 42 pp 205ndash226 2011

20 Advances in Evolutionary Biology

[2] D S Woodruff ldquoNeogene marine transgressions palaeogeog-raphy and biogeographic transitions on the Thai-Malay Penin-sulardquo Journal of Biogeography vol 30 no 4 pp 551ndash567 2003

[3] H K Voris ldquoMaps of Pleistocene sea levels in SoutheastAsia shorelines river systems and time durationsrdquo Journal ofBiogeography vol 27 no 5 pp 1153ndash1167 2000

[4] S Y Yap ldquoOn the distributional patterns of Southeast-EastAsian freshwater fish and their historyrdquo Journal of Biogeographyvol 29 no 9 pp 1187ndash1199 2002

[5] S K J McConnell ldquoMapping aquatic faunal exchanges acrossthe Sunda shelf South-East Asia using distributional andgenetic data sets from the cyprinid fish Barbodes gonionotus(Bleeker 1850)rdquo Journal of Natural History vol 38 no 5 pp651ndash670 2004

[6] T Mo Anatomy Relationships and Systematics of the Bagridae(Teleostei Siluroidei) with a Hypothesis of Siluroid Phylogenyvol 17 ofTheses Zoologicae Koeltz Scientific Books 1991

[7] H H Ng and M Kottelat ldquoRevision of the Asian catfish genusHemibagrus bleeker 1862 (Teleostei Siluriformes Bagridae)rdquoRaffles Bulletin of Zoology vol 61 no 1 pp 205ndash291 2013

[8] P K L Ng and H H Ng ldquoHemibagrus gracilis a new speciesof large riverine catfish (Teleostei Bagridae) from PeninsularMalaysiardquo Raffles Bulletin of Zoology vol 43 pp 133ndash142 1995

[9] H H Tan and M Kottelat ldquoRedescription of Betta picta(Teleostei Osphronemidae) and description of Betta falx sp nfrom central Sumatrardquo Revue Suisse de Zoologie vol 105 no 3pp 557ndash568 1998

[10] J J Dodson F Colombani and P K Ng ldquoPhylogeographicstructure in mitochondrial DNA of a South-east Asian fresh-water fish Hemibagrus nemurus (Siluroidei Bagridae) andPleistocene sea-level changes on the Sunda shelfrdquo MolecularEcology vol 4 no 3 pp 331ndash346 1995

[11] H H Ng and J J Dodson ldquoMorphological and genetic descrip-tions of a new species of catfish Hemibagrus chrysops fromSarawak East Malaysia with an assessment of phylogeneticrelationships (Teleostei Bagridae)rdquo Raffles Bulletin of Zoologyvol 47 no 1 pp 45ndash57 1999

[12] P D N Hebert A Cywinska S L Ball and J R DeWaardldquoBiological identifications through DNA barcodesrdquo Proceedingsof the Royal Society B Biological Sciences vol 270 no 1512 pp313ndash321 2003

[13] J C Carolan T EMurray U Fitzpatrick et al ldquoColour patternsdo not diagnose species quantitative evaluation of a DNAbarcoded cryptic bumblebee complexrdquo PLoS ONE vol 7 no 1Article ID e29251 2012

[14] M Kekkonen and P D N Hebert ldquoDNA barcode-baseddelineation of putative species efficient start for taxonomicworkflowsrdquoMolecular Ecology Resources vol 14 no 4 pp 706ndash715 2014

[15] DM Hillis CMoritz and B KMarbleMolecular SystematicsSinauer Sunderland Mass USA 2nd edition 1996

[16] T D Kocher W K Thomas A Meyer et al ldquoDynamics ofmitochondrial DNA evolution in animals amplification andsequencingwith conserved primersrdquoProceedings of theNationalAcademy of Sciences of the United States of America vol 86 no16 pp 6196ndash6200 1989

[17] T A Hall ldquoBioEdit a user-friendly biological sequence align-ment editor and analysis program for Windows 9598NTrdquoNucleic Acids Symposium Series vol 41 pp 95ndash98 1999

[18] D A Benson I Karsch-Mizrachi K Clark D J Lipman JOstell and EW Sayers ldquoGenBankrdquoNucleic Acids Research vol40 no D1 pp D48ndashD53 2012

[19] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[20] X Ku Z Peng R Diogo and S He ldquoMtDNA phylogenyprovides evidence of generic polyphyleticism for East Asianbagrid catfishesrdquoHydrobiologia vol 579 no 1 pp 147ndash159 2007

[21] L Yang and S He ldquoPhylogeography of the freshwater catfishHemibagrus guttatus (Siluriformes Bagridae) implications forSouth China biogeography and influence of sea-level changesrdquoMolecular Phylogenetics and Evolution vol 49 no 1 pp 393ndash398 2008

[22] B W Bowen A L Bass L A Rocha W S Grant and DR Robertson ldquoPhylogeography of the trumpetfishes (Aulosto-mus) ring species complex on a global scalerdquo Evolution vol 55no 5 pp 1029ndash1039 2001

[23] Y P Kartavtsev ldquoDivergence at Cyt-b and Co-1 mtDNA geneson different taxonomic levels and genetics of speciation inanimalsrdquoMitochondrial DNA vol 22 no 3 pp 55ndash65 2011

[24] SWada Y Kameda and S Chiba ldquoLong-term stasis and short-term divergence in the phenotypes of microsnails on oceanicislandsrdquoMolecular Ecology vol 22 no 18 pp 4801ndash4810 2013

[25] J Colborn R E Crabtree J B Shaklee E Pfeiler and B WBowen ldquoThe evolutionary enigma of bonefishes (Albula spp)cryptic species and ancient separations in a globally distributedshorefishrdquo Evolution vol 55 no 4 pp 807ndash820 2001

[26] J-D Durand K-N Shen W-J Chen et al ldquoSystematics of thegrey mullets (Teleostei Mugiliformes Mugilidae) molecularphylogenetic evidence challenges two centuries ofmorphology-based taxonomyrdquo Molecular Phylogenetics and Evolution vol64 no 1 pp 73ndash92 2012

[27] K-N Shen B W Jamandre C-C Hsu W-N Tzeng and J-D Durand ldquoPlio-Pleistocene sea level and temperature fluc-tuations in the northwestern Pacific promoted speciation inthe globally-distributed flathead mullet Mugil cephalusrdquo BMCEvolutionary Biology vol 11 no 1 article 83 2011

[28] R Hall ldquoThe plate tectonics of Cenozoic SE Asia and thedistribution of land and seardquo in Biogeography and GeologicalEvolution of SE Asia R Hall and J D Holloway Eds pp 99ndash131Backhuys Publishers Leiden The Netherlands 1998

[29] R Hall ldquoSoutheast Asiarsquos changing palaeogeographyrdquo Blumeavol 54 no 1ndash3 pp 148ndash161 2009

[30] R F Inger and P K Chen ldquoThe freshwater fishes of NorthBorneordquo Fieldiana Zoology vol 45 pp 1ndash268 1962

[31] J Bohlen V Slechtova H H Tan and R Britz ldquoPhylogenyof the Southeast Asian freshwater fish genus Pangio (Cyprini-formes Cobitidae)rdquoMolecular Phylogenetics and Evolution vol61 no 3 pp 854ndash865 2011

[32] M de Bruyn E Nugroho M M Hossain J C Wilson andP B Mather ldquoPhylogeographic evidence for the existence ofan ancient biogeographic barrier the Isthmus of Kra SeawayrdquoHeredity vol 94 no 3 pp 370ndash378 2005

[33] J Parnell ldquoThe biogeography of the Isthmus of Kra region areviewrdquoNordic Journal of Botany vol 31 no 1 pp 001ndash015 2013

[34] S Usmani S G Tan S S Siraj and K Yusoff ldquoPopulationstructure of the Southeast Asian river catfishMystus nemurusrdquoAnimal Genetics vol 34 no 6 pp 462ndash464 2003

[35] M P Tan A F J Jamsari and M N Siti Azizah ldquoPhylo-geographic pattern of the striped snakehead Channa striatain Sundaland ancient river connectivity geographical and

Advances in Evolutionary Biology 21

anthropogenic singnaturesrdquo PLoS ONE vol 7 no 12 Article IDe52089 2012

[36] R Abell M L Thieme C Revenga et al ldquoFreshwater ecore-gions of the world a new map of biogeographic units forfreshwater biodiversity conservationrdquo BioScience vol 58 no 5pp 403ndash414 2008

[37] P U Clark D Archer D Pollard et al ldquoThemiddle Pleistocenetransition characteristics mechanisms and implications forlong-term changes in atmospheric pCO

2rdquo Quaternary Science

Reviews vol 25 no 23-24 pp 3150ndash3184 2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 12: ResearchArticle - Université Laval Julian... · ResearchArticle A DNA Barcode-Based Evaluation of the Southeast Asian Catfish Genus Hemibagrus Bleeker, 1862 (Teleostei: Siluriformes;

12 Advances in Evolutionary Biology

110∘

SPCPCHMK12

SPMEK0202SPMEK0401

SPMEK0301SPMEK0103

SPMEK1401SPMEK0501

SPMEK1201SPMEK1301

SPMEK1601SPMEK1502

SPMEK0701SPMEK0801SPMEK1701

SPCHP0501SPCHA0201

SPCHA0304SPPAT0101

100∘

10∘

0∘

120∘

0

0 250 500

250 500

750(km)

(mi)

000499

99 98

60

47

48

54

96

7091

38

86

87

99

80

58

H spilopterus ldquoCrdquo

H spilopterus ldquoBrdquo

H spilopterus ldquoArdquo

Shelf minus75mShelf minus120 m

Figure 8 Phylogenetic relationships within H spilopterus comprised of 3 subclades identified as A B and C The subtree is extracted fromthe taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicate trees in whichthe associated sequences clustered together in the bootstrap test (500 replicates) is shown next to the branches The distribution of thesesequences is illustrated in the right panel

species group found throughout North Borneo (and toa lesser degree in eastern Peninsular Malaysia) that maybe considered as part of the H nemurus species groupHowever the morphological confusion of the unspecifiedspecies group with the sympatric highly divergent Hbaramensis species group suggests the existence of a crypticspecies assemblage (see Section 4)

mtDNA Clade 7 Clade 7 (Figures 2 and 3) is composed of atleast 3 distinct subclades that appear to reflect 3 geneticallydivergent populations ofH spilopterus Subclade A (Figure 8)encompasses 7 sequences obtained from 8 fish sampled inthe Mekong River Four fish bearing 3 of these sequenceswere identified as paratypes (ZRC 43324) of H spilopterusby NampK (Table S1) One additional fish was identified asH spilopterus by NampK (ZRC 46157 Table S1) Subclade Bencompasses 6 sequences obtained from 9 fish all sampledfrom the Mekong River (Table S1) The fish sampled at ThatPhanom (ZRC 46012) was identified as H spilopterus inthe analysis of HampK (Table S1) Subclade C encompasses 5sequences obtained from 19 fish sampled in the Chao PhrayaMae Khlong and Chanthaburi Rivers Thailand Onesequence (CPCHMK12) was found in all of these drainages(Table S1) A closely allied sequence was found in one fishsampled at Phatthalung just south of the Isthmus of Kra onthe Thai-Malay Peninsula None of these fish were examined

by NampK and thus we are unable to definitively identifythe species However given the widespread occurrence ofH spilopterus in the Chao Phraya and Mae Klong Riversreported by NampK we conclude that these fish are part of awidespread H spilopterus population complex The meanwithin-group (clade 7) genetic divergence (119901 = 29 (04))suggests intraspecific population-level divergence

mtDNA Clade 8 Clade 8 encompasses 3 sequencesobtained from 9 fish sampled in east Sabah specifically theKinabatangan Segama and Umas Umas Rivers (Figure 9)The Kinabatangan sequence is somewhat divergent withinthe clade and the specimen was not identified by NampK Onesequence (SGM0606) characterized 6 fish from the SegamaRiver 2 of which were identified as H fortis (ZEC 4052140522 Table S1) by NampK (Table S1) Finally one fish bearinga closely related genotype from the Umas Umas River wasidentified as H baramensis by NampK (Table S1)

mtDNA Clade 9 Clade 9 is a sister group to the remaining2 clades composing the H nemurus species group Thiswell-defined clade (Figure 10) clusters 6 sequences from42 fish that are clearly identified as H hoevenii (NampK)Some of these haplotypes are geographically widespread onesequence (JOMSKA11 Table S1) was found in the Johor River(East Peninsular Malaysia) the Musi River (Sumatra) and

Advances in Evolutionary Biology 13

100∘ 110∘

0∘

120∘

00 250

250500

500750

(km)

(mi)

FOKIN0102FOSGM0606

FOUMA0201H fortis

10∘

99

86

001

Shelf minus75mShelf minus120 m

Figure 9 Phylogenetic relationships within H fortis comprised of 3 sequences The subtree is extracted from the taxon identificationphenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicate trees in which the associated sequencesclustered together in the bootstrap test (500 replicates) is shown next to the branches The distribution of these sequences is illustrated in theright panel

the Kapuas River (West Borneo) and a second sequence(HMUBHKABM23 Table S1) was found inMuarRiver (WestPeninsular Malaysia) and the Batang Hari (Sumatra) theKapuas (Sarawak) and the Baram Rivers (Sarawak) Many ofthese fish were identified as H hoevenii by NampK (Table S1)The existence of twodistinct but very closely related subclades(Figure 10 119901 = 06 (03)) that are not geographicallysegregated suggests some recent isolation event followed bywidespread dispersal and secondary contact

33 The H capitulum Group The final two monophyleticgroups of the Hemibagrus gene tree are approximatelyequivalent to the remaining species composing the Hnemurus species group as defined by NampK Dodson etal [10] and Ng and Dodson [11] have previously referredto clade 10 as the Sundaic clade as most sequences wereobtained from fish sampled on the Greater Sunda Islandsand clade 11 as the Indochinese clade asmany sequences wereobtained from fish sampled on the Indochinese mainland(Figure 2 Table S1) Although most of these sequencesare assigned to H capitulum by NampK there are somesignificant phylogeographic differences between the 2 cladesIn particular there appears to be an east-west division in thedistribution of the two clades on the Thai-Malay Peninsulawith the Sundaic clade (clade 10) found on the west coast andthe Indochinese clade found on the east coast with sympatricoccurrence in the Muar River Nevertheless mean sequencedivergence between the two clades is 119901 = 29 (05) avalue that is indicative of population-level or subspecies

divergence The two clades are thus best considered ascomprising one species

mtDNA Clade 10 The Sundaic clade includes a number ofclosely related sequences that comprise part of H capitulumand H nemurus Very little genetic structure is apparentand bootstrap support for tree topology within this group isgenerally very weak (Figure 11) A widely spread genotype(NCLCTBA20) characterized 6 of 17 specimens from CiTarum at the Cirata reservoir (West Java) (Table S1) and5 were identified as H nemurus in the analysis of NampK(ZRC 42564) (Table S1) The remaining fish characterized bythis sequence were obtained from Ci Liwong at Sawangan(West Java) and one fish was obtained from the Barito Riverat Banjarmassin south Borneo This latter specimen (ZRC40553) was identified by NampK as H capitulum (Table S1)Another common genotype (NBHKPBR09) was identified aseither H capitulum H hoevenii or H nemurus dependingon the river of origin (Table S1) Many of the remainingsequences of the Sundaic clade appear to characterize fish thatNampK identified asH capitulum A geographically widespreadgenotype (CPEBEMUKA36) characterized 9 fish sampledon the Perak River at Gerik that were identified by NampKas H capitulum (ZRC 41150 Table S1) Another commongenotype within this clade (CABHMS20) included 2 fishfrom the Batang Hari at Jambi and identified by NampK asH capitulum (ZRC40534 Table S1) Another 2 sequences(CABAR0203 CABAR0305) characterized 8 fish sampledon the Barito River at Banjarmasin and were identified as

14 Advances in Evolutionary Biology

100∘

10∘

110∘

0∘

120∘

0

0 250

250

500

500

750(km)

(mi)

53

64

99

85

64

0002

HJOMSKA11

HOBAH0601

HOBHMS05

HMUBHKABM23

HOBAH0701

HOKAP0501

H hoevenii ldquoBrdquo

H hoevenii ldquoArdquo

Shelf minus75mShelf minus120 m

Figure 10 Phylogenetic relationships withinH hoevenii comprised of 2 subclades and 6 sequences The subtree is extracted from the taxonidentification phenogram inferred using theNeighbor-Joiningmethod (see Figure 2)The percentage of replicate trees in which the associatedsequences clustered together in the bootstrap test (500 replicates) is shown next to the branches The distribution of these sequences isillustrated in the right panel

110∘

XXMAH0110

CABAR0305

96 CAKPR0201

74CAKAP0401

5931 CAKAP0601

35 CPEBEMUKA36

646 CAKAP0903

33 CAKAP080114 CAKAP0701

CARAJ0210

4 CABAR0203CABRA0204

20

CBHKPBR0919

CABRA0301

15CABHMS20

24 NCLCTBA20

63 NECIT0201

H capitulumldquoSundaic claderdquo

H nemurus

0002 120∘

0 250 500 750

(km)

0 250 500

(mi)100

10∘

0∘

Shelf minus75mShelf minus120 m

Figure 11 Phylogenetic relationships within theH capitulum ldquoSundaicrdquo clade including 2 sequences associated withH nemurusThe subtreeis extracted from the taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicatetrees in which the associated sequences clustered together in the bootstrap test (500 replicates) is shown next to the branchesThe distributionof these sequences is illustrated in the right panel

Advances in Evolutionary Biology 15

H capitulum

0∘

10∘

100∘

110∘

120∘

66

63

88

59

38

39 68

71

6991

57

9754

88 4944

39

53

89

0002

ldquoIndochineserdquogroup ldquoErdquo

H capitulumldquoIndochineserdquo

group ldquoDrdquoH capitulumldquoIndochineserdquo

group ldquoCrdquo

H capitulumldquoIndochineserdquo

group ldquoBrdquo

H capitulumldquoIndochineserdquo

group ldquoArdquo0

0

250

250

500

500

750

(km)

(mi)

CATER0301CATERR0401

CKETEMU23

CAMEK0601CACHP0301

CAMKPA03

CAMEK0910CAMEK1003

XXMUBM02

CABMTU09

CABRM0601

CAKAP1201CAKAP1001

CAKAP1101CAKAP1301

CABRM0501

CASAD0102

CASAD0220

CAEND0301

CABAH0801

CAPABH05CATEEN03

CATER0701CATERR0801

CAKETE04CATER0501

CAPAH0303

Shelf minus75mShelf minus120 m

Figure 12 Phylogenetic relationships within theH capitulum ldquoIndochineserdquo clade encompassing 5 subclades identified as A B C D and EThe subtree is extracted from the taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2)The percentageof replicate trees in which the associated sequences clustered together in the bootstrap test (500 replicates) is shown next to the branchesThedistribution of these sequences is illustrated in the right panel

H capitulum by NampK (ZRC 40553 Table S1) Although notincluded in the analysis of NampK samples from the Sundaicclade were also found in the Kapuas River (West Borneo)Finally 10 fish sampled on the Mahakam River at Samarindaeast Borneo are all characterized by one relatively divergentgenotype (CAMAH0110) butwere considered asH capitulumin the analysis of NampK (ZRC 40533 Table S1)TheMahakampopulation sample is the most genetically distinct of theSundaic clade (119901 = 17 (04)) but nevertheless indicativeof intraspecific population-level divergence (Figure 12)

The topology of the Sundaic clade does not permit usto draw a clear distinction between H nemurus and theSundaic H capitulum The mean within-group divergenceof the Sundaic clade is only 119901 = 08 (02) well withinpopulation-level divergence values We postulate that Hnemurus may be one of several regional variants of awidespread Sundaic H capitulum clade that has radiatedrelatively recently This may explain the existence of manyshared sequences throughout the Sundaic islands assigned todifferent species in different regions

mtDNA Clade 11 Finally the Indochinese clade of Hcapitulum represents a geographically widespread andweakly diversified clade (within-group mean sequencedivergence 119901 = 11 (02) Figure 12) Although few fishfrom this genetic clade were included in the morphological

analysis of NampK most may be considered as H capitulumgiven their geographic distribution and close geneticrelationship to the Sundaic H capitulum Four sequencesin this clade were identified as H capitulum in the analysisof NampK 3 specimens characterized by one sequence(CABMTU09) found in the Tutong River of westernBorneo (ZRC 40482) 6 characterized by the same sequencefrom the Baram River (ZRC 40498) another 2 sequencesfrom the Baram River (ZRC 40498) and 20 specimenscharacterized by an abundant genotype (CASAD0220)found at 2 sites in the Sadong River also in western Borneo(ZRC 39526 5 specimens 39527 3 specimens and 38753 4specimens) (Table S1) Unfortunately none of the specimenscollected on the Indochinese mainland for genetic analysiswere examined by NampK Nevertheless the distribution ofspecimens analyzed for mtDNA resembles the distribution ofspecimens examined by NampK Although weakly structuredwe may recognize 5 subclades One genotype (CAPAH0303)is basal to the phenogram Subclade A encompasses 8sequences mainly found in eastern Peninsular Malaysia(Table S1 Figure 12) These rivers furnished many of thespecimens NampK examined to define H capitulum Howeversome sequences in this group occur in sympatry withsequences identified within the Sundaic H capitulum groupTwo sequences from subclade A occurred in the Batang HariSumatra (Table S1) Subclade E also encompasses sequences

16 Advances in Evolutionary Biology

found in Peninsular Malaysia as well as in the Mekong ChaoPhraya and Mea Khlong Rivers (Figure 12) In addition3 subclades encompass sequences found principally inwestern Borneo subclade B in the Baram and Sadong RiversSarawak subclade C in the Kapuas River West Kalimantanand subclade D in the Baram and Tutong Rivers of Sarawakand Brunei respectively (Figure 12) This latter subclade alsoincludes one genotype found in the Muar River of westernpeninsular Malaysia Finally subclade E encompasses 5sequences from the Mekong Chao Praya and Mae KhlongRivers all within the distribution area of H spilopterus

EvolutionaryHistoryThe evolutionary history of theHemiba-grus species groups analyzed here dates from at least theMiocene 15ndash20Ma (Figure 13) when considering the widelyused molecular clock of 2 sequence divergence per millionyears Bootstrap support for the sister group relationships ofH wyckioides theH guttatus species group andH peguensisare all weak (clearly due to undersampling) and little can besaid of their evolutionary history other than the fact that theyoccupy basal positions in the rooted trees The remainder ofboth the NJ and ML trees is composed of two major sistergroups the H planiceps + H baramensis group and the Hnemurus species group (Figures 2 3 and 13)TheH planiceps+ H baramensis group is strongly supported in both NJ andML analyses (84 and 90 bootstrap support resp Figures2 and 3) The H nemurus species group is also stronglysupported in both analyses (98 and 99 bootstrap supportFigures 2 and 3) These two major phylogenetic groups areestimated to have diverged at least 8 to 12Ma ago during themid to late Miocene (Figure 13)

Much of the evolutionary history of Hemibagrus hasoccurred during the Quaternary starting approximately26Ma ago This is particularly true of the H planicepsand H nemurus species groups The radiation of the 5species included in the H planiceps species group appearsto coincide with the middle Pleistocene transition period ofapproximately 1Ma ago Their high-elevation distributionhowever appears to have largely isolated them from theeffects of lowered sea levels during glacial maxima and theattendant opportunities for widespread dispersal (Figure 5)The evolutionary histories of theH baramensis species groupand the unspecified species group date back to the latePliocene In contrast the low-elevation distribution of theHnemurus species group in Sundaland appears to have favoredtheir widespread distribution during the Quaternary ice ageIn particular H hoevenii appears to be the most recentlydispersed of the species under study with diversification anddispersal occurring approximately 03Ma ago (Figure 13)

4 Discussion

41 The Congruence of Morphological and Genetic AnalysesCombining genetic and morphological analyses to elucidatetaxonomy allowed us to better understand the diversificationand evolutionary relationships within this speciose group offishes Given the rapid and relatively constant rate of mtDNAevolution the deeper the branches within the phylogenetictree are the greater our ability to distinguish valid taxa

should be Conversely the lack of complete congruencebetween morphological- and genetic-based identificationsis expected to increase among the shallower branches of thephylogenetic tree resulting in the appearance of polyphyleticspecies Although 2 of the 8 species groups defined by Ngand Kottelat [7] were not sampled in this study the validityof the remaining 6 species groups and their componentspecies defined on the basis of morphological similaritywas largely confirmed by the analysis of the mitochondrialCyt 119887 bar code As expected congruence is strongest at thebase of the NJ and ML trees where the H guttatus speciesgroup formed a well-defined monophyletic group The Hmenoda and H wyckii species group were each representedby only one species but they were clearly distinct relativeto sequences characterizing the neighboring species groupsThe H planiceps H baramensis and H nemurus speciesgroups and their component species were also generallycongruent in morphological and genetic analyses Also asexpected most cases of discrepancy between morphological-based species identification and genetic-based speciesidentification occurred on relatively shallow branches of thephylogenetic tree These involve principally H spilopterusand the two mtDNA subclades composing H capitulumHowever a far more unexpected lack of congruence involvesthe definition of a deeply divergent unspecified genetic groupconfounding members of theH baramensis species group inthe morphological analysis of Ng and Kottelat [7]

Interpreting Polyphyly The apparent polyphyletic origin ofa species may be due to incomplete lineage sorting amongrecently diverged species the introgression of mtDNA dueto species hybridization or the erroneous identificationof species due to the lumping of morphologically crypticspecies In the absence of nuclear DNA analyses it isoften difficult to distinguish between introgression andincomplete lineage sorting For example several sequencescharacterizing the Indochinese clade of H capitulum(subclade E) are found in the Mekong and Chao PhrayaRivers well within the distributional area of H spilopterusH spilopterus and H capitulum first diverged at least 3MaOn the one hand this apparent case of polyphyly mayhave resulted from incomplete lineage sorting betweenrecently divergedH spilopterus andH capitulum We wouldhowever have expected similar cases of polyphyly amongpopulations of the Indochinese clade of H capitulum butthis was not detected On the other hand the sympatricoccurrence of H spilopterus subclade A and subcladeB sequences with H capitulum Indochinese subclade Esequences in the Mekong River may represent a migrationevent and local introgression of H capitulum with Hspilopterus A similar event may have occurred in theChao Phraya and Mae Khlong Rivers where H spilopterussequences cooccur withH capitulum sequences Computingthe divergence of H capitulum sequences (Indochinesesubclade E) cooccurring with H spilopterus and Hcapitulum sequences occurring in eastern PeninsularMalaysia (subclade A) reveals a mean sequence divergence119901 = 15 (04) Thus the H capitulum sequences occurringin sympatry with H spilopterus diverged at most 750 ka ago

Advances in Evolutionary Biology 17

H fortis

XXRAJ0604H furcatus

H sabanusH semotusH divaricatus

H planicepsGREND0206

VEBAH0907

H bonganPESAL0102

H wyckioidesH macropterus

H guttatusXXRAJ0301Leiocassis longirostris

Ma

4971

92

47

51

9999

99

9999

99

99

99

9999

99

99

99

99

65

96

96

4946

93

79

78

70

91

93

98

86

87

91

95

97

71

85

9999

05101520

005010015020 000

H capitulum Indochinese clades AndashE

H capitulum Sundaic clade

H hoevenii B

H hoevenii A

H spilopterus C

H spilopterus B

H spilopterus AH spilopterus A

H spp CH spp B

H spp A

Figure 13 Linearized tree with branch lengths estimated with the molecular clock using a mutation rate of 1 per Ma (=divergence rate of2 per Ma) The tree topology is identical to the ML reconstruction illustrated in Figure 3 (see the specificities of the tree reconstruction inthe caption of Figure 3) Boxes represent 95 confidence intervals of divergence time estimates for each node Vertical dashed lines redirectto dates indicated on the timescale (Ma) Numbers below timescale indicate the estimated number of substitution changes using Hasegawa-Kishino-Yanomodel (HKY)with gamma distribution (+119866 = 13290) after linearizing the treeH furcatus is synonymizedwithH baramensis

from eastern Peninsular Malaysia populations suggestinga recent introgression event Similarly H capitulum in theKapuas River is characterized by sequences of both theSundaic and the Indochinese clades As both sequences arebroadly sympatric it is not possible to distinguish betweenincomplete lineage sorting and introgression of these closelyrelated intraspecific lineages

A Possible Case of Morphological Stasis Clade 6 of thepresent analysis defines a well-supported monophyletic cladefound in Sabah Sarawak and the Endau River of easternpeninsula Malaysia that has no equivalent taxonomic statusin the revision of Ng and Kottelat [7] In Borneo they occursympatrically with the H baramensis species group and areconfounded as either H baramensis or H semotus These

two clades are genetically highly divergent having divergedbetween 8 and 12Ma ago It thus seems highly unlikely thatthey represent retained polymorphisms due to incompletelineage sorting Sequences previously identified as character-izing H chrysops [11] are part of clade 6 and extend to thesouth (Rajang and Sadong Rivers of Sarawak West Borneo)with one closely related genotype found in eastern peninsularMalaysia (Endau River) where it may well be confoundedwith H capitulum H chrysops was synonymized with Hcapitulum by Ng and Kottelat [7] based on their analysis ofspecimens ofH chrysops from the Rajang RiverThus despitea long and apparently independent phylogenetic historythe unspecified species group appears largely cryptic andconfounded principally with members of the H baramensisspecies group

18 Advances in Evolutionary Biology

We suggest that this situation is an example of mor-phological stasis involving the coexistence of a number ofgenetically divergent yet morphologically cryptic species thatare lumped together [24] A striking example of morpho-logical and ecological stasis among fishes is provided by thecoastal marine bonefishes (Albula spp) Distinct evolution-ary lineages (separated by sequence divergence between 5 and30 [25]) of bonefish coexist in several sample locations yetthey show little morphological or ecological differentiationin sympatry [25] Similarly a proportion of the grey mulletspecies (Mugilidae) also appears to consist of cryptic species[26] For exampleMugil cephalus in Taiwan is characterizedby three divergent lineages that have been shown to charac-terize 3 reproductively isolated but morphologically crypticspecies [26 27]

Here we have discovered 2 highly distinct and structuredclades (each composed of 3 mtDNA subclades) that aremorphologically confounded within different species Forexample H spp C of the unspecified species group isidentified as H baramensis in the Baram River and as Hsemotus in the Padas River We speculate that North Borneoharbors a morphologically cryptic species complex possiblycomposed of several species pairs inhabiting different riversystems The reconstruction of this phylogenetic history andthe taxonomic status of its components remains a majorchallenge to the revision of the Hemibagrus genus

42 Phylogeographic Hypotheses The phylogeography ofthe genus appears to have been principally influenced bytwo geological histories one encompassing the formationof North Borneo the second the emergence of the SundaIslands and their connectivity with the Indochinesemainlandassociated with recurrent sea-level changes North Borneoappears to have acted as a species sink promoting speciationand locally high biodiversity within the genus but with littleevidence of subsequent dispersal out of North Borneo Incontrast the geological events influencing the Indochinesemainland and the Sunda Islands have permitted bothspeciation and differential dispersal depending on thebiology of the species For example the H planiceps speciesgroup is found throughout the Greater Sunda Islands andtheThai-Malay Peninsula as is theH nemurus species groupWhereas the H nemurus species group generally occupiesthe low altitude lower reaches of large rivers the speciescomposing the H planiceps group are found in the upperreaches of large rivers The H nemurus species group iscomposed of a number of widely distributed closely relatedspecies whereas theH planiceps group is composed of a largenumber of species that are highly localized geographicallyIt thus appears that sea-level changes have had little impacton the dispersal history of the H planiceps species group buthave deeply influenced that of the H nemurus species group

The Case of North Borneo From 15Ma to 5Ma greater areasof Borneo became emergent and the central mountainson the Sarawak-Kalimantan border extending into Sabahbecame wider and higher which may have represented newbarriers to dispersal [28 29] Borneo gradually developedinto the present large island by emergence of land in the

north and east The H baramensis species group is restrictedto North Borneo as is the unspecified species group of theHnemurus species group H fortis is also distributed in NorthBorneo The isolation of the freshwater fish communities ofNorth Borneo (particularly in the Labuk-Segama watershed)has long been recognized based on the number of endemicspecies and the concentration of relict species [30] The latePliocene-early Pleistocene diversification of H fortis theunspecified species group and the H baramensis speciesgroup may thus be associated with their dispersal to andisolation within the watersheds of North Borneo Only onesequence of the unspecified species group is found outsideof North Borneo and H fortis appears to be distributedthroughout central and east Borneo [7]

ThePhylogeography of the H nemurus Species Group Periodicmarine transgressions on the Sundaland platform during theearly and middle Miocene [2 29] may have been sufficientto isolate populations of freshwater organisms and promotecladogenesis [31] This may have been responsible for theinitial mid to late Miocene divergence of the H nemurusspecies group The majority of sequences assigned to thisspecies group belong to 3 species (H spilopterus H capitulum(including H nemurus) and H hoevenii) all of which havebeen influenced by the more recent geological history of theSunda Islands and their connectivity with the Indochinesemainland H spilopterus is known from river drainages inIndochina including the Mekong Dong Nai Ban PakongChao Phraya and Mae Klong where it is found in a widevariety of lotic and lentic habitats [7] H spilopterus divergedin the late Pliocene-early Pleistocene possibly isolated tothe north of the Kra Peninsula during the late Pliocenemarine transgression that is purported to have created amajor biogeographic transition zone between the Sundaicand Indochinese biotas ([32] but see [33] for a critique ofthis biogeographic scenario) The existence of 2 subclades(A and B) in the Mekong River separated by 2 sequencedivergence is consistent with intraspecific divergence Thetwo subclades possibly represent 2 historical mtDNA lineagesthat are retained within the species reflecting a Pleistoceneisolation event followed by secondary contact within thecontemporary Mekong River Subclade C from the ChaoPhraya and Mae Khlong Rivers exhibits a level of geneticdivergence consistent with sibling species-level divergenceshowing a 5 sequence divergence relative to Mekong RiverH spilopterus

H capitulum may be considered as part of the Sundaicbiota located principally to the south of the Kra marinetransgression zone that may have isolated H spilopterus tothe north on the Indochinese mainlandThe biogeographicalevent leading to the subsequent formation of two intraspecificgroups within H capitulum is not at all clear The onlyarea where the two clades are clearly geographically separateis on the Thai-Malay Peninsula with the ldquoSundaicrdquo cladeon the west coast and the ldquoIndochineserdquo clade on theeast coast separated by the central Titiwangsa mountainrange The genetic distinctiveness of east and west coastpopulations of H capitulum has also been documented fornuclear DNA [34] A similar pattern of separation has been

Advances in Evolutionary Biology 19

reported for 5 additional freshwater fishes within this region[35] However as the divergence of the two H capitulumclades postdates the formation of the mountain range byapproximately 200Ma the geographic separation of the twoclades most likely represents differential dispersal rather thanancient vicariance The presence of the Sundaic clade on thewest coast of the Thai-Malay Peninsula as well as on theSundaic Islands reflects the historical connections betweeneast Sumatra and west Peninsular Malaysia across the Straitof Malacca This area encompasses the Northern CentralSumatra-Western Malaysia freshwater ecoregion [36] TheMuar River forms its southern border coincident with thesouthernmost distribution of the Sundaic clade on the Thai-Malay Peninsula It is important to note however that mostof Sumatra and Java emerged to achieve their present size andelevation only 5Ma ago and much of East Java continuedto be the site of marine deposition until late Pliocene-earlyPleistocene [29] We may speculate that the Sundaic clade ofH capitulum resulted from the relatively recent colonizationof these islands and Borneo The radiation within the cladeonly dates back to approximately 1Ma ago H nemurus fromWest Java may thus best be considered an isolated regionalpopulation of very recent origin

The diversification of the Indochinese clade dates fromat least 12Ma ago coincident with the mid-Pleistocenetransition at which time low-frequency high-amplitude andquasiperiodic (100 ka) glacial variability emerged It began125Ma ago and was complete by 07Ma ago [37] The tran-sition marked the beginning of major changes in sea levelsduring which the Sunda Shelf periodically emerged and wastraversed by enormous fluvial systems [3] The contributionof these events to the diversification of this clade cannotbe satisfactorily reconstructed One possible explanation isthat high sea levels produced barriers to gene flow and thepopulations of West Borneo (subclades B C and D) wereisolated from those of east Peninsular Malaysia This couldbe due to one or more dispersal events from Borneo via theGreat Sunda River (Figure 1) to east Peninsular Malaysia orlineage sorting whereby sequences of subclades A and E havebeen lost from West Borneo As in the case of the sympatricoccurrence of H spilopterus and H capitulum subclade E(see above) in the Mekong River we are unable to clearlydefine the relative roles of incomplete lineage sorting andintrogression followingmigration Given the recent origins ofthe Indochinese H capitulum sequences incomplete lineagesorting most probably blurs patterns of recent isolation

The last species of the H nemurus species group is Hhoevenii The species is widespread throughout PeninsularMalaysia and the Sundaic Islands It is found primarily inthe lower reaches of rivers and is apparently capable ofwithstanding low salt concentration [7] H hoevenii thusappears to be the most liable of all members of the Hnemurus species group to be impacted by Pleistocene sea-level changes This is clearly reflected in the widespreaddistribution of the sequences of this species Two lineages areevident having diverged no more than 03Ma ago suggest-ing a late Pleistocene isolation event followed by extensivedispersal and admixture in recent times This represents theclearest example of the dispersal opportunities afforded by

Pleistocene sea level changes on the Sunda Shelf In additionthe relatively low genetic diversity detected (only 6 sequencesin 42 fish) suggests a population bottleneck in the recent pastpossibly related to population declines in the lower reaches ofrivers flooded by increasing sea levels

43 Future Directions We have provided a global picture ofthe evolutionary taxonomy of the genus in Southeast AsiaClearly a more rigorous phylogenetic reconstruction of thegenus Hemibagrus requiring the construction of multiplegene trees based on the analysis of both mitochondrial andnuclear DNA markers from a wider taxonomic array ofhemibagrids will involve an even greater effort of samplingthroughout the region In particular we require samplingat smaller spatial and temporal scales For example smallerscale sampling in the Padas River and the Segama Riverof North Borneo using both mitochondrial and nucleargenetic markers would contribute to clarifying the natureof the cryptic species complex believed to inhabit theserivers The demonstration of reproductive isolation andormicroallopatric distribution among morphologically crypticspecies may result in the description of greater speciesrichness within the genus than presently described Thespecies status of H chrysops merits reconsideration givenits highly divergent evolutionary history Nuclear geneticmarkers in conjunction with mtDNA markers could revealcases of historical introgression particularly in the KapuasRiver and the Mekong River The hemibagrid catfishes con-tinue to provide multiple opportunities for the study of theevolutionary taxonomy and biogeography of a major faunalcomponent of the fluvial ecosystems of Southeast Asia

Disclosure

Ethanol-preserved tissue samples of many of the specimensidentified in Table S1 are freely available upon request fromJJD

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Goh Yan Yih for assistance in collectingspecimens and molecular analyses Mrs Francoise Colom-bani for molecular analyses and Dr Heok Hee Ng forspecies identification This work was funded by the NationalUniversity of Singapore grant to P K L Ng and a CanadianNatural Sciences and Engineering Research Council grant toJ J Dodson

References

[1] D J Lohman M de Bruyn T Page et al ldquoBiogeography ofthe Indo-Australian archipelagordquo Annual Review of EcologyEvolution and Systematics vol 42 pp 205ndash226 2011

20 Advances in Evolutionary Biology

[2] D S Woodruff ldquoNeogene marine transgressions palaeogeog-raphy and biogeographic transitions on the Thai-Malay Penin-sulardquo Journal of Biogeography vol 30 no 4 pp 551ndash567 2003

[3] H K Voris ldquoMaps of Pleistocene sea levels in SoutheastAsia shorelines river systems and time durationsrdquo Journal ofBiogeography vol 27 no 5 pp 1153ndash1167 2000

[4] S Y Yap ldquoOn the distributional patterns of Southeast-EastAsian freshwater fish and their historyrdquo Journal of Biogeographyvol 29 no 9 pp 1187ndash1199 2002

[5] S K J McConnell ldquoMapping aquatic faunal exchanges acrossthe Sunda shelf South-East Asia using distributional andgenetic data sets from the cyprinid fish Barbodes gonionotus(Bleeker 1850)rdquo Journal of Natural History vol 38 no 5 pp651ndash670 2004

[6] T Mo Anatomy Relationships and Systematics of the Bagridae(Teleostei Siluroidei) with a Hypothesis of Siluroid Phylogenyvol 17 ofTheses Zoologicae Koeltz Scientific Books 1991

[7] H H Ng and M Kottelat ldquoRevision of the Asian catfish genusHemibagrus bleeker 1862 (Teleostei Siluriformes Bagridae)rdquoRaffles Bulletin of Zoology vol 61 no 1 pp 205ndash291 2013

[8] P K L Ng and H H Ng ldquoHemibagrus gracilis a new speciesof large riverine catfish (Teleostei Bagridae) from PeninsularMalaysiardquo Raffles Bulletin of Zoology vol 43 pp 133ndash142 1995

[9] H H Tan and M Kottelat ldquoRedescription of Betta picta(Teleostei Osphronemidae) and description of Betta falx sp nfrom central Sumatrardquo Revue Suisse de Zoologie vol 105 no 3pp 557ndash568 1998

[10] J J Dodson F Colombani and P K Ng ldquoPhylogeographicstructure in mitochondrial DNA of a South-east Asian fresh-water fish Hemibagrus nemurus (Siluroidei Bagridae) andPleistocene sea-level changes on the Sunda shelfrdquo MolecularEcology vol 4 no 3 pp 331ndash346 1995

[11] H H Ng and J J Dodson ldquoMorphological and genetic descrip-tions of a new species of catfish Hemibagrus chrysops fromSarawak East Malaysia with an assessment of phylogeneticrelationships (Teleostei Bagridae)rdquo Raffles Bulletin of Zoologyvol 47 no 1 pp 45ndash57 1999

[12] P D N Hebert A Cywinska S L Ball and J R DeWaardldquoBiological identifications through DNA barcodesrdquo Proceedingsof the Royal Society B Biological Sciences vol 270 no 1512 pp313ndash321 2003

[13] J C Carolan T EMurray U Fitzpatrick et al ldquoColour patternsdo not diagnose species quantitative evaluation of a DNAbarcoded cryptic bumblebee complexrdquo PLoS ONE vol 7 no 1Article ID e29251 2012

[14] M Kekkonen and P D N Hebert ldquoDNA barcode-baseddelineation of putative species efficient start for taxonomicworkflowsrdquoMolecular Ecology Resources vol 14 no 4 pp 706ndash715 2014

[15] DM Hillis CMoritz and B KMarbleMolecular SystematicsSinauer Sunderland Mass USA 2nd edition 1996

[16] T D Kocher W K Thomas A Meyer et al ldquoDynamics ofmitochondrial DNA evolution in animals amplification andsequencingwith conserved primersrdquoProceedings of theNationalAcademy of Sciences of the United States of America vol 86 no16 pp 6196ndash6200 1989

[17] T A Hall ldquoBioEdit a user-friendly biological sequence align-ment editor and analysis program for Windows 9598NTrdquoNucleic Acids Symposium Series vol 41 pp 95ndash98 1999

[18] D A Benson I Karsch-Mizrachi K Clark D J Lipman JOstell and EW Sayers ldquoGenBankrdquoNucleic Acids Research vol40 no D1 pp D48ndashD53 2012

[19] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[20] X Ku Z Peng R Diogo and S He ldquoMtDNA phylogenyprovides evidence of generic polyphyleticism for East Asianbagrid catfishesrdquoHydrobiologia vol 579 no 1 pp 147ndash159 2007

[21] L Yang and S He ldquoPhylogeography of the freshwater catfishHemibagrus guttatus (Siluriformes Bagridae) implications forSouth China biogeography and influence of sea-level changesrdquoMolecular Phylogenetics and Evolution vol 49 no 1 pp 393ndash398 2008

[22] B W Bowen A L Bass L A Rocha W S Grant and DR Robertson ldquoPhylogeography of the trumpetfishes (Aulosto-mus) ring species complex on a global scalerdquo Evolution vol 55no 5 pp 1029ndash1039 2001

[23] Y P Kartavtsev ldquoDivergence at Cyt-b and Co-1 mtDNA geneson different taxonomic levels and genetics of speciation inanimalsrdquoMitochondrial DNA vol 22 no 3 pp 55ndash65 2011

[24] SWada Y Kameda and S Chiba ldquoLong-term stasis and short-term divergence in the phenotypes of microsnails on oceanicislandsrdquoMolecular Ecology vol 22 no 18 pp 4801ndash4810 2013

[25] J Colborn R E Crabtree J B Shaklee E Pfeiler and B WBowen ldquoThe evolutionary enigma of bonefishes (Albula spp)cryptic species and ancient separations in a globally distributedshorefishrdquo Evolution vol 55 no 4 pp 807ndash820 2001

[26] J-D Durand K-N Shen W-J Chen et al ldquoSystematics of thegrey mullets (Teleostei Mugiliformes Mugilidae) molecularphylogenetic evidence challenges two centuries ofmorphology-based taxonomyrdquo Molecular Phylogenetics and Evolution vol64 no 1 pp 73ndash92 2012

[27] K-N Shen B W Jamandre C-C Hsu W-N Tzeng and J-D Durand ldquoPlio-Pleistocene sea level and temperature fluc-tuations in the northwestern Pacific promoted speciation inthe globally-distributed flathead mullet Mugil cephalusrdquo BMCEvolutionary Biology vol 11 no 1 article 83 2011

[28] R Hall ldquoThe plate tectonics of Cenozoic SE Asia and thedistribution of land and seardquo in Biogeography and GeologicalEvolution of SE Asia R Hall and J D Holloway Eds pp 99ndash131Backhuys Publishers Leiden The Netherlands 1998

[29] R Hall ldquoSoutheast Asiarsquos changing palaeogeographyrdquo Blumeavol 54 no 1ndash3 pp 148ndash161 2009

[30] R F Inger and P K Chen ldquoThe freshwater fishes of NorthBorneordquo Fieldiana Zoology vol 45 pp 1ndash268 1962

[31] J Bohlen V Slechtova H H Tan and R Britz ldquoPhylogenyof the Southeast Asian freshwater fish genus Pangio (Cyprini-formes Cobitidae)rdquoMolecular Phylogenetics and Evolution vol61 no 3 pp 854ndash865 2011

[32] M de Bruyn E Nugroho M M Hossain J C Wilson andP B Mather ldquoPhylogeographic evidence for the existence ofan ancient biogeographic barrier the Isthmus of Kra SeawayrdquoHeredity vol 94 no 3 pp 370ndash378 2005

[33] J Parnell ldquoThe biogeography of the Isthmus of Kra region areviewrdquoNordic Journal of Botany vol 31 no 1 pp 001ndash015 2013

[34] S Usmani S G Tan S S Siraj and K Yusoff ldquoPopulationstructure of the Southeast Asian river catfishMystus nemurusrdquoAnimal Genetics vol 34 no 6 pp 462ndash464 2003

[35] M P Tan A F J Jamsari and M N Siti Azizah ldquoPhylo-geographic pattern of the striped snakehead Channa striatain Sundaland ancient river connectivity geographical and

Advances in Evolutionary Biology 21

anthropogenic singnaturesrdquo PLoS ONE vol 7 no 12 Article IDe52089 2012

[36] R Abell M L Thieme C Revenga et al ldquoFreshwater ecore-gions of the world a new map of biogeographic units forfreshwater biodiversity conservationrdquo BioScience vol 58 no 5pp 403ndash414 2008

[37] P U Clark D Archer D Pollard et al ldquoThemiddle Pleistocenetransition characteristics mechanisms and implications forlong-term changes in atmospheric pCO

2rdquo Quaternary Science

Reviews vol 25 no 23-24 pp 3150ndash3184 2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

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International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

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BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

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Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

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Enzyme Research

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International Journal of

Microbiology

Page 13: ResearchArticle - Université Laval Julian... · ResearchArticle A DNA Barcode-Based Evaluation of the Southeast Asian Catfish Genus Hemibagrus Bleeker, 1862 (Teleostei: Siluriformes;

Advances in Evolutionary Biology 13

100∘ 110∘

0∘

120∘

00 250

250500

500750

(km)

(mi)

FOKIN0102FOSGM0606

FOUMA0201H fortis

10∘

99

86

001

Shelf minus75mShelf minus120 m

Figure 9 Phylogenetic relationships within H fortis comprised of 3 sequences The subtree is extracted from the taxon identificationphenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicate trees in which the associated sequencesclustered together in the bootstrap test (500 replicates) is shown next to the branches The distribution of these sequences is illustrated in theright panel

the Kapuas River (West Borneo) and a second sequence(HMUBHKABM23 Table S1) was found inMuarRiver (WestPeninsular Malaysia) and the Batang Hari (Sumatra) theKapuas (Sarawak) and the Baram Rivers (Sarawak) Many ofthese fish were identified as H hoevenii by NampK (Table S1)The existence of twodistinct but very closely related subclades(Figure 10 119901 = 06 (03)) that are not geographicallysegregated suggests some recent isolation event followed bywidespread dispersal and secondary contact

33 The H capitulum Group The final two monophyleticgroups of the Hemibagrus gene tree are approximatelyequivalent to the remaining species composing the Hnemurus species group as defined by NampK Dodson etal [10] and Ng and Dodson [11] have previously referredto clade 10 as the Sundaic clade as most sequences wereobtained from fish sampled on the Greater Sunda Islandsand clade 11 as the Indochinese clade asmany sequences wereobtained from fish sampled on the Indochinese mainland(Figure 2 Table S1) Although most of these sequencesare assigned to H capitulum by NampK there are somesignificant phylogeographic differences between the 2 cladesIn particular there appears to be an east-west division in thedistribution of the two clades on the Thai-Malay Peninsulawith the Sundaic clade (clade 10) found on the west coast andthe Indochinese clade found on the east coast with sympatricoccurrence in the Muar River Nevertheless mean sequencedivergence between the two clades is 119901 = 29 (05) avalue that is indicative of population-level or subspecies

divergence The two clades are thus best considered ascomprising one species

mtDNA Clade 10 The Sundaic clade includes a number ofclosely related sequences that comprise part of H capitulumand H nemurus Very little genetic structure is apparentand bootstrap support for tree topology within this group isgenerally very weak (Figure 11) A widely spread genotype(NCLCTBA20) characterized 6 of 17 specimens from CiTarum at the Cirata reservoir (West Java) (Table S1) and5 were identified as H nemurus in the analysis of NampK(ZRC 42564) (Table S1) The remaining fish characterized bythis sequence were obtained from Ci Liwong at Sawangan(West Java) and one fish was obtained from the Barito Riverat Banjarmassin south Borneo This latter specimen (ZRC40553) was identified by NampK as H capitulum (Table S1)Another common genotype (NBHKPBR09) was identified aseither H capitulum H hoevenii or H nemurus dependingon the river of origin (Table S1) Many of the remainingsequences of the Sundaic clade appear to characterize fish thatNampK identified asH capitulum A geographically widespreadgenotype (CPEBEMUKA36) characterized 9 fish sampledon the Perak River at Gerik that were identified by NampKas H capitulum (ZRC 41150 Table S1) Another commongenotype within this clade (CABHMS20) included 2 fishfrom the Batang Hari at Jambi and identified by NampK asH capitulum (ZRC40534 Table S1) Another 2 sequences(CABAR0203 CABAR0305) characterized 8 fish sampledon the Barito River at Banjarmasin and were identified as

14 Advances in Evolutionary Biology

100∘

10∘

110∘

0∘

120∘

0

0 250

250

500

500

750(km)

(mi)

53

64

99

85

64

0002

HJOMSKA11

HOBAH0601

HOBHMS05

HMUBHKABM23

HOBAH0701

HOKAP0501

H hoevenii ldquoBrdquo

H hoevenii ldquoArdquo

Shelf minus75mShelf minus120 m

Figure 10 Phylogenetic relationships withinH hoevenii comprised of 2 subclades and 6 sequences The subtree is extracted from the taxonidentification phenogram inferred using theNeighbor-Joiningmethod (see Figure 2)The percentage of replicate trees in which the associatedsequences clustered together in the bootstrap test (500 replicates) is shown next to the branches The distribution of these sequences isillustrated in the right panel

110∘

XXMAH0110

CABAR0305

96 CAKPR0201

74CAKAP0401

5931 CAKAP0601

35 CPEBEMUKA36

646 CAKAP0903

33 CAKAP080114 CAKAP0701

CARAJ0210

4 CABAR0203CABRA0204

20

CBHKPBR0919

CABRA0301

15CABHMS20

24 NCLCTBA20

63 NECIT0201

H capitulumldquoSundaic claderdquo

H nemurus

0002 120∘

0 250 500 750

(km)

0 250 500

(mi)100

10∘

0∘

Shelf minus75mShelf minus120 m

Figure 11 Phylogenetic relationships within theH capitulum ldquoSundaicrdquo clade including 2 sequences associated withH nemurusThe subtreeis extracted from the taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicatetrees in which the associated sequences clustered together in the bootstrap test (500 replicates) is shown next to the branchesThe distributionof these sequences is illustrated in the right panel

Advances in Evolutionary Biology 15

H capitulum

0∘

10∘

100∘

110∘

120∘

66

63

88

59

38

39 68

71

6991

57

9754

88 4944

39

53

89

0002

ldquoIndochineserdquogroup ldquoErdquo

H capitulumldquoIndochineserdquo

group ldquoDrdquoH capitulumldquoIndochineserdquo

group ldquoCrdquo

H capitulumldquoIndochineserdquo

group ldquoBrdquo

H capitulumldquoIndochineserdquo

group ldquoArdquo0

0

250

250

500

500

750

(km)

(mi)

CATER0301CATERR0401

CKETEMU23

CAMEK0601CACHP0301

CAMKPA03

CAMEK0910CAMEK1003

XXMUBM02

CABMTU09

CABRM0601

CAKAP1201CAKAP1001

CAKAP1101CAKAP1301

CABRM0501

CASAD0102

CASAD0220

CAEND0301

CABAH0801

CAPABH05CATEEN03

CATER0701CATERR0801

CAKETE04CATER0501

CAPAH0303

Shelf minus75mShelf minus120 m

Figure 12 Phylogenetic relationships within theH capitulum ldquoIndochineserdquo clade encompassing 5 subclades identified as A B C D and EThe subtree is extracted from the taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2)The percentageof replicate trees in which the associated sequences clustered together in the bootstrap test (500 replicates) is shown next to the branchesThedistribution of these sequences is illustrated in the right panel

H capitulum by NampK (ZRC 40553 Table S1) Although notincluded in the analysis of NampK samples from the Sundaicclade were also found in the Kapuas River (West Borneo)Finally 10 fish sampled on the Mahakam River at Samarindaeast Borneo are all characterized by one relatively divergentgenotype (CAMAH0110) butwere considered asH capitulumin the analysis of NampK (ZRC 40533 Table S1)TheMahakampopulation sample is the most genetically distinct of theSundaic clade (119901 = 17 (04)) but nevertheless indicativeof intraspecific population-level divergence (Figure 12)

The topology of the Sundaic clade does not permit usto draw a clear distinction between H nemurus and theSundaic H capitulum The mean within-group divergenceof the Sundaic clade is only 119901 = 08 (02) well withinpopulation-level divergence values We postulate that Hnemurus may be one of several regional variants of awidespread Sundaic H capitulum clade that has radiatedrelatively recently This may explain the existence of manyshared sequences throughout the Sundaic islands assigned todifferent species in different regions

mtDNA Clade 11 Finally the Indochinese clade of Hcapitulum represents a geographically widespread andweakly diversified clade (within-group mean sequencedivergence 119901 = 11 (02) Figure 12) Although few fishfrom this genetic clade were included in the morphological

analysis of NampK most may be considered as H capitulumgiven their geographic distribution and close geneticrelationship to the Sundaic H capitulum Four sequencesin this clade were identified as H capitulum in the analysisof NampK 3 specimens characterized by one sequence(CABMTU09) found in the Tutong River of westernBorneo (ZRC 40482) 6 characterized by the same sequencefrom the Baram River (ZRC 40498) another 2 sequencesfrom the Baram River (ZRC 40498) and 20 specimenscharacterized by an abundant genotype (CASAD0220)found at 2 sites in the Sadong River also in western Borneo(ZRC 39526 5 specimens 39527 3 specimens and 38753 4specimens) (Table S1) Unfortunately none of the specimenscollected on the Indochinese mainland for genetic analysiswere examined by NampK Nevertheless the distribution ofspecimens analyzed for mtDNA resembles the distribution ofspecimens examined by NampK Although weakly structuredwe may recognize 5 subclades One genotype (CAPAH0303)is basal to the phenogram Subclade A encompasses 8sequences mainly found in eastern Peninsular Malaysia(Table S1 Figure 12) These rivers furnished many of thespecimens NampK examined to define H capitulum Howeversome sequences in this group occur in sympatry withsequences identified within the Sundaic H capitulum groupTwo sequences from subclade A occurred in the Batang HariSumatra (Table S1) Subclade E also encompasses sequences

16 Advances in Evolutionary Biology

found in Peninsular Malaysia as well as in the Mekong ChaoPhraya and Mea Khlong Rivers (Figure 12) In addition3 subclades encompass sequences found principally inwestern Borneo subclade B in the Baram and Sadong RiversSarawak subclade C in the Kapuas River West Kalimantanand subclade D in the Baram and Tutong Rivers of Sarawakand Brunei respectively (Figure 12) This latter subclade alsoincludes one genotype found in the Muar River of westernpeninsular Malaysia Finally subclade E encompasses 5sequences from the Mekong Chao Praya and Mae KhlongRivers all within the distribution area of H spilopterus

EvolutionaryHistoryThe evolutionary history of theHemiba-grus species groups analyzed here dates from at least theMiocene 15ndash20Ma (Figure 13) when considering the widelyused molecular clock of 2 sequence divergence per millionyears Bootstrap support for the sister group relationships ofH wyckioides theH guttatus species group andH peguensisare all weak (clearly due to undersampling) and little can besaid of their evolutionary history other than the fact that theyoccupy basal positions in the rooted trees The remainder ofboth the NJ and ML trees is composed of two major sistergroups the H planiceps + H baramensis group and the Hnemurus species group (Figures 2 3 and 13)TheH planiceps+ H baramensis group is strongly supported in both NJ andML analyses (84 and 90 bootstrap support resp Figures2 and 3) The H nemurus species group is also stronglysupported in both analyses (98 and 99 bootstrap supportFigures 2 and 3) These two major phylogenetic groups areestimated to have diverged at least 8 to 12Ma ago during themid to late Miocene (Figure 13)

Much of the evolutionary history of Hemibagrus hasoccurred during the Quaternary starting approximately26Ma ago This is particularly true of the H planicepsand H nemurus species groups The radiation of the 5species included in the H planiceps species group appearsto coincide with the middle Pleistocene transition period ofapproximately 1Ma ago Their high-elevation distributionhowever appears to have largely isolated them from theeffects of lowered sea levels during glacial maxima and theattendant opportunities for widespread dispersal (Figure 5)The evolutionary histories of theH baramensis species groupand the unspecified species group date back to the latePliocene In contrast the low-elevation distribution of theHnemurus species group in Sundaland appears to have favoredtheir widespread distribution during the Quaternary ice ageIn particular H hoevenii appears to be the most recentlydispersed of the species under study with diversification anddispersal occurring approximately 03Ma ago (Figure 13)

4 Discussion

41 The Congruence of Morphological and Genetic AnalysesCombining genetic and morphological analyses to elucidatetaxonomy allowed us to better understand the diversificationand evolutionary relationships within this speciose group offishes Given the rapid and relatively constant rate of mtDNAevolution the deeper the branches within the phylogenetictree are the greater our ability to distinguish valid taxa

should be Conversely the lack of complete congruencebetween morphological- and genetic-based identificationsis expected to increase among the shallower branches of thephylogenetic tree resulting in the appearance of polyphyleticspecies Although 2 of the 8 species groups defined by Ngand Kottelat [7] were not sampled in this study the validityof the remaining 6 species groups and their componentspecies defined on the basis of morphological similaritywas largely confirmed by the analysis of the mitochondrialCyt 119887 bar code As expected congruence is strongest at thebase of the NJ and ML trees where the H guttatus speciesgroup formed a well-defined monophyletic group The Hmenoda and H wyckii species group were each representedby only one species but they were clearly distinct relativeto sequences characterizing the neighboring species groupsThe H planiceps H baramensis and H nemurus speciesgroups and their component species were also generallycongruent in morphological and genetic analyses Also asexpected most cases of discrepancy between morphological-based species identification and genetic-based speciesidentification occurred on relatively shallow branches of thephylogenetic tree These involve principally H spilopterusand the two mtDNA subclades composing H capitulumHowever a far more unexpected lack of congruence involvesthe definition of a deeply divergent unspecified genetic groupconfounding members of theH baramensis species group inthe morphological analysis of Ng and Kottelat [7]

Interpreting Polyphyly The apparent polyphyletic origin ofa species may be due to incomplete lineage sorting amongrecently diverged species the introgression of mtDNA dueto species hybridization or the erroneous identificationof species due to the lumping of morphologically crypticspecies In the absence of nuclear DNA analyses it isoften difficult to distinguish between introgression andincomplete lineage sorting For example several sequencescharacterizing the Indochinese clade of H capitulum(subclade E) are found in the Mekong and Chao PhrayaRivers well within the distributional area of H spilopterusH spilopterus and H capitulum first diverged at least 3MaOn the one hand this apparent case of polyphyly mayhave resulted from incomplete lineage sorting betweenrecently divergedH spilopterus andH capitulum We wouldhowever have expected similar cases of polyphyly amongpopulations of the Indochinese clade of H capitulum butthis was not detected On the other hand the sympatricoccurrence of H spilopterus subclade A and subcladeB sequences with H capitulum Indochinese subclade Esequences in the Mekong River may represent a migrationevent and local introgression of H capitulum with Hspilopterus A similar event may have occurred in theChao Phraya and Mae Khlong Rivers where H spilopterussequences cooccur withH capitulum sequences Computingthe divergence of H capitulum sequences (Indochinesesubclade E) cooccurring with H spilopterus and Hcapitulum sequences occurring in eastern PeninsularMalaysia (subclade A) reveals a mean sequence divergence119901 = 15 (04) Thus the H capitulum sequences occurringin sympatry with H spilopterus diverged at most 750 ka ago

Advances in Evolutionary Biology 17

H fortis

XXRAJ0604H furcatus

H sabanusH semotusH divaricatus

H planicepsGREND0206

VEBAH0907

H bonganPESAL0102

H wyckioidesH macropterus

H guttatusXXRAJ0301Leiocassis longirostris

Ma

4971

92

47

51

9999

99

9999

99

99

99

9999

99

99

99

99

65

96

96

4946

93

79

78

70

91

93

98

86

87

91

95

97

71

85

9999

05101520

005010015020 000

H capitulum Indochinese clades AndashE

H capitulum Sundaic clade

H hoevenii B

H hoevenii A

H spilopterus C

H spilopterus B

H spilopterus AH spilopterus A

H spp CH spp B

H spp A

Figure 13 Linearized tree with branch lengths estimated with the molecular clock using a mutation rate of 1 per Ma (=divergence rate of2 per Ma) The tree topology is identical to the ML reconstruction illustrated in Figure 3 (see the specificities of the tree reconstruction inthe caption of Figure 3) Boxes represent 95 confidence intervals of divergence time estimates for each node Vertical dashed lines redirectto dates indicated on the timescale (Ma) Numbers below timescale indicate the estimated number of substitution changes using Hasegawa-Kishino-Yanomodel (HKY)with gamma distribution (+119866 = 13290) after linearizing the treeH furcatus is synonymizedwithH baramensis

from eastern Peninsular Malaysia populations suggestinga recent introgression event Similarly H capitulum in theKapuas River is characterized by sequences of both theSundaic and the Indochinese clades As both sequences arebroadly sympatric it is not possible to distinguish betweenincomplete lineage sorting and introgression of these closelyrelated intraspecific lineages

A Possible Case of Morphological Stasis Clade 6 of thepresent analysis defines a well-supported monophyletic cladefound in Sabah Sarawak and the Endau River of easternpeninsula Malaysia that has no equivalent taxonomic statusin the revision of Ng and Kottelat [7] In Borneo they occursympatrically with the H baramensis species group and areconfounded as either H baramensis or H semotus These

two clades are genetically highly divergent having divergedbetween 8 and 12Ma ago It thus seems highly unlikely thatthey represent retained polymorphisms due to incompletelineage sorting Sequences previously identified as character-izing H chrysops [11] are part of clade 6 and extend to thesouth (Rajang and Sadong Rivers of Sarawak West Borneo)with one closely related genotype found in eastern peninsularMalaysia (Endau River) where it may well be confoundedwith H capitulum H chrysops was synonymized with Hcapitulum by Ng and Kottelat [7] based on their analysis ofspecimens ofH chrysops from the Rajang RiverThus despitea long and apparently independent phylogenetic historythe unspecified species group appears largely cryptic andconfounded principally with members of the H baramensisspecies group

18 Advances in Evolutionary Biology

We suggest that this situation is an example of mor-phological stasis involving the coexistence of a number ofgenetically divergent yet morphologically cryptic species thatare lumped together [24] A striking example of morpho-logical and ecological stasis among fishes is provided by thecoastal marine bonefishes (Albula spp) Distinct evolution-ary lineages (separated by sequence divergence between 5 and30 [25]) of bonefish coexist in several sample locations yetthey show little morphological or ecological differentiationin sympatry [25] Similarly a proportion of the grey mulletspecies (Mugilidae) also appears to consist of cryptic species[26] For exampleMugil cephalus in Taiwan is characterizedby three divergent lineages that have been shown to charac-terize 3 reproductively isolated but morphologically crypticspecies [26 27]

Here we have discovered 2 highly distinct and structuredclades (each composed of 3 mtDNA subclades) that aremorphologically confounded within different species Forexample H spp C of the unspecified species group isidentified as H baramensis in the Baram River and as Hsemotus in the Padas River We speculate that North Borneoharbors a morphologically cryptic species complex possiblycomposed of several species pairs inhabiting different riversystems The reconstruction of this phylogenetic history andthe taxonomic status of its components remains a majorchallenge to the revision of the Hemibagrus genus

42 Phylogeographic Hypotheses The phylogeography ofthe genus appears to have been principally influenced bytwo geological histories one encompassing the formationof North Borneo the second the emergence of the SundaIslands and their connectivity with the Indochinesemainlandassociated with recurrent sea-level changes North Borneoappears to have acted as a species sink promoting speciationand locally high biodiversity within the genus but with littleevidence of subsequent dispersal out of North Borneo Incontrast the geological events influencing the Indochinesemainland and the Sunda Islands have permitted bothspeciation and differential dispersal depending on thebiology of the species For example the H planiceps speciesgroup is found throughout the Greater Sunda Islands andtheThai-Malay Peninsula as is theH nemurus species groupWhereas the H nemurus species group generally occupiesthe low altitude lower reaches of large rivers the speciescomposing the H planiceps group are found in the upperreaches of large rivers The H nemurus species group iscomposed of a number of widely distributed closely relatedspecies whereas theH planiceps group is composed of a largenumber of species that are highly localized geographicallyIt thus appears that sea-level changes have had little impacton the dispersal history of the H planiceps species group buthave deeply influenced that of the H nemurus species group

The Case of North Borneo From 15Ma to 5Ma greater areasof Borneo became emergent and the central mountainson the Sarawak-Kalimantan border extending into Sabahbecame wider and higher which may have represented newbarriers to dispersal [28 29] Borneo gradually developedinto the present large island by emergence of land in the

north and east The H baramensis species group is restrictedto North Borneo as is the unspecified species group of theHnemurus species group H fortis is also distributed in NorthBorneo The isolation of the freshwater fish communities ofNorth Borneo (particularly in the Labuk-Segama watershed)has long been recognized based on the number of endemicspecies and the concentration of relict species [30] The latePliocene-early Pleistocene diversification of H fortis theunspecified species group and the H baramensis speciesgroup may thus be associated with their dispersal to andisolation within the watersheds of North Borneo Only onesequence of the unspecified species group is found outsideof North Borneo and H fortis appears to be distributedthroughout central and east Borneo [7]

ThePhylogeography of the H nemurus Species Group Periodicmarine transgressions on the Sundaland platform during theearly and middle Miocene [2 29] may have been sufficientto isolate populations of freshwater organisms and promotecladogenesis [31] This may have been responsible for theinitial mid to late Miocene divergence of the H nemurusspecies group The majority of sequences assigned to thisspecies group belong to 3 species (H spilopterus H capitulum(including H nemurus) and H hoevenii) all of which havebeen influenced by the more recent geological history of theSunda Islands and their connectivity with the Indochinesemainland H spilopterus is known from river drainages inIndochina including the Mekong Dong Nai Ban PakongChao Phraya and Mae Klong where it is found in a widevariety of lotic and lentic habitats [7] H spilopterus divergedin the late Pliocene-early Pleistocene possibly isolated tothe north of the Kra Peninsula during the late Pliocenemarine transgression that is purported to have created amajor biogeographic transition zone between the Sundaicand Indochinese biotas ([32] but see [33] for a critique ofthis biogeographic scenario) The existence of 2 subclades(A and B) in the Mekong River separated by 2 sequencedivergence is consistent with intraspecific divergence Thetwo subclades possibly represent 2 historical mtDNA lineagesthat are retained within the species reflecting a Pleistoceneisolation event followed by secondary contact within thecontemporary Mekong River Subclade C from the ChaoPhraya and Mae Khlong Rivers exhibits a level of geneticdivergence consistent with sibling species-level divergenceshowing a 5 sequence divergence relative to Mekong RiverH spilopterus

H capitulum may be considered as part of the Sundaicbiota located principally to the south of the Kra marinetransgression zone that may have isolated H spilopterus tothe north on the Indochinese mainlandThe biogeographicalevent leading to the subsequent formation of two intraspecificgroups within H capitulum is not at all clear The onlyarea where the two clades are clearly geographically separateis on the Thai-Malay Peninsula with the ldquoSundaicrdquo cladeon the west coast and the ldquoIndochineserdquo clade on theeast coast separated by the central Titiwangsa mountainrange The genetic distinctiveness of east and west coastpopulations of H capitulum has also been documented fornuclear DNA [34] A similar pattern of separation has been

Advances in Evolutionary Biology 19

reported for 5 additional freshwater fishes within this region[35] However as the divergence of the two H capitulumclades postdates the formation of the mountain range byapproximately 200Ma the geographic separation of the twoclades most likely represents differential dispersal rather thanancient vicariance The presence of the Sundaic clade on thewest coast of the Thai-Malay Peninsula as well as on theSundaic Islands reflects the historical connections betweeneast Sumatra and west Peninsular Malaysia across the Straitof Malacca This area encompasses the Northern CentralSumatra-Western Malaysia freshwater ecoregion [36] TheMuar River forms its southern border coincident with thesouthernmost distribution of the Sundaic clade on the Thai-Malay Peninsula It is important to note however that mostof Sumatra and Java emerged to achieve their present size andelevation only 5Ma ago and much of East Java continuedto be the site of marine deposition until late Pliocene-earlyPleistocene [29] We may speculate that the Sundaic clade ofH capitulum resulted from the relatively recent colonizationof these islands and Borneo The radiation within the cladeonly dates back to approximately 1Ma ago H nemurus fromWest Java may thus best be considered an isolated regionalpopulation of very recent origin

The diversification of the Indochinese clade dates fromat least 12Ma ago coincident with the mid-Pleistocenetransition at which time low-frequency high-amplitude andquasiperiodic (100 ka) glacial variability emerged It began125Ma ago and was complete by 07Ma ago [37] The tran-sition marked the beginning of major changes in sea levelsduring which the Sunda Shelf periodically emerged and wastraversed by enormous fluvial systems [3] The contributionof these events to the diversification of this clade cannotbe satisfactorily reconstructed One possible explanation isthat high sea levels produced barriers to gene flow and thepopulations of West Borneo (subclades B C and D) wereisolated from those of east Peninsular Malaysia This couldbe due to one or more dispersal events from Borneo via theGreat Sunda River (Figure 1) to east Peninsular Malaysia orlineage sorting whereby sequences of subclades A and E havebeen lost from West Borneo As in the case of the sympatricoccurrence of H spilopterus and H capitulum subclade E(see above) in the Mekong River we are unable to clearlydefine the relative roles of incomplete lineage sorting andintrogression followingmigration Given the recent origins ofthe Indochinese H capitulum sequences incomplete lineagesorting most probably blurs patterns of recent isolation

The last species of the H nemurus species group is Hhoevenii The species is widespread throughout PeninsularMalaysia and the Sundaic Islands It is found primarily inthe lower reaches of rivers and is apparently capable ofwithstanding low salt concentration [7] H hoevenii thusappears to be the most liable of all members of the Hnemurus species group to be impacted by Pleistocene sea-level changes This is clearly reflected in the widespreaddistribution of the sequences of this species Two lineages areevident having diverged no more than 03Ma ago suggest-ing a late Pleistocene isolation event followed by extensivedispersal and admixture in recent times This represents theclearest example of the dispersal opportunities afforded by

Pleistocene sea level changes on the Sunda Shelf In additionthe relatively low genetic diversity detected (only 6 sequencesin 42 fish) suggests a population bottleneck in the recent pastpossibly related to population declines in the lower reaches ofrivers flooded by increasing sea levels

43 Future Directions We have provided a global picture ofthe evolutionary taxonomy of the genus in Southeast AsiaClearly a more rigorous phylogenetic reconstruction of thegenus Hemibagrus requiring the construction of multiplegene trees based on the analysis of both mitochondrial andnuclear DNA markers from a wider taxonomic array ofhemibagrids will involve an even greater effort of samplingthroughout the region In particular we require samplingat smaller spatial and temporal scales For example smallerscale sampling in the Padas River and the Segama Riverof North Borneo using both mitochondrial and nucleargenetic markers would contribute to clarifying the natureof the cryptic species complex believed to inhabit theserivers The demonstration of reproductive isolation andormicroallopatric distribution among morphologically crypticspecies may result in the description of greater speciesrichness within the genus than presently described Thespecies status of H chrysops merits reconsideration givenits highly divergent evolutionary history Nuclear geneticmarkers in conjunction with mtDNA markers could revealcases of historical introgression particularly in the KapuasRiver and the Mekong River The hemibagrid catfishes con-tinue to provide multiple opportunities for the study of theevolutionary taxonomy and biogeography of a major faunalcomponent of the fluvial ecosystems of Southeast Asia

Disclosure

Ethanol-preserved tissue samples of many of the specimensidentified in Table S1 are freely available upon request fromJJD

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Goh Yan Yih for assistance in collectingspecimens and molecular analyses Mrs Francoise Colom-bani for molecular analyses and Dr Heok Hee Ng forspecies identification This work was funded by the NationalUniversity of Singapore grant to P K L Ng and a CanadianNatural Sciences and Engineering Research Council grant toJ J Dodson

References

[1] D J Lohman M de Bruyn T Page et al ldquoBiogeography ofthe Indo-Australian archipelagordquo Annual Review of EcologyEvolution and Systematics vol 42 pp 205ndash226 2011

20 Advances in Evolutionary Biology

[2] D S Woodruff ldquoNeogene marine transgressions palaeogeog-raphy and biogeographic transitions on the Thai-Malay Penin-sulardquo Journal of Biogeography vol 30 no 4 pp 551ndash567 2003

[3] H K Voris ldquoMaps of Pleistocene sea levels in SoutheastAsia shorelines river systems and time durationsrdquo Journal ofBiogeography vol 27 no 5 pp 1153ndash1167 2000

[4] S Y Yap ldquoOn the distributional patterns of Southeast-EastAsian freshwater fish and their historyrdquo Journal of Biogeographyvol 29 no 9 pp 1187ndash1199 2002

[5] S K J McConnell ldquoMapping aquatic faunal exchanges acrossthe Sunda shelf South-East Asia using distributional andgenetic data sets from the cyprinid fish Barbodes gonionotus(Bleeker 1850)rdquo Journal of Natural History vol 38 no 5 pp651ndash670 2004

[6] T Mo Anatomy Relationships and Systematics of the Bagridae(Teleostei Siluroidei) with a Hypothesis of Siluroid Phylogenyvol 17 ofTheses Zoologicae Koeltz Scientific Books 1991

[7] H H Ng and M Kottelat ldquoRevision of the Asian catfish genusHemibagrus bleeker 1862 (Teleostei Siluriformes Bagridae)rdquoRaffles Bulletin of Zoology vol 61 no 1 pp 205ndash291 2013

[8] P K L Ng and H H Ng ldquoHemibagrus gracilis a new speciesof large riverine catfish (Teleostei Bagridae) from PeninsularMalaysiardquo Raffles Bulletin of Zoology vol 43 pp 133ndash142 1995

[9] H H Tan and M Kottelat ldquoRedescription of Betta picta(Teleostei Osphronemidae) and description of Betta falx sp nfrom central Sumatrardquo Revue Suisse de Zoologie vol 105 no 3pp 557ndash568 1998

[10] J J Dodson F Colombani and P K Ng ldquoPhylogeographicstructure in mitochondrial DNA of a South-east Asian fresh-water fish Hemibagrus nemurus (Siluroidei Bagridae) andPleistocene sea-level changes on the Sunda shelfrdquo MolecularEcology vol 4 no 3 pp 331ndash346 1995

[11] H H Ng and J J Dodson ldquoMorphological and genetic descrip-tions of a new species of catfish Hemibagrus chrysops fromSarawak East Malaysia with an assessment of phylogeneticrelationships (Teleostei Bagridae)rdquo Raffles Bulletin of Zoologyvol 47 no 1 pp 45ndash57 1999

[12] P D N Hebert A Cywinska S L Ball and J R DeWaardldquoBiological identifications through DNA barcodesrdquo Proceedingsof the Royal Society B Biological Sciences vol 270 no 1512 pp313ndash321 2003

[13] J C Carolan T EMurray U Fitzpatrick et al ldquoColour patternsdo not diagnose species quantitative evaluation of a DNAbarcoded cryptic bumblebee complexrdquo PLoS ONE vol 7 no 1Article ID e29251 2012

[14] M Kekkonen and P D N Hebert ldquoDNA barcode-baseddelineation of putative species efficient start for taxonomicworkflowsrdquoMolecular Ecology Resources vol 14 no 4 pp 706ndash715 2014

[15] DM Hillis CMoritz and B KMarbleMolecular SystematicsSinauer Sunderland Mass USA 2nd edition 1996

[16] T D Kocher W K Thomas A Meyer et al ldquoDynamics ofmitochondrial DNA evolution in animals amplification andsequencingwith conserved primersrdquoProceedings of theNationalAcademy of Sciences of the United States of America vol 86 no16 pp 6196ndash6200 1989

[17] T A Hall ldquoBioEdit a user-friendly biological sequence align-ment editor and analysis program for Windows 9598NTrdquoNucleic Acids Symposium Series vol 41 pp 95ndash98 1999

[18] D A Benson I Karsch-Mizrachi K Clark D J Lipman JOstell and EW Sayers ldquoGenBankrdquoNucleic Acids Research vol40 no D1 pp D48ndashD53 2012

[19] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[20] X Ku Z Peng R Diogo and S He ldquoMtDNA phylogenyprovides evidence of generic polyphyleticism for East Asianbagrid catfishesrdquoHydrobiologia vol 579 no 1 pp 147ndash159 2007

[21] L Yang and S He ldquoPhylogeography of the freshwater catfishHemibagrus guttatus (Siluriformes Bagridae) implications forSouth China biogeography and influence of sea-level changesrdquoMolecular Phylogenetics and Evolution vol 49 no 1 pp 393ndash398 2008

[22] B W Bowen A L Bass L A Rocha W S Grant and DR Robertson ldquoPhylogeography of the trumpetfishes (Aulosto-mus) ring species complex on a global scalerdquo Evolution vol 55no 5 pp 1029ndash1039 2001

[23] Y P Kartavtsev ldquoDivergence at Cyt-b and Co-1 mtDNA geneson different taxonomic levels and genetics of speciation inanimalsrdquoMitochondrial DNA vol 22 no 3 pp 55ndash65 2011

[24] SWada Y Kameda and S Chiba ldquoLong-term stasis and short-term divergence in the phenotypes of microsnails on oceanicislandsrdquoMolecular Ecology vol 22 no 18 pp 4801ndash4810 2013

[25] J Colborn R E Crabtree J B Shaklee E Pfeiler and B WBowen ldquoThe evolutionary enigma of bonefishes (Albula spp)cryptic species and ancient separations in a globally distributedshorefishrdquo Evolution vol 55 no 4 pp 807ndash820 2001

[26] J-D Durand K-N Shen W-J Chen et al ldquoSystematics of thegrey mullets (Teleostei Mugiliformes Mugilidae) molecularphylogenetic evidence challenges two centuries ofmorphology-based taxonomyrdquo Molecular Phylogenetics and Evolution vol64 no 1 pp 73ndash92 2012

[27] K-N Shen B W Jamandre C-C Hsu W-N Tzeng and J-D Durand ldquoPlio-Pleistocene sea level and temperature fluc-tuations in the northwestern Pacific promoted speciation inthe globally-distributed flathead mullet Mugil cephalusrdquo BMCEvolutionary Biology vol 11 no 1 article 83 2011

[28] R Hall ldquoThe plate tectonics of Cenozoic SE Asia and thedistribution of land and seardquo in Biogeography and GeologicalEvolution of SE Asia R Hall and J D Holloway Eds pp 99ndash131Backhuys Publishers Leiden The Netherlands 1998

[29] R Hall ldquoSoutheast Asiarsquos changing palaeogeographyrdquo Blumeavol 54 no 1ndash3 pp 148ndash161 2009

[30] R F Inger and P K Chen ldquoThe freshwater fishes of NorthBorneordquo Fieldiana Zoology vol 45 pp 1ndash268 1962

[31] J Bohlen V Slechtova H H Tan and R Britz ldquoPhylogenyof the Southeast Asian freshwater fish genus Pangio (Cyprini-formes Cobitidae)rdquoMolecular Phylogenetics and Evolution vol61 no 3 pp 854ndash865 2011

[32] M de Bruyn E Nugroho M M Hossain J C Wilson andP B Mather ldquoPhylogeographic evidence for the existence ofan ancient biogeographic barrier the Isthmus of Kra SeawayrdquoHeredity vol 94 no 3 pp 370ndash378 2005

[33] J Parnell ldquoThe biogeography of the Isthmus of Kra region areviewrdquoNordic Journal of Botany vol 31 no 1 pp 001ndash015 2013

[34] S Usmani S G Tan S S Siraj and K Yusoff ldquoPopulationstructure of the Southeast Asian river catfishMystus nemurusrdquoAnimal Genetics vol 34 no 6 pp 462ndash464 2003

[35] M P Tan A F J Jamsari and M N Siti Azizah ldquoPhylo-geographic pattern of the striped snakehead Channa striatain Sundaland ancient river connectivity geographical and

Advances in Evolutionary Biology 21

anthropogenic singnaturesrdquo PLoS ONE vol 7 no 12 Article IDe52089 2012

[36] R Abell M L Thieme C Revenga et al ldquoFreshwater ecore-gions of the world a new map of biogeographic units forfreshwater biodiversity conservationrdquo BioScience vol 58 no 5pp 403ndash414 2008

[37] P U Clark D Archer D Pollard et al ldquoThemiddle Pleistocenetransition characteristics mechanisms and implications forlong-term changes in atmospheric pCO

2rdquo Quaternary Science

Reviews vol 25 no 23-24 pp 3150ndash3184 2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

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BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

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Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 14: ResearchArticle - Université Laval Julian... · ResearchArticle A DNA Barcode-Based Evaluation of the Southeast Asian Catfish Genus Hemibagrus Bleeker, 1862 (Teleostei: Siluriformes;

14 Advances in Evolutionary Biology

100∘

10∘

110∘

0∘

120∘

0

0 250

250

500

500

750(km)

(mi)

53

64

99

85

64

0002

HJOMSKA11

HOBAH0601

HOBHMS05

HMUBHKABM23

HOBAH0701

HOKAP0501

H hoevenii ldquoBrdquo

H hoevenii ldquoArdquo

Shelf minus75mShelf minus120 m

Figure 10 Phylogenetic relationships withinH hoevenii comprised of 2 subclades and 6 sequences The subtree is extracted from the taxonidentification phenogram inferred using theNeighbor-Joiningmethod (see Figure 2)The percentage of replicate trees in which the associatedsequences clustered together in the bootstrap test (500 replicates) is shown next to the branches The distribution of these sequences isillustrated in the right panel

110∘

XXMAH0110

CABAR0305

96 CAKPR0201

74CAKAP0401

5931 CAKAP0601

35 CPEBEMUKA36

646 CAKAP0903

33 CAKAP080114 CAKAP0701

CARAJ0210

4 CABAR0203CABRA0204

20

CBHKPBR0919

CABRA0301

15CABHMS20

24 NCLCTBA20

63 NECIT0201

H capitulumldquoSundaic claderdquo

H nemurus

0002 120∘

0 250 500 750

(km)

0 250 500

(mi)100

10∘

0∘

Shelf minus75mShelf minus120 m

Figure 11 Phylogenetic relationships within theH capitulum ldquoSundaicrdquo clade including 2 sequences associated withH nemurusThe subtreeis extracted from the taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2) The percentage of replicatetrees in which the associated sequences clustered together in the bootstrap test (500 replicates) is shown next to the branchesThe distributionof these sequences is illustrated in the right panel

Advances in Evolutionary Biology 15

H capitulum

0∘

10∘

100∘

110∘

120∘

66

63

88

59

38

39 68

71

6991

57

9754

88 4944

39

53

89

0002

ldquoIndochineserdquogroup ldquoErdquo

H capitulumldquoIndochineserdquo

group ldquoDrdquoH capitulumldquoIndochineserdquo

group ldquoCrdquo

H capitulumldquoIndochineserdquo

group ldquoBrdquo

H capitulumldquoIndochineserdquo

group ldquoArdquo0

0

250

250

500

500

750

(km)

(mi)

CATER0301CATERR0401

CKETEMU23

CAMEK0601CACHP0301

CAMKPA03

CAMEK0910CAMEK1003

XXMUBM02

CABMTU09

CABRM0601

CAKAP1201CAKAP1001

CAKAP1101CAKAP1301

CABRM0501

CASAD0102

CASAD0220

CAEND0301

CABAH0801

CAPABH05CATEEN03

CATER0701CATERR0801

CAKETE04CATER0501

CAPAH0303

Shelf minus75mShelf minus120 m

Figure 12 Phylogenetic relationships within theH capitulum ldquoIndochineserdquo clade encompassing 5 subclades identified as A B C D and EThe subtree is extracted from the taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2)The percentageof replicate trees in which the associated sequences clustered together in the bootstrap test (500 replicates) is shown next to the branchesThedistribution of these sequences is illustrated in the right panel

H capitulum by NampK (ZRC 40553 Table S1) Although notincluded in the analysis of NampK samples from the Sundaicclade were also found in the Kapuas River (West Borneo)Finally 10 fish sampled on the Mahakam River at Samarindaeast Borneo are all characterized by one relatively divergentgenotype (CAMAH0110) butwere considered asH capitulumin the analysis of NampK (ZRC 40533 Table S1)TheMahakampopulation sample is the most genetically distinct of theSundaic clade (119901 = 17 (04)) but nevertheless indicativeof intraspecific population-level divergence (Figure 12)

The topology of the Sundaic clade does not permit usto draw a clear distinction between H nemurus and theSundaic H capitulum The mean within-group divergenceof the Sundaic clade is only 119901 = 08 (02) well withinpopulation-level divergence values We postulate that Hnemurus may be one of several regional variants of awidespread Sundaic H capitulum clade that has radiatedrelatively recently This may explain the existence of manyshared sequences throughout the Sundaic islands assigned todifferent species in different regions

mtDNA Clade 11 Finally the Indochinese clade of Hcapitulum represents a geographically widespread andweakly diversified clade (within-group mean sequencedivergence 119901 = 11 (02) Figure 12) Although few fishfrom this genetic clade were included in the morphological

analysis of NampK most may be considered as H capitulumgiven their geographic distribution and close geneticrelationship to the Sundaic H capitulum Four sequencesin this clade were identified as H capitulum in the analysisof NampK 3 specimens characterized by one sequence(CABMTU09) found in the Tutong River of westernBorneo (ZRC 40482) 6 characterized by the same sequencefrom the Baram River (ZRC 40498) another 2 sequencesfrom the Baram River (ZRC 40498) and 20 specimenscharacterized by an abundant genotype (CASAD0220)found at 2 sites in the Sadong River also in western Borneo(ZRC 39526 5 specimens 39527 3 specimens and 38753 4specimens) (Table S1) Unfortunately none of the specimenscollected on the Indochinese mainland for genetic analysiswere examined by NampK Nevertheless the distribution ofspecimens analyzed for mtDNA resembles the distribution ofspecimens examined by NampK Although weakly structuredwe may recognize 5 subclades One genotype (CAPAH0303)is basal to the phenogram Subclade A encompasses 8sequences mainly found in eastern Peninsular Malaysia(Table S1 Figure 12) These rivers furnished many of thespecimens NampK examined to define H capitulum Howeversome sequences in this group occur in sympatry withsequences identified within the Sundaic H capitulum groupTwo sequences from subclade A occurred in the Batang HariSumatra (Table S1) Subclade E also encompasses sequences

16 Advances in Evolutionary Biology

found in Peninsular Malaysia as well as in the Mekong ChaoPhraya and Mea Khlong Rivers (Figure 12) In addition3 subclades encompass sequences found principally inwestern Borneo subclade B in the Baram and Sadong RiversSarawak subclade C in the Kapuas River West Kalimantanand subclade D in the Baram and Tutong Rivers of Sarawakand Brunei respectively (Figure 12) This latter subclade alsoincludes one genotype found in the Muar River of westernpeninsular Malaysia Finally subclade E encompasses 5sequences from the Mekong Chao Praya and Mae KhlongRivers all within the distribution area of H spilopterus

EvolutionaryHistoryThe evolutionary history of theHemiba-grus species groups analyzed here dates from at least theMiocene 15ndash20Ma (Figure 13) when considering the widelyused molecular clock of 2 sequence divergence per millionyears Bootstrap support for the sister group relationships ofH wyckioides theH guttatus species group andH peguensisare all weak (clearly due to undersampling) and little can besaid of their evolutionary history other than the fact that theyoccupy basal positions in the rooted trees The remainder ofboth the NJ and ML trees is composed of two major sistergroups the H planiceps + H baramensis group and the Hnemurus species group (Figures 2 3 and 13)TheH planiceps+ H baramensis group is strongly supported in both NJ andML analyses (84 and 90 bootstrap support resp Figures2 and 3) The H nemurus species group is also stronglysupported in both analyses (98 and 99 bootstrap supportFigures 2 and 3) These two major phylogenetic groups areestimated to have diverged at least 8 to 12Ma ago during themid to late Miocene (Figure 13)

Much of the evolutionary history of Hemibagrus hasoccurred during the Quaternary starting approximately26Ma ago This is particularly true of the H planicepsand H nemurus species groups The radiation of the 5species included in the H planiceps species group appearsto coincide with the middle Pleistocene transition period ofapproximately 1Ma ago Their high-elevation distributionhowever appears to have largely isolated them from theeffects of lowered sea levels during glacial maxima and theattendant opportunities for widespread dispersal (Figure 5)The evolutionary histories of theH baramensis species groupand the unspecified species group date back to the latePliocene In contrast the low-elevation distribution of theHnemurus species group in Sundaland appears to have favoredtheir widespread distribution during the Quaternary ice ageIn particular H hoevenii appears to be the most recentlydispersed of the species under study with diversification anddispersal occurring approximately 03Ma ago (Figure 13)

4 Discussion

41 The Congruence of Morphological and Genetic AnalysesCombining genetic and morphological analyses to elucidatetaxonomy allowed us to better understand the diversificationand evolutionary relationships within this speciose group offishes Given the rapid and relatively constant rate of mtDNAevolution the deeper the branches within the phylogenetictree are the greater our ability to distinguish valid taxa

should be Conversely the lack of complete congruencebetween morphological- and genetic-based identificationsis expected to increase among the shallower branches of thephylogenetic tree resulting in the appearance of polyphyleticspecies Although 2 of the 8 species groups defined by Ngand Kottelat [7] were not sampled in this study the validityof the remaining 6 species groups and their componentspecies defined on the basis of morphological similaritywas largely confirmed by the analysis of the mitochondrialCyt 119887 bar code As expected congruence is strongest at thebase of the NJ and ML trees where the H guttatus speciesgroup formed a well-defined monophyletic group The Hmenoda and H wyckii species group were each representedby only one species but they were clearly distinct relativeto sequences characterizing the neighboring species groupsThe H planiceps H baramensis and H nemurus speciesgroups and their component species were also generallycongruent in morphological and genetic analyses Also asexpected most cases of discrepancy between morphological-based species identification and genetic-based speciesidentification occurred on relatively shallow branches of thephylogenetic tree These involve principally H spilopterusand the two mtDNA subclades composing H capitulumHowever a far more unexpected lack of congruence involvesthe definition of a deeply divergent unspecified genetic groupconfounding members of theH baramensis species group inthe morphological analysis of Ng and Kottelat [7]

Interpreting Polyphyly The apparent polyphyletic origin ofa species may be due to incomplete lineage sorting amongrecently diverged species the introgression of mtDNA dueto species hybridization or the erroneous identificationof species due to the lumping of morphologically crypticspecies In the absence of nuclear DNA analyses it isoften difficult to distinguish between introgression andincomplete lineage sorting For example several sequencescharacterizing the Indochinese clade of H capitulum(subclade E) are found in the Mekong and Chao PhrayaRivers well within the distributional area of H spilopterusH spilopterus and H capitulum first diverged at least 3MaOn the one hand this apparent case of polyphyly mayhave resulted from incomplete lineage sorting betweenrecently divergedH spilopterus andH capitulum We wouldhowever have expected similar cases of polyphyly amongpopulations of the Indochinese clade of H capitulum butthis was not detected On the other hand the sympatricoccurrence of H spilopterus subclade A and subcladeB sequences with H capitulum Indochinese subclade Esequences in the Mekong River may represent a migrationevent and local introgression of H capitulum with Hspilopterus A similar event may have occurred in theChao Phraya and Mae Khlong Rivers where H spilopterussequences cooccur withH capitulum sequences Computingthe divergence of H capitulum sequences (Indochinesesubclade E) cooccurring with H spilopterus and Hcapitulum sequences occurring in eastern PeninsularMalaysia (subclade A) reveals a mean sequence divergence119901 = 15 (04) Thus the H capitulum sequences occurringin sympatry with H spilopterus diverged at most 750 ka ago

Advances in Evolutionary Biology 17

H fortis

XXRAJ0604H furcatus

H sabanusH semotusH divaricatus

H planicepsGREND0206

VEBAH0907

H bonganPESAL0102

H wyckioidesH macropterus

H guttatusXXRAJ0301Leiocassis longirostris

Ma

4971

92

47

51

9999

99

9999

99

99

99

9999

99

99

99

99

65

96

96

4946

93

79

78

70

91

93

98

86

87

91

95

97

71

85

9999

05101520

005010015020 000

H capitulum Indochinese clades AndashE

H capitulum Sundaic clade

H hoevenii B

H hoevenii A

H spilopterus C

H spilopterus B

H spilopterus AH spilopterus A

H spp CH spp B

H spp A

Figure 13 Linearized tree with branch lengths estimated with the molecular clock using a mutation rate of 1 per Ma (=divergence rate of2 per Ma) The tree topology is identical to the ML reconstruction illustrated in Figure 3 (see the specificities of the tree reconstruction inthe caption of Figure 3) Boxes represent 95 confidence intervals of divergence time estimates for each node Vertical dashed lines redirectto dates indicated on the timescale (Ma) Numbers below timescale indicate the estimated number of substitution changes using Hasegawa-Kishino-Yanomodel (HKY)with gamma distribution (+119866 = 13290) after linearizing the treeH furcatus is synonymizedwithH baramensis

from eastern Peninsular Malaysia populations suggestinga recent introgression event Similarly H capitulum in theKapuas River is characterized by sequences of both theSundaic and the Indochinese clades As both sequences arebroadly sympatric it is not possible to distinguish betweenincomplete lineage sorting and introgression of these closelyrelated intraspecific lineages

A Possible Case of Morphological Stasis Clade 6 of thepresent analysis defines a well-supported monophyletic cladefound in Sabah Sarawak and the Endau River of easternpeninsula Malaysia that has no equivalent taxonomic statusin the revision of Ng and Kottelat [7] In Borneo they occursympatrically with the H baramensis species group and areconfounded as either H baramensis or H semotus These

two clades are genetically highly divergent having divergedbetween 8 and 12Ma ago It thus seems highly unlikely thatthey represent retained polymorphisms due to incompletelineage sorting Sequences previously identified as character-izing H chrysops [11] are part of clade 6 and extend to thesouth (Rajang and Sadong Rivers of Sarawak West Borneo)with one closely related genotype found in eastern peninsularMalaysia (Endau River) where it may well be confoundedwith H capitulum H chrysops was synonymized with Hcapitulum by Ng and Kottelat [7] based on their analysis ofspecimens ofH chrysops from the Rajang RiverThus despitea long and apparently independent phylogenetic historythe unspecified species group appears largely cryptic andconfounded principally with members of the H baramensisspecies group

18 Advances in Evolutionary Biology

We suggest that this situation is an example of mor-phological stasis involving the coexistence of a number ofgenetically divergent yet morphologically cryptic species thatare lumped together [24] A striking example of morpho-logical and ecological stasis among fishes is provided by thecoastal marine bonefishes (Albula spp) Distinct evolution-ary lineages (separated by sequence divergence between 5 and30 [25]) of bonefish coexist in several sample locations yetthey show little morphological or ecological differentiationin sympatry [25] Similarly a proportion of the grey mulletspecies (Mugilidae) also appears to consist of cryptic species[26] For exampleMugil cephalus in Taiwan is characterizedby three divergent lineages that have been shown to charac-terize 3 reproductively isolated but morphologically crypticspecies [26 27]

Here we have discovered 2 highly distinct and structuredclades (each composed of 3 mtDNA subclades) that aremorphologically confounded within different species Forexample H spp C of the unspecified species group isidentified as H baramensis in the Baram River and as Hsemotus in the Padas River We speculate that North Borneoharbors a morphologically cryptic species complex possiblycomposed of several species pairs inhabiting different riversystems The reconstruction of this phylogenetic history andthe taxonomic status of its components remains a majorchallenge to the revision of the Hemibagrus genus

42 Phylogeographic Hypotheses The phylogeography ofthe genus appears to have been principally influenced bytwo geological histories one encompassing the formationof North Borneo the second the emergence of the SundaIslands and their connectivity with the Indochinesemainlandassociated with recurrent sea-level changes North Borneoappears to have acted as a species sink promoting speciationand locally high biodiversity within the genus but with littleevidence of subsequent dispersal out of North Borneo Incontrast the geological events influencing the Indochinesemainland and the Sunda Islands have permitted bothspeciation and differential dispersal depending on thebiology of the species For example the H planiceps speciesgroup is found throughout the Greater Sunda Islands andtheThai-Malay Peninsula as is theH nemurus species groupWhereas the H nemurus species group generally occupiesthe low altitude lower reaches of large rivers the speciescomposing the H planiceps group are found in the upperreaches of large rivers The H nemurus species group iscomposed of a number of widely distributed closely relatedspecies whereas theH planiceps group is composed of a largenumber of species that are highly localized geographicallyIt thus appears that sea-level changes have had little impacton the dispersal history of the H planiceps species group buthave deeply influenced that of the H nemurus species group

The Case of North Borneo From 15Ma to 5Ma greater areasof Borneo became emergent and the central mountainson the Sarawak-Kalimantan border extending into Sabahbecame wider and higher which may have represented newbarriers to dispersal [28 29] Borneo gradually developedinto the present large island by emergence of land in the

north and east The H baramensis species group is restrictedto North Borneo as is the unspecified species group of theHnemurus species group H fortis is also distributed in NorthBorneo The isolation of the freshwater fish communities ofNorth Borneo (particularly in the Labuk-Segama watershed)has long been recognized based on the number of endemicspecies and the concentration of relict species [30] The latePliocene-early Pleistocene diversification of H fortis theunspecified species group and the H baramensis speciesgroup may thus be associated with their dispersal to andisolation within the watersheds of North Borneo Only onesequence of the unspecified species group is found outsideof North Borneo and H fortis appears to be distributedthroughout central and east Borneo [7]

ThePhylogeography of the H nemurus Species Group Periodicmarine transgressions on the Sundaland platform during theearly and middle Miocene [2 29] may have been sufficientto isolate populations of freshwater organisms and promotecladogenesis [31] This may have been responsible for theinitial mid to late Miocene divergence of the H nemurusspecies group The majority of sequences assigned to thisspecies group belong to 3 species (H spilopterus H capitulum(including H nemurus) and H hoevenii) all of which havebeen influenced by the more recent geological history of theSunda Islands and their connectivity with the Indochinesemainland H spilopterus is known from river drainages inIndochina including the Mekong Dong Nai Ban PakongChao Phraya and Mae Klong where it is found in a widevariety of lotic and lentic habitats [7] H spilopterus divergedin the late Pliocene-early Pleistocene possibly isolated tothe north of the Kra Peninsula during the late Pliocenemarine transgression that is purported to have created amajor biogeographic transition zone between the Sundaicand Indochinese biotas ([32] but see [33] for a critique ofthis biogeographic scenario) The existence of 2 subclades(A and B) in the Mekong River separated by 2 sequencedivergence is consistent with intraspecific divergence Thetwo subclades possibly represent 2 historical mtDNA lineagesthat are retained within the species reflecting a Pleistoceneisolation event followed by secondary contact within thecontemporary Mekong River Subclade C from the ChaoPhraya and Mae Khlong Rivers exhibits a level of geneticdivergence consistent with sibling species-level divergenceshowing a 5 sequence divergence relative to Mekong RiverH spilopterus

H capitulum may be considered as part of the Sundaicbiota located principally to the south of the Kra marinetransgression zone that may have isolated H spilopterus tothe north on the Indochinese mainlandThe biogeographicalevent leading to the subsequent formation of two intraspecificgroups within H capitulum is not at all clear The onlyarea where the two clades are clearly geographically separateis on the Thai-Malay Peninsula with the ldquoSundaicrdquo cladeon the west coast and the ldquoIndochineserdquo clade on theeast coast separated by the central Titiwangsa mountainrange The genetic distinctiveness of east and west coastpopulations of H capitulum has also been documented fornuclear DNA [34] A similar pattern of separation has been

Advances in Evolutionary Biology 19

reported for 5 additional freshwater fishes within this region[35] However as the divergence of the two H capitulumclades postdates the formation of the mountain range byapproximately 200Ma the geographic separation of the twoclades most likely represents differential dispersal rather thanancient vicariance The presence of the Sundaic clade on thewest coast of the Thai-Malay Peninsula as well as on theSundaic Islands reflects the historical connections betweeneast Sumatra and west Peninsular Malaysia across the Straitof Malacca This area encompasses the Northern CentralSumatra-Western Malaysia freshwater ecoregion [36] TheMuar River forms its southern border coincident with thesouthernmost distribution of the Sundaic clade on the Thai-Malay Peninsula It is important to note however that mostof Sumatra and Java emerged to achieve their present size andelevation only 5Ma ago and much of East Java continuedto be the site of marine deposition until late Pliocene-earlyPleistocene [29] We may speculate that the Sundaic clade ofH capitulum resulted from the relatively recent colonizationof these islands and Borneo The radiation within the cladeonly dates back to approximately 1Ma ago H nemurus fromWest Java may thus best be considered an isolated regionalpopulation of very recent origin

The diversification of the Indochinese clade dates fromat least 12Ma ago coincident with the mid-Pleistocenetransition at which time low-frequency high-amplitude andquasiperiodic (100 ka) glacial variability emerged It began125Ma ago and was complete by 07Ma ago [37] The tran-sition marked the beginning of major changes in sea levelsduring which the Sunda Shelf periodically emerged and wastraversed by enormous fluvial systems [3] The contributionof these events to the diversification of this clade cannotbe satisfactorily reconstructed One possible explanation isthat high sea levels produced barriers to gene flow and thepopulations of West Borneo (subclades B C and D) wereisolated from those of east Peninsular Malaysia This couldbe due to one or more dispersal events from Borneo via theGreat Sunda River (Figure 1) to east Peninsular Malaysia orlineage sorting whereby sequences of subclades A and E havebeen lost from West Borneo As in the case of the sympatricoccurrence of H spilopterus and H capitulum subclade E(see above) in the Mekong River we are unable to clearlydefine the relative roles of incomplete lineage sorting andintrogression followingmigration Given the recent origins ofthe Indochinese H capitulum sequences incomplete lineagesorting most probably blurs patterns of recent isolation

The last species of the H nemurus species group is Hhoevenii The species is widespread throughout PeninsularMalaysia and the Sundaic Islands It is found primarily inthe lower reaches of rivers and is apparently capable ofwithstanding low salt concentration [7] H hoevenii thusappears to be the most liable of all members of the Hnemurus species group to be impacted by Pleistocene sea-level changes This is clearly reflected in the widespreaddistribution of the sequences of this species Two lineages areevident having diverged no more than 03Ma ago suggest-ing a late Pleistocene isolation event followed by extensivedispersal and admixture in recent times This represents theclearest example of the dispersal opportunities afforded by

Pleistocene sea level changes on the Sunda Shelf In additionthe relatively low genetic diversity detected (only 6 sequencesin 42 fish) suggests a population bottleneck in the recent pastpossibly related to population declines in the lower reaches ofrivers flooded by increasing sea levels

43 Future Directions We have provided a global picture ofthe evolutionary taxonomy of the genus in Southeast AsiaClearly a more rigorous phylogenetic reconstruction of thegenus Hemibagrus requiring the construction of multiplegene trees based on the analysis of both mitochondrial andnuclear DNA markers from a wider taxonomic array ofhemibagrids will involve an even greater effort of samplingthroughout the region In particular we require samplingat smaller spatial and temporal scales For example smallerscale sampling in the Padas River and the Segama Riverof North Borneo using both mitochondrial and nucleargenetic markers would contribute to clarifying the natureof the cryptic species complex believed to inhabit theserivers The demonstration of reproductive isolation andormicroallopatric distribution among morphologically crypticspecies may result in the description of greater speciesrichness within the genus than presently described Thespecies status of H chrysops merits reconsideration givenits highly divergent evolutionary history Nuclear geneticmarkers in conjunction with mtDNA markers could revealcases of historical introgression particularly in the KapuasRiver and the Mekong River The hemibagrid catfishes con-tinue to provide multiple opportunities for the study of theevolutionary taxonomy and biogeography of a major faunalcomponent of the fluvial ecosystems of Southeast Asia

Disclosure

Ethanol-preserved tissue samples of many of the specimensidentified in Table S1 are freely available upon request fromJJD

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Goh Yan Yih for assistance in collectingspecimens and molecular analyses Mrs Francoise Colom-bani for molecular analyses and Dr Heok Hee Ng forspecies identification This work was funded by the NationalUniversity of Singapore grant to P K L Ng and a CanadianNatural Sciences and Engineering Research Council grant toJ J Dodson

References

[1] D J Lohman M de Bruyn T Page et al ldquoBiogeography ofthe Indo-Australian archipelagordquo Annual Review of EcologyEvolution and Systematics vol 42 pp 205ndash226 2011

20 Advances in Evolutionary Biology

[2] D S Woodruff ldquoNeogene marine transgressions palaeogeog-raphy and biogeographic transitions on the Thai-Malay Penin-sulardquo Journal of Biogeography vol 30 no 4 pp 551ndash567 2003

[3] H K Voris ldquoMaps of Pleistocene sea levels in SoutheastAsia shorelines river systems and time durationsrdquo Journal ofBiogeography vol 27 no 5 pp 1153ndash1167 2000

[4] S Y Yap ldquoOn the distributional patterns of Southeast-EastAsian freshwater fish and their historyrdquo Journal of Biogeographyvol 29 no 9 pp 1187ndash1199 2002

[5] S K J McConnell ldquoMapping aquatic faunal exchanges acrossthe Sunda shelf South-East Asia using distributional andgenetic data sets from the cyprinid fish Barbodes gonionotus(Bleeker 1850)rdquo Journal of Natural History vol 38 no 5 pp651ndash670 2004

[6] T Mo Anatomy Relationships and Systematics of the Bagridae(Teleostei Siluroidei) with a Hypothesis of Siluroid Phylogenyvol 17 ofTheses Zoologicae Koeltz Scientific Books 1991

[7] H H Ng and M Kottelat ldquoRevision of the Asian catfish genusHemibagrus bleeker 1862 (Teleostei Siluriformes Bagridae)rdquoRaffles Bulletin of Zoology vol 61 no 1 pp 205ndash291 2013

[8] P K L Ng and H H Ng ldquoHemibagrus gracilis a new speciesof large riverine catfish (Teleostei Bagridae) from PeninsularMalaysiardquo Raffles Bulletin of Zoology vol 43 pp 133ndash142 1995

[9] H H Tan and M Kottelat ldquoRedescription of Betta picta(Teleostei Osphronemidae) and description of Betta falx sp nfrom central Sumatrardquo Revue Suisse de Zoologie vol 105 no 3pp 557ndash568 1998

[10] J J Dodson F Colombani and P K Ng ldquoPhylogeographicstructure in mitochondrial DNA of a South-east Asian fresh-water fish Hemibagrus nemurus (Siluroidei Bagridae) andPleistocene sea-level changes on the Sunda shelfrdquo MolecularEcology vol 4 no 3 pp 331ndash346 1995

[11] H H Ng and J J Dodson ldquoMorphological and genetic descrip-tions of a new species of catfish Hemibagrus chrysops fromSarawak East Malaysia with an assessment of phylogeneticrelationships (Teleostei Bagridae)rdquo Raffles Bulletin of Zoologyvol 47 no 1 pp 45ndash57 1999

[12] P D N Hebert A Cywinska S L Ball and J R DeWaardldquoBiological identifications through DNA barcodesrdquo Proceedingsof the Royal Society B Biological Sciences vol 270 no 1512 pp313ndash321 2003

[13] J C Carolan T EMurray U Fitzpatrick et al ldquoColour patternsdo not diagnose species quantitative evaluation of a DNAbarcoded cryptic bumblebee complexrdquo PLoS ONE vol 7 no 1Article ID e29251 2012

[14] M Kekkonen and P D N Hebert ldquoDNA barcode-baseddelineation of putative species efficient start for taxonomicworkflowsrdquoMolecular Ecology Resources vol 14 no 4 pp 706ndash715 2014

[15] DM Hillis CMoritz and B KMarbleMolecular SystematicsSinauer Sunderland Mass USA 2nd edition 1996

[16] T D Kocher W K Thomas A Meyer et al ldquoDynamics ofmitochondrial DNA evolution in animals amplification andsequencingwith conserved primersrdquoProceedings of theNationalAcademy of Sciences of the United States of America vol 86 no16 pp 6196ndash6200 1989

[17] T A Hall ldquoBioEdit a user-friendly biological sequence align-ment editor and analysis program for Windows 9598NTrdquoNucleic Acids Symposium Series vol 41 pp 95ndash98 1999

[18] D A Benson I Karsch-Mizrachi K Clark D J Lipman JOstell and EW Sayers ldquoGenBankrdquoNucleic Acids Research vol40 no D1 pp D48ndashD53 2012

[19] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[20] X Ku Z Peng R Diogo and S He ldquoMtDNA phylogenyprovides evidence of generic polyphyleticism for East Asianbagrid catfishesrdquoHydrobiologia vol 579 no 1 pp 147ndash159 2007

[21] L Yang and S He ldquoPhylogeography of the freshwater catfishHemibagrus guttatus (Siluriformes Bagridae) implications forSouth China biogeography and influence of sea-level changesrdquoMolecular Phylogenetics and Evolution vol 49 no 1 pp 393ndash398 2008

[22] B W Bowen A L Bass L A Rocha W S Grant and DR Robertson ldquoPhylogeography of the trumpetfishes (Aulosto-mus) ring species complex on a global scalerdquo Evolution vol 55no 5 pp 1029ndash1039 2001

[23] Y P Kartavtsev ldquoDivergence at Cyt-b and Co-1 mtDNA geneson different taxonomic levels and genetics of speciation inanimalsrdquoMitochondrial DNA vol 22 no 3 pp 55ndash65 2011

[24] SWada Y Kameda and S Chiba ldquoLong-term stasis and short-term divergence in the phenotypes of microsnails on oceanicislandsrdquoMolecular Ecology vol 22 no 18 pp 4801ndash4810 2013

[25] J Colborn R E Crabtree J B Shaklee E Pfeiler and B WBowen ldquoThe evolutionary enigma of bonefishes (Albula spp)cryptic species and ancient separations in a globally distributedshorefishrdquo Evolution vol 55 no 4 pp 807ndash820 2001

[26] J-D Durand K-N Shen W-J Chen et al ldquoSystematics of thegrey mullets (Teleostei Mugiliformes Mugilidae) molecularphylogenetic evidence challenges two centuries ofmorphology-based taxonomyrdquo Molecular Phylogenetics and Evolution vol64 no 1 pp 73ndash92 2012

[27] K-N Shen B W Jamandre C-C Hsu W-N Tzeng and J-D Durand ldquoPlio-Pleistocene sea level and temperature fluc-tuations in the northwestern Pacific promoted speciation inthe globally-distributed flathead mullet Mugil cephalusrdquo BMCEvolutionary Biology vol 11 no 1 article 83 2011

[28] R Hall ldquoThe plate tectonics of Cenozoic SE Asia and thedistribution of land and seardquo in Biogeography and GeologicalEvolution of SE Asia R Hall and J D Holloway Eds pp 99ndash131Backhuys Publishers Leiden The Netherlands 1998

[29] R Hall ldquoSoutheast Asiarsquos changing palaeogeographyrdquo Blumeavol 54 no 1ndash3 pp 148ndash161 2009

[30] R F Inger and P K Chen ldquoThe freshwater fishes of NorthBorneordquo Fieldiana Zoology vol 45 pp 1ndash268 1962

[31] J Bohlen V Slechtova H H Tan and R Britz ldquoPhylogenyof the Southeast Asian freshwater fish genus Pangio (Cyprini-formes Cobitidae)rdquoMolecular Phylogenetics and Evolution vol61 no 3 pp 854ndash865 2011

[32] M de Bruyn E Nugroho M M Hossain J C Wilson andP B Mather ldquoPhylogeographic evidence for the existence ofan ancient biogeographic barrier the Isthmus of Kra SeawayrdquoHeredity vol 94 no 3 pp 370ndash378 2005

[33] J Parnell ldquoThe biogeography of the Isthmus of Kra region areviewrdquoNordic Journal of Botany vol 31 no 1 pp 001ndash015 2013

[34] S Usmani S G Tan S S Siraj and K Yusoff ldquoPopulationstructure of the Southeast Asian river catfishMystus nemurusrdquoAnimal Genetics vol 34 no 6 pp 462ndash464 2003

[35] M P Tan A F J Jamsari and M N Siti Azizah ldquoPhylo-geographic pattern of the striped snakehead Channa striatain Sundaland ancient river connectivity geographical and

Advances in Evolutionary Biology 21

anthropogenic singnaturesrdquo PLoS ONE vol 7 no 12 Article IDe52089 2012

[36] R Abell M L Thieme C Revenga et al ldquoFreshwater ecore-gions of the world a new map of biogeographic units forfreshwater biodiversity conservationrdquo BioScience vol 58 no 5pp 403ndash414 2008

[37] P U Clark D Archer D Pollard et al ldquoThemiddle Pleistocenetransition characteristics mechanisms and implications forlong-term changes in atmospheric pCO

2rdquo Quaternary Science

Reviews vol 25 no 23-24 pp 3150ndash3184 2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 15: ResearchArticle - Université Laval Julian... · ResearchArticle A DNA Barcode-Based Evaluation of the Southeast Asian Catfish Genus Hemibagrus Bleeker, 1862 (Teleostei: Siluriformes;

Advances in Evolutionary Biology 15

H capitulum

0∘

10∘

100∘

110∘

120∘

66

63

88

59

38

39 68

71

6991

57

9754

88 4944

39

53

89

0002

ldquoIndochineserdquogroup ldquoErdquo

H capitulumldquoIndochineserdquo

group ldquoDrdquoH capitulumldquoIndochineserdquo

group ldquoCrdquo

H capitulumldquoIndochineserdquo

group ldquoBrdquo

H capitulumldquoIndochineserdquo

group ldquoArdquo0

0

250

250

500

500

750

(km)

(mi)

CATER0301CATERR0401

CKETEMU23

CAMEK0601CACHP0301

CAMKPA03

CAMEK0910CAMEK1003

XXMUBM02

CABMTU09

CABRM0601

CAKAP1201CAKAP1001

CAKAP1101CAKAP1301

CABRM0501

CASAD0102

CASAD0220

CAEND0301

CABAH0801

CAPABH05CATEEN03

CATER0701CATERR0801

CAKETE04CATER0501

CAPAH0303

Shelf minus75mShelf minus120 m

Figure 12 Phylogenetic relationships within theH capitulum ldquoIndochineserdquo clade encompassing 5 subclades identified as A B C D and EThe subtree is extracted from the taxon identification phenogram inferred using the Neighbor-Joining method (see Figure 2)The percentageof replicate trees in which the associated sequences clustered together in the bootstrap test (500 replicates) is shown next to the branchesThedistribution of these sequences is illustrated in the right panel

H capitulum by NampK (ZRC 40553 Table S1) Although notincluded in the analysis of NampK samples from the Sundaicclade were also found in the Kapuas River (West Borneo)Finally 10 fish sampled on the Mahakam River at Samarindaeast Borneo are all characterized by one relatively divergentgenotype (CAMAH0110) butwere considered asH capitulumin the analysis of NampK (ZRC 40533 Table S1)TheMahakampopulation sample is the most genetically distinct of theSundaic clade (119901 = 17 (04)) but nevertheless indicativeof intraspecific population-level divergence (Figure 12)

The topology of the Sundaic clade does not permit usto draw a clear distinction between H nemurus and theSundaic H capitulum The mean within-group divergenceof the Sundaic clade is only 119901 = 08 (02) well withinpopulation-level divergence values We postulate that Hnemurus may be one of several regional variants of awidespread Sundaic H capitulum clade that has radiatedrelatively recently This may explain the existence of manyshared sequences throughout the Sundaic islands assigned todifferent species in different regions

mtDNA Clade 11 Finally the Indochinese clade of Hcapitulum represents a geographically widespread andweakly diversified clade (within-group mean sequencedivergence 119901 = 11 (02) Figure 12) Although few fishfrom this genetic clade were included in the morphological

analysis of NampK most may be considered as H capitulumgiven their geographic distribution and close geneticrelationship to the Sundaic H capitulum Four sequencesin this clade were identified as H capitulum in the analysisof NampK 3 specimens characterized by one sequence(CABMTU09) found in the Tutong River of westernBorneo (ZRC 40482) 6 characterized by the same sequencefrom the Baram River (ZRC 40498) another 2 sequencesfrom the Baram River (ZRC 40498) and 20 specimenscharacterized by an abundant genotype (CASAD0220)found at 2 sites in the Sadong River also in western Borneo(ZRC 39526 5 specimens 39527 3 specimens and 38753 4specimens) (Table S1) Unfortunately none of the specimenscollected on the Indochinese mainland for genetic analysiswere examined by NampK Nevertheless the distribution ofspecimens analyzed for mtDNA resembles the distribution ofspecimens examined by NampK Although weakly structuredwe may recognize 5 subclades One genotype (CAPAH0303)is basal to the phenogram Subclade A encompasses 8sequences mainly found in eastern Peninsular Malaysia(Table S1 Figure 12) These rivers furnished many of thespecimens NampK examined to define H capitulum Howeversome sequences in this group occur in sympatry withsequences identified within the Sundaic H capitulum groupTwo sequences from subclade A occurred in the Batang HariSumatra (Table S1) Subclade E also encompasses sequences

16 Advances in Evolutionary Biology

found in Peninsular Malaysia as well as in the Mekong ChaoPhraya and Mea Khlong Rivers (Figure 12) In addition3 subclades encompass sequences found principally inwestern Borneo subclade B in the Baram and Sadong RiversSarawak subclade C in the Kapuas River West Kalimantanand subclade D in the Baram and Tutong Rivers of Sarawakand Brunei respectively (Figure 12) This latter subclade alsoincludes one genotype found in the Muar River of westernpeninsular Malaysia Finally subclade E encompasses 5sequences from the Mekong Chao Praya and Mae KhlongRivers all within the distribution area of H spilopterus

EvolutionaryHistoryThe evolutionary history of theHemiba-grus species groups analyzed here dates from at least theMiocene 15ndash20Ma (Figure 13) when considering the widelyused molecular clock of 2 sequence divergence per millionyears Bootstrap support for the sister group relationships ofH wyckioides theH guttatus species group andH peguensisare all weak (clearly due to undersampling) and little can besaid of their evolutionary history other than the fact that theyoccupy basal positions in the rooted trees The remainder ofboth the NJ and ML trees is composed of two major sistergroups the H planiceps + H baramensis group and the Hnemurus species group (Figures 2 3 and 13)TheH planiceps+ H baramensis group is strongly supported in both NJ andML analyses (84 and 90 bootstrap support resp Figures2 and 3) The H nemurus species group is also stronglysupported in both analyses (98 and 99 bootstrap supportFigures 2 and 3) These two major phylogenetic groups areestimated to have diverged at least 8 to 12Ma ago during themid to late Miocene (Figure 13)

Much of the evolutionary history of Hemibagrus hasoccurred during the Quaternary starting approximately26Ma ago This is particularly true of the H planicepsand H nemurus species groups The radiation of the 5species included in the H planiceps species group appearsto coincide with the middle Pleistocene transition period ofapproximately 1Ma ago Their high-elevation distributionhowever appears to have largely isolated them from theeffects of lowered sea levels during glacial maxima and theattendant opportunities for widespread dispersal (Figure 5)The evolutionary histories of theH baramensis species groupand the unspecified species group date back to the latePliocene In contrast the low-elevation distribution of theHnemurus species group in Sundaland appears to have favoredtheir widespread distribution during the Quaternary ice ageIn particular H hoevenii appears to be the most recentlydispersed of the species under study with diversification anddispersal occurring approximately 03Ma ago (Figure 13)

4 Discussion

41 The Congruence of Morphological and Genetic AnalysesCombining genetic and morphological analyses to elucidatetaxonomy allowed us to better understand the diversificationand evolutionary relationships within this speciose group offishes Given the rapid and relatively constant rate of mtDNAevolution the deeper the branches within the phylogenetictree are the greater our ability to distinguish valid taxa

should be Conversely the lack of complete congruencebetween morphological- and genetic-based identificationsis expected to increase among the shallower branches of thephylogenetic tree resulting in the appearance of polyphyleticspecies Although 2 of the 8 species groups defined by Ngand Kottelat [7] were not sampled in this study the validityof the remaining 6 species groups and their componentspecies defined on the basis of morphological similaritywas largely confirmed by the analysis of the mitochondrialCyt 119887 bar code As expected congruence is strongest at thebase of the NJ and ML trees where the H guttatus speciesgroup formed a well-defined monophyletic group The Hmenoda and H wyckii species group were each representedby only one species but they were clearly distinct relativeto sequences characterizing the neighboring species groupsThe H planiceps H baramensis and H nemurus speciesgroups and their component species were also generallycongruent in morphological and genetic analyses Also asexpected most cases of discrepancy between morphological-based species identification and genetic-based speciesidentification occurred on relatively shallow branches of thephylogenetic tree These involve principally H spilopterusand the two mtDNA subclades composing H capitulumHowever a far more unexpected lack of congruence involvesthe definition of a deeply divergent unspecified genetic groupconfounding members of theH baramensis species group inthe morphological analysis of Ng and Kottelat [7]

Interpreting Polyphyly The apparent polyphyletic origin ofa species may be due to incomplete lineage sorting amongrecently diverged species the introgression of mtDNA dueto species hybridization or the erroneous identificationof species due to the lumping of morphologically crypticspecies In the absence of nuclear DNA analyses it isoften difficult to distinguish between introgression andincomplete lineage sorting For example several sequencescharacterizing the Indochinese clade of H capitulum(subclade E) are found in the Mekong and Chao PhrayaRivers well within the distributional area of H spilopterusH spilopterus and H capitulum first diverged at least 3MaOn the one hand this apparent case of polyphyly mayhave resulted from incomplete lineage sorting betweenrecently divergedH spilopterus andH capitulum We wouldhowever have expected similar cases of polyphyly amongpopulations of the Indochinese clade of H capitulum butthis was not detected On the other hand the sympatricoccurrence of H spilopterus subclade A and subcladeB sequences with H capitulum Indochinese subclade Esequences in the Mekong River may represent a migrationevent and local introgression of H capitulum with Hspilopterus A similar event may have occurred in theChao Phraya and Mae Khlong Rivers where H spilopterussequences cooccur withH capitulum sequences Computingthe divergence of H capitulum sequences (Indochinesesubclade E) cooccurring with H spilopterus and Hcapitulum sequences occurring in eastern PeninsularMalaysia (subclade A) reveals a mean sequence divergence119901 = 15 (04) Thus the H capitulum sequences occurringin sympatry with H spilopterus diverged at most 750 ka ago

Advances in Evolutionary Biology 17

H fortis

XXRAJ0604H furcatus

H sabanusH semotusH divaricatus

H planicepsGREND0206

VEBAH0907

H bonganPESAL0102

H wyckioidesH macropterus

H guttatusXXRAJ0301Leiocassis longirostris

Ma

4971

92

47

51

9999

99

9999

99

99

99

9999

99

99

99

99

65

96

96

4946

93

79

78

70

91

93

98

86

87

91

95

97

71

85

9999

05101520

005010015020 000

H capitulum Indochinese clades AndashE

H capitulum Sundaic clade

H hoevenii B

H hoevenii A

H spilopterus C

H spilopterus B

H spilopterus AH spilopterus A

H spp CH spp B

H spp A

Figure 13 Linearized tree with branch lengths estimated with the molecular clock using a mutation rate of 1 per Ma (=divergence rate of2 per Ma) The tree topology is identical to the ML reconstruction illustrated in Figure 3 (see the specificities of the tree reconstruction inthe caption of Figure 3) Boxes represent 95 confidence intervals of divergence time estimates for each node Vertical dashed lines redirectto dates indicated on the timescale (Ma) Numbers below timescale indicate the estimated number of substitution changes using Hasegawa-Kishino-Yanomodel (HKY)with gamma distribution (+119866 = 13290) after linearizing the treeH furcatus is synonymizedwithH baramensis

from eastern Peninsular Malaysia populations suggestinga recent introgression event Similarly H capitulum in theKapuas River is characterized by sequences of both theSundaic and the Indochinese clades As both sequences arebroadly sympatric it is not possible to distinguish betweenincomplete lineage sorting and introgression of these closelyrelated intraspecific lineages

A Possible Case of Morphological Stasis Clade 6 of thepresent analysis defines a well-supported monophyletic cladefound in Sabah Sarawak and the Endau River of easternpeninsula Malaysia that has no equivalent taxonomic statusin the revision of Ng and Kottelat [7] In Borneo they occursympatrically with the H baramensis species group and areconfounded as either H baramensis or H semotus These

two clades are genetically highly divergent having divergedbetween 8 and 12Ma ago It thus seems highly unlikely thatthey represent retained polymorphisms due to incompletelineage sorting Sequences previously identified as character-izing H chrysops [11] are part of clade 6 and extend to thesouth (Rajang and Sadong Rivers of Sarawak West Borneo)with one closely related genotype found in eastern peninsularMalaysia (Endau River) where it may well be confoundedwith H capitulum H chrysops was synonymized with Hcapitulum by Ng and Kottelat [7] based on their analysis ofspecimens ofH chrysops from the Rajang RiverThus despitea long and apparently independent phylogenetic historythe unspecified species group appears largely cryptic andconfounded principally with members of the H baramensisspecies group

18 Advances in Evolutionary Biology

We suggest that this situation is an example of mor-phological stasis involving the coexistence of a number ofgenetically divergent yet morphologically cryptic species thatare lumped together [24] A striking example of morpho-logical and ecological stasis among fishes is provided by thecoastal marine bonefishes (Albula spp) Distinct evolution-ary lineages (separated by sequence divergence between 5 and30 [25]) of bonefish coexist in several sample locations yetthey show little morphological or ecological differentiationin sympatry [25] Similarly a proportion of the grey mulletspecies (Mugilidae) also appears to consist of cryptic species[26] For exampleMugil cephalus in Taiwan is characterizedby three divergent lineages that have been shown to charac-terize 3 reproductively isolated but morphologically crypticspecies [26 27]

Here we have discovered 2 highly distinct and structuredclades (each composed of 3 mtDNA subclades) that aremorphologically confounded within different species Forexample H spp C of the unspecified species group isidentified as H baramensis in the Baram River and as Hsemotus in the Padas River We speculate that North Borneoharbors a morphologically cryptic species complex possiblycomposed of several species pairs inhabiting different riversystems The reconstruction of this phylogenetic history andthe taxonomic status of its components remains a majorchallenge to the revision of the Hemibagrus genus

42 Phylogeographic Hypotheses The phylogeography ofthe genus appears to have been principally influenced bytwo geological histories one encompassing the formationof North Borneo the second the emergence of the SundaIslands and their connectivity with the Indochinesemainlandassociated with recurrent sea-level changes North Borneoappears to have acted as a species sink promoting speciationand locally high biodiversity within the genus but with littleevidence of subsequent dispersal out of North Borneo Incontrast the geological events influencing the Indochinesemainland and the Sunda Islands have permitted bothspeciation and differential dispersal depending on thebiology of the species For example the H planiceps speciesgroup is found throughout the Greater Sunda Islands andtheThai-Malay Peninsula as is theH nemurus species groupWhereas the H nemurus species group generally occupiesthe low altitude lower reaches of large rivers the speciescomposing the H planiceps group are found in the upperreaches of large rivers The H nemurus species group iscomposed of a number of widely distributed closely relatedspecies whereas theH planiceps group is composed of a largenumber of species that are highly localized geographicallyIt thus appears that sea-level changes have had little impacton the dispersal history of the H planiceps species group buthave deeply influenced that of the H nemurus species group

The Case of North Borneo From 15Ma to 5Ma greater areasof Borneo became emergent and the central mountainson the Sarawak-Kalimantan border extending into Sabahbecame wider and higher which may have represented newbarriers to dispersal [28 29] Borneo gradually developedinto the present large island by emergence of land in the

north and east The H baramensis species group is restrictedto North Borneo as is the unspecified species group of theHnemurus species group H fortis is also distributed in NorthBorneo The isolation of the freshwater fish communities ofNorth Borneo (particularly in the Labuk-Segama watershed)has long been recognized based on the number of endemicspecies and the concentration of relict species [30] The latePliocene-early Pleistocene diversification of H fortis theunspecified species group and the H baramensis speciesgroup may thus be associated with their dispersal to andisolation within the watersheds of North Borneo Only onesequence of the unspecified species group is found outsideof North Borneo and H fortis appears to be distributedthroughout central and east Borneo [7]

ThePhylogeography of the H nemurus Species Group Periodicmarine transgressions on the Sundaland platform during theearly and middle Miocene [2 29] may have been sufficientto isolate populations of freshwater organisms and promotecladogenesis [31] This may have been responsible for theinitial mid to late Miocene divergence of the H nemurusspecies group The majority of sequences assigned to thisspecies group belong to 3 species (H spilopterus H capitulum(including H nemurus) and H hoevenii) all of which havebeen influenced by the more recent geological history of theSunda Islands and their connectivity with the Indochinesemainland H spilopterus is known from river drainages inIndochina including the Mekong Dong Nai Ban PakongChao Phraya and Mae Klong where it is found in a widevariety of lotic and lentic habitats [7] H spilopterus divergedin the late Pliocene-early Pleistocene possibly isolated tothe north of the Kra Peninsula during the late Pliocenemarine transgression that is purported to have created amajor biogeographic transition zone between the Sundaicand Indochinese biotas ([32] but see [33] for a critique ofthis biogeographic scenario) The existence of 2 subclades(A and B) in the Mekong River separated by 2 sequencedivergence is consistent with intraspecific divergence Thetwo subclades possibly represent 2 historical mtDNA lineagesthat are retained within the species reflecting a Pleistoceneisolation event followed by secondary contact within thecontemporary Mekong River Subclade C from the ChaoPhraya and Mae Khlong Rivers exhibits a level of geneticdivergence consistent with sibling species-level divergenceshowing a 5 sequence divergence relative to Mekong RiverH spilopterus

H capitulum may be considered as part of the Sundaicbiota located principally to the south of the Kra marinetransgression zone that may have isolated H spilopterus tothe north on the Indochinese mainlandThe biogeographicalevent leading to the subsequent formation of two intraspecificgroups within H capitulum is not at all clear The onlyarea where the two clades are clearly geographically separateis on the Thai-Malay Peninsula with the ldquoSundaicrdquo cladeon the west coast and the ldquoIndochineserdquo clade on theeast coast separated by the central Titiwangsa mountainrange The genetic distinctiveness of east and west coastpopulations of H capitulum has also been documented fornuclear DNA [34] A similar pattern of separation has been

Advances in Evolutionary Biology 19

reported for 5 additional freshwater fishes within this region[35] However as the divergence of the two H capitulumclades postdates the formation of the mountain range byapproximately 200Ma the geographic separation of the twoclades most likely represents differential dispersal rather thanancient vicariance The presence of the Sundaic clade on thewest coast of the Thai-Malay Peninsula as well as on theSundaic Islands reflects the historical connections betweeneast Sumatra and west Peninsular Malaysia across the Straitof Malacca This area encompasses the Northern CentralSumatra-Western Malaysia freshwater ecoregion [36] TheMuar River forms its southern border coincident with thesouthernmost distribution of the Sundaic clade on the Thai-Malay Peninsula It is important to note however that mostof Sumatra and Java emerged to achieve their present size andelevation only 5Ma ago and much of East Java continuedto be the site of marine deposition until late Pliocene-earlyPleistocene [29] We may speculate that the Sundaic clade ofH capitulum resulted from the relatively recent colonizationof these islands and Borneo The radiation within the cladeonly dates back to approximately 1Ma ago H nemurus fromWest Java may thus best be considered an isolated regionalpopulation of very recent origin

The diversification of the Indochinese clade dates fromat least 12Ma ago coincident with the mid-Pleistocenetransition at which time low-frequency high-amplitude andquasiperiodic (100 ka) glacial variability emerged It began125Ma ago and was complete by 07Ma ago [37] The tran-sition marked the beginning of major changes in sea levelsduring which the Sunda Shelf periodically emerged and wastraversed by enormous fluvial systems [3] The contributionof these events to the diversification of this clade cannotbe satisfactorily reconstructed One possible explanation isthat high sea levels produced barriers to gene flow and thepopulations of West Borneo (subclades B C and D) wereisolated from those of east Peninsular Malaysia This couldbe due to one or more dispersal events from Borneo via theGreat Sunda River (Figure 1) to east Peninsular Malaysia orlineage sorting whereby sequences of subclades A and E havebeen lost from West Borneo As in the case of the sympatricoccurrence of H spilopterus and H capitulum subclade E(see above) in the Mekong River we are unable to clearlydefine the relative roles of incomplete lineage sorting andintrogression followingmigration Given the recent origins ofthe Indochinese H capitulum sequences incomplete lineagesorting most probably blurs patterns of recent isolation

The last species of the H nemurus species group is Hhoevenii The species is widespread throughout PeninsularMalaysia and the Sundaic Islands It is found primarily inthe lower reaches of rivers and is apparently capable ofwithstanding low salt concentration [7] H hoevenii thusappears to be the most liable of all members of the Hnemurus species group to be impacted by Pleistocene sea-level changes This is clearly reflected in the widespreaddistribution of the sequences of this species Two lineages areevident having diverged no more than 03Ma ago suggest-ing a late Pleistocene isolation event followed by extensivedispersal and admixture in recent times This represents theclearest example of the dispersal opportunities afforded by

Pleistocene sea level changes on the Sunda Shelf In additionthe relatively low genetic diversity detected (only 6 sequencesin 42 fish) suggests a population bottleneck in the recent pastpossibly related to population declines in the lower reaches ofrivers flooded by increasing sea levels

43 Future Directions We have provided a global picture ofthe evolutionary taxonomy of the genus in Southeast AsiaClearly a more rigorous phylogenetic reconstruction of thegenus Hemibagrus requiring the construction of multiplegene trees based on the analysis of both mitochondrial andnuclear DNA markers from a wider taxonomic array ofhemibagrids will involve an even greater effort of samplingthroughout the region In particular we require samplingat smaller spatial and temporal scales For example smallerscale sampling in the Padas River and the Segama Riverof North Borneo using both mitochondrial and nucleargenetic markers would contribute to clarifying the natureof the cryptic species complex believed to inhabit theserivers The demonstration of reproductive isolation andormicroallopatric distribution among morphologically crypticspecies may result in the description of greater speciesrichness within the genus than presently described Thespecies status of H chrysops merits reconsideration givenits highly divergent evolutionary history Nuclear geneticmarkers in conjunction with mtDNA markers could revealcases of historical introgression particularly in the KapuasRiver and the Mekong River The hemibagrid catfishes con-tinue to provide multiple opportunities for the study of theevolutionary taxonomy and biogeography of a major faunalcomponent of the fluvial ecosystems of Southeast Asia

Disclosure

Ethanol-preserved tissue samples of many of the specimensidentified in Table S1 are freely available upon request fromJJD

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Goh Yan Yih for assistance in collectingspecimens and molecular analyses Mrs Francoise Colom-bani for molecular analyses and Dr Heok Hee Ng forspecies identification This work was funded by the NationalUniversity of Singapore grant to P K L Ng and a CanadianNatural Sciences and Engineering Research Council grant toJ J Dodson

References

[1] D J Lohman M de Bruyn T Page et al ldquoBiogeography ofthe Indo-Australian archipelagordquo Annual Review of EcologyEvolution and Systematics vol 42 pp 205ndash226 2011

20 Advances in Evolutionary Biology

[2] D S Woodruff ldquoNeogene marine transgressions palaeogeog-raphy and biogeographic transitions on the Thai-Malay Penin-sulardquo Journal of Biogeography vol 30 no 4 pp 551ndash567 2003

[3] H K Voris ldquoMaps of Pleistocene sea levels in SoutheastAsia shorelines river systems and time durationsrdquo Journal ofBiogeography vol 27 no 5 pp 1153ndash1167 2000

[4] S Y Yap ldquoOn the distributional patterns of Southeast-EastAsian freshwater fish and their historyrdquo Journal of Biogeographyvol 29 no 9 pp 1187ndash1199 2002

[5] S K J McConnell ldquoMapping aquatic faunal exchanges acrossthe Sunda shelf South-East Asia using distributional andgenetic data sets from the cyprinid fish Barbodes gonionotus(Bleeker 1850)rdquo Journal of Natural History vol 38 no 5 pp651ndash670 2004

[6] T Mo Anatomy Relationships and Systematics of the Bagridae(Teleostei Siluroidei) with a Hypothesis of Siluroid Phylogenyvol 17 ofTheses Zoologicae Koeltz Scientific Books 1991

[7] H H Ng and M Kottelat ldquoRevision of the Asian catfish genusHemibagrus bleeker 1862 (Teleostei Siluriformes Bagridae)rdquoRaffles Bulletin of Zoology vol 61 no 1 pp 205ndash291 2013

[8] P K L Ng and H H Ng ldquoHemibagrus gracilis a new speciesof large riverine catfish (Teleostei Bagridae) from PeninsularMalaysiardquo Raffles Bulletin of Zoology vol 43 pp 133ndash142 1995

[9] H H Tan and M Kottelat ldquoRedescription of Betta picta(Teleostei Osphronemidae) and description of Betta falx sp nfrom central Sumatrardquo Revue Suisse de Zoologie vol 105 no 3pp 557ndash568 1998

[10] J J Dodson F Colombani and P K Ng ldquoPhylogeographicstructure in mitochondrial DNA of a South-east Asian fresh-water fish Hemibagrus nemurus (Siluroidei Bagridae) andPleistocene sea-level changes on the Sunda shelfrdquo MolecularEcology vol 4 no 3 pp 331ndash346 1995

[11] H H Ng and J J Dodson ldquoMorphological and genetic descrip-tions of a new species of catfish Hemibagrus chrysops fromSarawak East Malaysia with an assessment of phylogeneticrelationships (Teleostei Bagridae)rdquo Raffles Bulletin of Zoologyvol 47 no 1 pp 45ndash57 1999

[12] P D N Hebert A Cywinska S L Ball and J R DeWaardldquoBiological identifications through DNA barcodesrdquo Proceedingsof the Royal Society B Biological Sciences vol 270 no 1512 pp313ndash321 2003

[13] J C Carolan T EMurray U Fitzpatrick et al ldquoColour patternsdo not diagnose species quantitative evaluation of a DNAbarcoded cryptic bumblebee complexrdquo PLoS ONE vol 7 no 1Article ID e29251 2012

[14] M Kekkonen and P D N Hebert ldquoDNA barcode-baseddelineation of putative species efficient start for taxonomicworkflowsrdquoMolecular Ecology Resources vol 14 no 4 pp 706ndash715 2014

[15] DM Hillis CMoritz and B KMarbleMolecular SystematicsSinauer Sunderland Mass USA 2nd edition 1996

[16] T D Kocher W K Thomas A Meyer et al ldquoDynamics ofmitochondrial DNA evolution in animals amplification andsequencingwith conserved primersrdquoProceedings of theNationalAcademy of Sciences of the United States of America vol 86 no16 pp 6196ndash6200 1989

[17] T A Hall ldquoBioEdit a user-friendly biological sequence align-ment editor and analysis program for Windows 9598NTrdquoNucleic Acids Symposium Series vol 41 pp 95ndash98 1999

[18] D A Benson I Karsch-Mizrachi K Clark D J Lipman JOstell and EW Sayers ldquoGenBankrdquoNucleic Acids Research vol40 no D1 pp D48ndashD53 2012

[19] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[20] X Ku Z Peng R Diogo and S He ldquoMtDNA phylogenyprovides evidence of generic polyphyleticism for East Asianbagrid catfishesrdquoHydrobiologia vol 579 no 1 pp 147ndash159 2007

[21] L Yang and S He ldquoPhylogeography of the freshwater catfishHemibagrus guttatus (Siluriformes Bagridae) implications forSouth China biogeography and influence of sea-level changesrdquoMolecular Phylogenetics and Evolution vol 49 no 1 pp 393ndash398 2008

[22] B W Bowen A L Bass L A Rocha W S Grant and DR Robertson ldquoPhylogeography of the trumpetfishes (Aulosto-mus) ring species complex on a global scalerdquo Evolution vol 55no 5 pp 1029ndash1039 2001

[23] Y P Kartavtsev ldquoDivergence at Cyt-b and Co-1 mtDNA geneson different taxonomic levels and genetics of speciation inanimalsrdquoMitochondrial DNA vol 22 no 3 pp 55ndash65 2011

[24] SWada Y Kameda and S Chiba ldquoLong-term stasis and short-term divergence in the phenotypes of microsnails on oceanicislandsrdquoMolecular Ecology vol 22 no 18 pp 4801ndash4810 2013

[25] J Colborn R E Crabtree J B Shaklee E Pfeiler and B WBowen ldquoThe evolutionary enigma of bonefishes (Albula spp)cryptic species and ancient separations in a globally distributedshorefishrdquo Evolution vol 55 no 4 pp 807ndash820 2001

[26] J-D Durand K-N Shen W-J Chen et al ldquoSystematics of thegrey mullets (Teleostei Mugiliformes Mugilidae) molecularphylogenetic evidence challenges two centuries ofmorphology-based taxonomyrdquo Molecular Phylogenetics and Evolution vol64 no 1 pp 73ndash92 2012

[27] K-N Shen B W Jamandre C-C Hsu W-N Tzeng and J-D Durand ldquoPlio-Pleistocene sea level and temperature fluc-tuations in the northwestern Pacific promoted speciation inthe globally-distributed flathead mullet Mugil cephalusrdquo BMCEvolutionary Biology vol 11 no 1 article 83 2011

[28] R Hall ldquoThe plate tectonics of Cenozoic SE Asia and thedistribution of land and seardquo in Biogeography and GeologicalEvolution of SE Asia R Hall and J D Holloway Eds pp 99ndash131Backhuys Publishers Leiden The Netherlands 1998

[29] R Hall ldquoSoutheast Asiarsquos changing palaeogeographyrdquo Blumeavol 54 no 1ndash3 pp 148ndash161 2009

[30] R F Inger and P K Chen ldquoThe freshwater fishes of NorthBorneordquo Fieldiana Zoology vol 45 pp 1ndash268 1962

[31] J Bohlen V Slechtova H H Tan and R Britz ldquoPhylogenyof the Southeast Asian freshwater fish genus Pangio (Cyprini-formes Cobitidae)rdquoMolecular Phylogenetics and Evolution vol61 no 3 pp 854ndash865 2011

[32] M de Bruyn E Nugroho M M Hossain J C Wilson andP B Mather ldquoPhylogeographic evidence for the existence ofan ancient biogeographic barrier the Isthmus of Kra SeawayrdquoHeredity vol 94 no 3 pp 370ndash378 2005

[33] J Parnell ldquoThe biogeography of the Isthmus of Kra region areviewrdquoNordic Journal of Botany vol 31 no 1 pp 001ndash015 2013

[34] S Usmani S G Tan S S Siraj and K Yusoff ldquoPopulationstructure of the Southeast Asian river catfishMystus nemurusrdquoAnimal Genetics vol 34 no 6 pp 462ndash464 2003

[35] M P Tan A F J Jamsari and M N Siti Azizah ldquoPhylo-geographic pattern of the striped snakehead Channa striatain Sundaland ancient river connectivity geographical and

Advances in Evolutionary Biology 21

anthropogenic singnaturesrdquo PLoS ONE vol 7 no 12 Article IDe52089 2012

[36] R Abell M L Thieme C Revenga et al ldquoFreshwater ecore-gions of the world a new map of biogeographic units forfreshwater biodiversity conservationrdquo BioScience vol 58 no 5pp 403ndash414 2008

[37] P U Clark D Archer D Pollard et al ldquoThemiddle Pleistocenetransition characteristics mechanisms and implications forlong-term changes in atmospheric pCO

2rdquo Quaternary Science

Reviews vol 25 no 23-24 pp 3150ndash3184 2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 16: ResearchArticle - Université Laval Julian... · ResearchArticle A DNA Barcode-Based Evaluation of the Southeast Asian Catfish Genus Hemibagrus Bleeker, 1862 (Teleostei: Siluriformes;

16 Advances in Evolutionary Biology

found in Peninsular Malaysia as well as in the Mekong ChaoPhraya and Mea Khlong Rivers (Figure 12) In addition3 subclades encompass sequences found principally inwestern Borneo subclade B in the Baram and Sadong RiversSarawak subclade C in the Kapuas River West Kalimantanand subclade D in the Baram and Tutong Rivers of Sarawakand Brunei respectively (Figure 12) This latter subclade alsoincludes one genotype found in the Muar River of westernpeninsular Malaysia Finally subclade E encompasses 5sequences from the Mekong Chao Praya and Mae KhlongRivers all within the distribution area of H spilopterus

EvolutionaryHistoryThe evolutionary history of theHemiba-grus species groups analyzed here dates from at least theMiocene 15ndash20Ma (Figure 13) when considering the widelyused molecular clock of 2 sequence divergence per millionyears Bootstrap support for the sister group relationships ofH wyckioides theH guttatus species group andH peguensisare all weak (clearly due to undersampling) and little can besaid of their evolutionary history other than the fact that theyoccupy basal positions in the rooted trees The remainder ofboth the NJ and ML trees is composed of two major sistergroups the H planiceps + H baramensis group and the Hnemurus species group (Figures 2 3 and 13)TheH planiceps+ H baramensis group is strongly supported in both NJ andML analyses (84 and 90 bootstrap support resp Figures2 and 3) The H nemurus species group is also stronglysupported in both analyses (98 and 99 bootstrap supportFigures 2 and 3) These two major phylogenetic groups areestimated to have diverged at least 8 to 12Ma ago during themid to late Miocene (Figure 13)

Much of the evolutionary history of Hemibagrus hasoccurred during the Quaternary starting approximately26Ma ago This is particularly true of the H planicepsand H nemurus species groups The radiation of the 5species included in the H planiceps species group appearsto coincide with the middle Pleistocene transition period ofapproximately 1Ma ago Their high-elevation distributionhowever appears to have largely isolated them from theeffects of lowered sea levels during glacial maxima and theattendant opportunities for widespread dispersal (Figure 5)The evolutionary histories of theH baramensis species groupand the unspecified species group date back to the latePliocene In contrast the low-elevation distribution of theHnemurus species group in Sundaland appears to have favoredtheir widespread distribution during the Quaternary ice ageIn particular H hoevenii appears to be the most recentlydispersed of the species under study with diversification anddispersal occurring approximately 03Ma ago (Figure 13)

4 Discussion

41 The Congruence of Morphological and Genetic AnalysesCombining genetic and morphological analyses to elucidatetaxonomy allowed us to better understand the diversificationand evolutionary relationships within this speciose group offishes Given the rapid and relatively constant rate of mtDNAevolution the deeper the branches within the phylogenetictree are the greater our ability to distinguish valid taxa

should be Conversely the lack of complete congruencebetween morphological- and genetic-based identificationsis expected to increase among the shallower branches of thephylogenetic tree resulting in the appearance of polyphyleticspecies Although 2 of the 8 species groups defined by Ngand Kottelat [7] were not sampled in this study the validityof the remaining 6 species groups and their componentspecies defined on the basis of morphological similaritywas largely confirmed by the analysis of the mitochondrialCyt 119887 bar code As expected congruence is strongest at thebase of the NJ and ML trees where the H guttatus speciesgroup formed a well-defined monophyletic group The Hmenoda and H wyckii species group were each representedby only one species but they were clearly distinct relativeto sequences characterizing the neighboring species groupsThe H planiceps H baramensis and H nemurus speciesgroups and their component species were also generallycongruent in morphological and genetic analyses Also asexpected most cases of discrepancy between morphological-based species identification and genetic-based speciesidentification occurred on relatively shallow branches of thephylogenetic tree These involve principally H spilopterusand the two mtDNA subclades composing H capitulumHowever a far more unexpected lack of congruence involvesthe definition of a deeply divergent unspecified genetic groupconfounding members of theH baramensis species group inthe morphological analysis of Ng and Kottelat [7]

Interpreting Polyphyly The apparent polyphyletic origin ofa species may be due to incomplete lineage sorting amongrecently diverged species the introgression of mtDNA dueto species hybridization or the erroneous identificationof species due to the lumping of morphologically crypticspecies In the absence of nuclear DNA analyses it isoften difficult to distinguish between introgression andincomplete lineage sorting For example several sequencescharacterizing the Indochinese clade of H capitulum(subclade E) are found in the Mekong and Chao PhrayaRivers well within the distributional area of H spilopterusH spilopterus and H capitulum first diverged at least 3MaOn the one hand this apparent case of polyphyly mayhave resulted from incomplete lineage sorting betweenrecently divergedH spilopterus andH capitulum We wouldhowever have expected similar cases of polyphyly amongpopulations of the Indochinese clade of H capitulum butthis was not detected On the other hand the sympatricoccurrence of H spilopterus subclade A and subcladeB sequences with H capitulum Indochinese subclade Esequences in the Mekong River may represent a migrationevent and local introgression of H capitulum with Hspilopterus A similar event may have occurred in theChao Phraya and Mae Khlong Rivers where H spilopterussequences cooccur withH capitulum sequences Computingthe divergence of H capitulum sequences (Indochinesesubclade E) cooccurring with H spilopterus and Hcapitulum sequences occurring in eastern PeninsularMalaysia (subclade A) reveals a mean sequence divergence119901 = 15 (04) Thus the H capitulum sequences occurringin sympatry with H spilopterus diverged at most 750 ka ago

Advances in Evolutionary Biology 17

H fortis

XXRAJ0604H furcatus

H sabanusH semotusH divaricatus

H planicepsGREND0206

VEBAH0907

H bonganPESAL0102

H wyckioidesH macropterus

H guttatusXXRAJ0301Leiocassis longirostris

Ma

4971

92

47

51

9999

99

9999

99

99

99

9999

99

99

99

99

65

96

96

4946

93

79

78

70

91

93

98

86

87

91

95

97

71

85

9999

05101520

005010015020 000

H capitulum Indochinese clades AndashE

H capitulum Sundaic clade

H hoevenii B

H hoevenii A

H spilopterus C

H spilopterus B

H spilopterus AH spilopterus A

H spp CH spp B

H spp A

Figure 13 Linearized tree with branch lengths estimated with the molecular clock using a mutation rate of 1 per Ma (=divergence rate of2 per Ma) The tree topology is identical to the ML reconstruction illustrated in Figure 3 (see the specificities of the tree reconstruction inthe caption of Figure 3) Boxes represent 95 confidence intervals of divergence time estimates for each node Vertical dashed lines redirectto dates indicated on the timescale (Ma) Numbers below timescale indicate the estimated number of substitution changes using Hasegawa-Kishino-Yanomodel (HKY)with gamma distribution (+119866 = 13290) after linearizing the treeH furcatus is synonymizedwithH baramensis

from eastern Peninsular Malaysia populations suggestinga recent introgression event Similarly H capitulum in theKapuas River is characterized by sequences of both theSundaic and the Indochinese clades As both sequences arebroadly sympatric it is not possible to distinguish betweenincomplete lineage sorting and introgression of these closelyrelated intraspecific lineages

A Possible Case of Morphological Stasis Clade 6 of thepresent analysis defines a well-supported monophyletic cladefound in Sabah Sarawak and the Endau River of easternpeninsula Malaysia that has no equivalent taxonomic statusin the revision of Ng and Kottelat [7] In Borneo they occursympatrically with the H baramensis species group and areconfounded as either H baramensis or H semotus These

two clades are genetically highly divergent having divergedbetween 8 and 12Ma ago It thus seems highly unlikely thatthey represent retained polymorphisms due to incompletelineage sorting Sequences previously identified as character-izing H chrysops [11] are part of clade 6 and extend to thesouth (Rajang and Sadong Rivers of Sarawak West Borneo)with one closely related genotype found in eastern peninsularMalaysia (Endau River) where it may well be confoundedwith H capitulum H chrysops was synonymized with Hcapitulum by Ng and Kottelat [7] based on their analysis ofspecimens ofH chrysops from the Rajang RiverThus despitea long and apparently independent phylogenetic historythe unspecified species group appears largely cryptic andconfounded principally with members of the H baramensisspecies group

18 Advances in Evolutionary Biology

We suggest that this situation is an example of mor-phological stasis involving the coexistence of a number ofgenetically divergent yet morphologically cryptic species thatare lumped together [24] A striking example of morpho-logical and ecological stasis among fishes is provided by thecoastal marine bonefishes (Albula spp) Distinct evolution-ary lineages (separated by sequence divergence between 5 and30 [25]) of bonefish coexist in several sample locations yetthey show little morphological or ecological differentiationin sympatry [25] Similarly a proportion of the grey mulletspecies (Mugilidae) also appears to consist of cryptic species[26] For exampleMugil cephalus in Taiwan is characterizedby three divergent lineages that have been shown to charac-terize 3 reproductively isolated but morphologically crypticspecies [26 27]

Here we have discovered 2 highly distinct and structuredclades (each composed of 3 mtDNA subclades) that aremorphologically confounded within different species Forexample H spp C of the unspecified species group isidentified as H baramensis in the Baram River and as Hsemotus in the Padas River We speculate that North Borneoharbors a morphologically cryptic species complex possiblycomposed of several species pairs inhabiting different riversystems The reconstruction of this phylogenetic history andthe taxonomic status of its components remains a majorchallenge to the revision of the Hemibagrus genus

42 Phylogeographic Hypotheses The phylogeography ofthe genus appears to have been principally influenced bytwo geological histories one encompassing the formationof North Borneo the second the emergence of the SundaIslands and their connectivity with the Indochinesemainlandassociated with recurrent sea-level changes North Borneoappears to have acted as a species sink promoting speciationand locally high biodiversity within the genus but with littleevidence of subsequent dispersal out of North Borneo Incontrast the geological events influencing the Indochinesemainland and the Sunda Islands have permitted bothspeciation and differential dispersal depending on thebiology of the species For example the H planiceps speciesgroup is found throughout the Greater Sunda Islands andtheThai-Malay Peninsula as is theH nemurus species groupWhereas the H nemurus species group generally occupiesthe low altitude lower reaches of large rivers the speciescomposing the H planiceps group are found in the upperreaches of large rivers The H nemurus species group iscomposed of a number of widely distributed closely relatedspecies whereas theH planiceps group is composed of a largenumber of species that are highly localized geographicallyIt thus appears that sea-level changes have had little impacton the dispersal history of the H planiceps species group buthave deeply influenced that of the H nemurus species group

The Case of North Borneo From 15Ma to 5Ma greater areasof Borneo became emergent and the central mountainson the Sarawak-Kalimantan border extending into Sabahbecame wider and higher which may have represented newbarriers to dispersal [28 29] Borneo gradually developedinto the present large island by emergence of land in the

north and east The H baramensis species group is restrictedto North Borneo as is the unspecified species group of theHnemurus species group H fortis is also distributed in NorthBorneo The isolation of the freshwater fish communities ofNorth Borneo (particularly in the Labuk-Segama watershed)has long been recognized based on the number of endemicspecies and the concentration of relict species [30] The latePliocene-early Pleistocene diversification of H fortis theunspecified species group and the H baramensis speciesgroup may thus be associated with their dispersal to andisolation within the watersheds of North Borneo Only onesequence of the unspecified species group is found outsideof North Borneo and H fortis appears to be distributedthroughout central and east Borneo [7]

ThePhylogeography of the H nemurus Species Group Periodicmarine transgressions on the Sundaland platform during theearly and middle Miocene [2 29] may have been sufficientto isolate populations of freshwater organisms and promotecladogenesis [31] This may have been responsible for theinitial mid to late Miocene divergence of the H nemurusspecies group The majority of sequences assigned to thisspecies group belong to 3 species (H spilopterus H capitulum(including H nemurus) and H hoevenii) all of which havebeen influenced by the more recent geological history of theSunda Islands and their connectivity with the Indochinesemainland H spilopterus is known from river drainages inIndochina including the Mekong Dong Nai Ban PakongChao Phraya and Mae Klong where it is found in a widevariety of lotic and lentic habitats [7] H spilopterus divergedin the late Pliocene-early Pleistocene possibly isolated tothe north of the Kra Peninsula during the late Pliocenemarine transgression that is purported to have created amajor biogeographic transition zone between the Sundaicand Indochinese biotas ([32] but see [33] for a critique ofthis biogeographic scenario) The existence of 2 subclades(A and B) in the Mekong River separated by 2 sequencedivergence is consistent with intraspecific divergence Thetwo subclades possibly represent 2 historical mtDNA lineagesthat are retained within the species reflecting a Pleistoceneisolation event followed by secondary contact within thecontemporary Mekong River Subclade C from the ChaoPhraya and Mae Khlong Rivers exhibits a level of geneticdivergence consistent with sibling species-level divergenceshowing a 5 sequence divergence relative to Mekong RiverH spilopterus

H capitulum may be considered as part of the Sundaicbiota located principally to the south of the Kra marinetransgression zone that may have isolated H spilopterus tothe north on the Indochinese mainlandThe biogeographicalevent leading to the subsequent formation of two intraspecificgroups within H capitulum is not at all clear The onlyarea where the two clades are clearly geographically separateis on the Thai-Malay Peninsula with the ldquoSundaicrdquo cladeon the west coast and the ldquoIndochineserdquo clade on theeast coast separated by the central Titiwangsa mountainrange The genetic distinctiveness of east and west coastpopulations of H capitulum has also been documented fornuclear DNA [34] A similar pattern of separation has been

Advances in Evolutionary Biology 19

reported for 5 additional freshwater fishes within this region[35] However as the divergence of the two H capitulumclades postdates the formation of the mountain range byapproximately 200Ma the geographic separation of the twoclades most likely represents differential dispersal rather thanancient vicariance The presence of the Sundaic clade on thewest coast of the Thai-Malay Peninsula as well as on theSundaic Islands reflects the historical connections betweeneast Sumatra and west Peninsular Malaysia across the Straitof Malacca This area encompasses the Northern CentralSumatra-Western Malaysia freshwater ecoregion [36] TheMuar River forms its southern border coincident with thesouthernmost distribution of the Sundaic clade on the Thai-Malay Peninsula It is important to note however that mostof Sumatra and Java emerged to achieve their present size andelevation only 5Ma ago and much of East Java continuedto be the site of marine deposition until late Pliocene-earlyPleistocene [29] We may speculate that the Sundaic clade ofH capitulum resulted from the relatively recent colonizationof these islands and Borneo The radiation within the cladeonly dates back to approximately 1Ma ago H nemurus fromWest Java may thus best be considered an isolated regionalpopulation of very recent origin

The diversification of the Indochinese clade dates fromat least 12Ma ago coincident with the mid-Pleistocenetransition at which time low-frequency high-amplitude andquasiperiodic (100 ka) glacial variability emerged It began125Ma ago and was complete by 07Ma ago [37] The tran-sition marked the beginning of major changes in sea levelsduring which the Sunda Shelf periodically emerged and wastraversed by enormous fluvial systems [3] The contributionof these events to the diversification of this clade cannotbe satisfactorily reconstructed One possible explanation isthat high sea levels produced barriers to gene flow and thepopulations of West Borneo (subclades B C and D) wereisolated from those of east Peninsular Malaysia This couldbe due to one or more dispersal events from Borneo via theGreat Sunda River (Figure 1) to east Peninsular Malaysia orlineage sorting whereby sequences of subclades A and E havebeen lost from West Borneo As in the case of the sympatricoccurrence of H spilopterus and H capitulum subclade E(see above) in the Mekong River we are unable to clearlydefine the relative roles of incomplete lineage sorting andintrogression followingmigration Given the recent origins ofthe Indochinese H capitulum sequences incomplete lineagesorting most probably blurs patterns of recent isolation

The last species of the H nemurus species group is Hhoevenii The species is widespread throughout PeninsularMalaysia and the Sundaic Islands It is found primarily inthe lower reaches of rivers and is apparently capable ofwithstanding low salt concentration [7] H hoevenii thusappears to be the most liable of all members of the Hnemurus species group to be impacted by Pleistocene sea-level changes This is clearly reflected in the widespreaddistribution of the sequences of this species Two lineages areevident having diverged no more than 03Ma ago suggest-ing a late Pleistocene isolation event followed by extensivedispersal and admixture in recent times This represents theclearest example of the dispersal opportunities afforded by

Pleistocene sea level changes on the Sunda Shelf In additionthe relatively low genetic diversity detected (only 6 sequencesin 42 fish) suggests a population bottleneck in the recent pastpossibly related to population declines in the lower reaches ofrivers flooded by increasing sea levels

43 Future Directions We have provided a global picture ofthe evolutionary taxonomy of the genus in Southeast AsiaClearly a more rigorous phylogenetic reconstruction of thegenus Hemibagrus requiring the construction of multiplegene trees based on the analysis of both mitochondrial andnuclear DNA markers from a wider taxonomic array ofhemibagrids will involve an even greater effort of samplingthroughout the region In particular we require samplingat smaller spatial and temporal scales For example smallerscale sampling in the Padas River and the Segama Riverof North Borneo using both mitochondrial and nucleargenetic markers would contribute to clarifying the natureof the cryptic species complex believed to inhabit theserivers The demonstration of reproductive isolation andormicroallopatric distribution among morphologically crypticspecies may result in the description of greater speciesrichness within the genus than presently described Thespecies status of H chrysops merits reconsideration givenits highly divergent evolutionary history Nuclear geneticmarkers in conjunction with mtDNA markers could revealcases of historical introgression particularly in the KapuasRiver and the Mekong River The hemibagrid catfishes con-tinue to provide multiple opportunities for the study of theevolutionary taxonomy and biogeography of a major faunalcomponent of the fluvial ecosystems of Southeast Asia

Disclosure

Ethanol-preserved tissue samples of many of the specimensidentified in Table S1 are freely available upon request fromJJD

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Goh Yan Yih for assistance in collectingspecimens and molecular analyses Mrs Francoise Colom-bani for molecular analyses and Dr Heok Hee Ng forspecies identification This work was funded by the NationalUniversity of Singapore grant to P K L Ng and a CanadianNatural Sciences and Engineering Research Council grant toJ J Dodson

References

[1] D J Lohman M de Bruyn T Page et al ldquoBiogeography ofthe Indo-Australian archipelagordquo Annual Review of EcologyEvolution and Systematics vol 42 pp 205ndash226 2011

20 Advances in Evolutionary Biology

[2] D S Woodruff ldquoNeogene marine transgressions palaeogeog-raphy and biogeographic transitions on the Thai-Malay Penin-sulardquo Journal of Biogeography vol 30 no 4 pp 551ndash567 2003

[3] H K Voris ldquoMaps of Pleistocene sea levels in SoutheastAsia shorelines river systems and time durationsrdquo Journal ofBiogeography vol 27 no 5 pp 1153ndash1167 2000

[4] S Y Yap ldquoOn the distributional patterns of Southeast-EastAsian freshwater fish and their historyrdquo Journal of Biogeographyvol 29 no 9 pp 1187ndash1199 2002

[5] S K J McConnell ldquoMapping aquatic faunal exchanges acrossthe Sunda shelf South-East Asia using distributional andgenetic data sets from the cyprinid fish Barbodes gonionotus(Bleeker 1850)rdquo Journal of Natural History vol 38 no 5 pp651ndash670 2004

[6] T Mo Anatomy Relationships and Systematics of the Bagridae(Teleostei Siluroidei) with a Hypothesis of Siluroid Phylogenyvol 17 ofTheses Zoologicae Koeltz Scientific Books 1991

[7] H H Ng and M Kottelat ldquoRevision of the Asian catfish genusHemibagrus bleeker 1862 (Teleostei Siluriformes Bagridae)rdquoRaffles Bulletin of Zoology vol 61 no 1 pp 205ndash291 2013

[8] P K L Ng and H H Ng ldquoHemibagrus gracilis a new speciesof large riverine catfish (Teleostei Bagridae) from PeninsularMalaysiardquo Raffles Bulletin of Zoology vol 43 pp 133ndash142 1995

[9] H H Tan and M Kottelat ldquoRedescription of Betta picta(Teleostei Osphronemidae) and description of Betta falx sp nfrom central Sumatrardquo Revue Suisse de Zoologie vol 105 no 3pp 557ndash568 1998

[10] J J Dodson F Colombani and P K Ng ldquoPhylogeographicstructure in mitochondrial DNA of a South-east Asian fresh-water fish Hemibagrus nemurus (Siluroidei Bagridae) andPleistocene sea-level changes on the Sunda shelfrdquo MolecularEcology vol 4 no 3 pp 331ndash346 1995

[11] H H Ng and J J Dodson ldquoMorphological and genetic descrip-tions of a new species of catfish Hemibagrus chrysops fromSarawak East Malaysia with an assessment of phylogeneticrelationships (Teleostei Bagridae)rdquo Raffles Bulletin of Zoologyvol 47 no 1 pp 45ndash57 1999

[12] P D N Hebert A Cywinska S L Ball and J R DeWaardldquoBiological identifications through DNA barcodesrdquo Proceedingsof the Royal Society B Biological Sciences vol 270 no 1512 pp313ndash321 2003

[13] J C Carolan T EMurray U Fitzpatrick et al ldquoColour patternsdo not diagnose species quantitative evaluation of a DNAbarcoded cryptic bumblebee complexrdquo PLoS ONE vol 7 no 1Article ID e29251 2012

[14] M Kekkonen and P D N Hebert ldquoDNA barcode-baseddelineation of putative species efficient start for taxonomicworkflowsrdquoMolecular Ecology Resources vol 14 no 4 pp 706ndash715 2014

[15] DM Hillis CMoritz and B KMarbleMolecular SystematicsSinauer Sunderland Mass USA 2nd edition 1996

[16] T D Kocher W K Thomas A Meyer et al ldquoDynamics ofmitochondrial DNA evolution in animals amplification andsequencingwith conserved primersrdquoProceedings of theNationalAcademy of Sciences of the United States of America vol 86 no16 pp 6196ndash6200 1989

[17] T A Hall ldquoBioEdit a user-friendly biological sequence align-ment editor and analysis program for Windows 9598NTrdquoNucleic Acids Symposium Series vol 41 pp 95ndash98 1999

[18] D A Benson I Karsch-Mizrachi K Clark D J Lipman JOstell and EW Sayers ldquoGenBankrdquoNucleic Acids Research vol40 no D1 pp D48ndashD53 2012

[19] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[20] X Ku Z Peng R Diogo and S He ldquoMtDNA phylogenyprovides evidence of generic polyphyleticism for East Asianbagrid catfishesrdquoHydrobiologia vol 579 no 1 pp 147ndash159 2007

[21] L Yang and S He ldquoPhylogeography of the freshwater catfishHemibagrus guttatus (Siluriformes Bagridae) implications forSouth China biogeography and influence of sea-level changesrdquoMolecular Phylogenetics and Evolution vol 49 no 1 pp 393ndash398 2008

[22] B W Bowen A L Bass L A Rocha W S Grant and DR Robertson ldquoPhylogeography of the trumpetfishes (Aulosto-mus) ring species complex on a global scalerdquo Evolution vol 55no 5 pp 1029ndash1039 2001

[23] Y P Kartavtsev ldquoDivergence at Cyt-b and Co-1 mtDNA geneson different taxonomic levels and genetics of speciation inanimalsrdquoMitochondrial DNA vol 22 no 3 pp 55ndash65 2011

[24] SWada Y Kameda and S Chiba ldquoLong-term stasis and short-term divergence in the phenotypes of microsnails on oceanicislandsrdquoMolecular Ecology vol 22 no 18 pp 4801ndash4810 2013

[25] J Colborn R E Crabtree J B Shaklee E Pfeiler and B WBowen ldquoThe evolutionary enigma of bonefishes (Albula spp)cryptic species and ancient separations in a globally distributedshorefishrdquo Evolution vol 55 no 4 pp 807ndash820 2001

[26] J-D Durand K-N Shen W-J Chen et al ldquoSystematics of thegrey mullets (Teleostei Mugiliformes Mugilidae) molecularphylogenetic evidence challenges two centuries ofmorphology-based taxonomyrdquo Molecular Phylogenetics and Evolution vol64 no 1 pp 73ndash92 2012

[27] K-N Shen B W Jamandre C-C Hsu W-N Tzeng and J-D Durand ldquoPlio-Pleistocene sea level and temperature fluc-tuations in the northwestern Pacific promoted speciation inthe globally-distributed flathead mullet Mugil cephalusrdquo BMCEvolutionary Biology vol 11 no 1 article 83 2011

[28] R Hall ldquoThe plate tectonics of Cenozoic SE Asia and thedistribution of land and seardquo in Biogeography and GeologicalEvolution of SE Asia R Hall and J D Holloway Eds pp 99ndash131Backhuys Publishers Leiden The Netherlands 1998

[29] R Hall ldquoSoutheast Asiarsquos changing palaeogeographyrdquo Blumeavol 54 no 1ndash3 pp 148ndash161 2009

[30] R F Inger and P K Chen ldquoThe freshwater fishes of NorthBorneordquo Fieldiana Zoology vol 45 pp 1ndash268 1962

[31] J Bohlen V Slechtova H H Tan and R Britz ldquoPhylogenyof the Southeast Asian freshwater fish genus Pangio (Cyprini-formes Cobitidae)rdquoMolecular Phylogenetics and Evolution vol61 no 3 pp 854ndash865 2011

[32] M de Bruyn E Nugroho M M Hossain J C Wilson andP B Mather ldquoPhylogeographic evidence for the existence ofan ancient biogeographic barrier the Isthmus of Kra SeawayrdquoHeredity vol 94 no 3 pp 370ndash378 2005

[33] J Parnell ldquoThe biogeography of the Isthmus of Kra region areviewrdquoNordic Journal of Botany vol 31 no 1 pp 001ndash015 2013

[34] S Usmani S G Tan S S Siraj and K Yusoff ldquoPopulationstructure of the Southeast Asian river catfishMystus nemurusrdquoAnimal Genetics vol 34 no 6 pp 462ndash464 2003

[35] M P Tan A F J Jamsari and M N Siti Azizah ldquoPhylo-geographic pattern of the striped snakehead Channa striatain Sundaland ancient river connectivity geographical and

Advances in Evolutionary Biology 21

anthropogenic singnaturesrdquo PLoS ONE vol 7 no 12 Article IDe52089 2012

[36] R Abell M L Thieme C Revenga et al ldquoFreshwater ecore-gions of the world a new map of biogeographic units forfreshwater biodiversity conservationrdquo BioScience vol 58 no 5pp 403ndash414 2008

[37] P U Clark D Archer D Pollard et al ldquoThemiddle Pleistocenetransition characteristics mechanisms and implications forlong-term changes in atmospheric pCO

2rdquo Quaternary Science

Reviews vol 25 no 23-24 pp 3150ndash3184 2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 17: ResearchArticle - Université Laval Julian... · ResearchArticle A DNA Barcode-Based Evaluation of the Southeast Asian Catfish Genus Hemibagrus Bleeker, 1862 (Teleostei: Siluriformes;

Advances in Evolutionary Biology 17

H fortis

XXRAJ0604H furcatus

H sabanusH semotusH divaricatus

H planicepsGREND0206

VEBAH0907

H bonganPESAL0102

H wyckioidesH macropterus

H guttatusXXRAJ0301Leiocassis longirostris

Ma

4971

92

47

51

9999

99

9999

99

99

99

9999

99

99

99

99

65

96

96

4946

93

79

78

70

91

93

98

86

87

91

95

97

71

85

9999

05101520

005010015020 000

H capitulum Indochinese clades AndashE

H capitulum Sundaic clade

H hoevenii B

H hoevenii A

H spilopterus C

H spilopterus B

H spilopterus AH spilopterus A

H spp CH spp B

H spp A

Figure 13 Linearized tree with branch lengths estimated with the molecular clock using a mutation rate of 1 per Ma (=divergence rate of2 per Ma) The tree topology is identical to the ML reconstruction illustrated in Figure 3 (see the specificities of the tree reconstruction inthe caption of Figure 3) Boxes represent 95 confidence intervals of divergence time estimates for each node Vertical dashed lines redirectto dates indicated on the timescale (Ma) Numbers below timescale indicate the estimated number of substitution changes using Hasegawa-Kishino-Yanomodel (HKY)with gamma distribution (+119866 = 13290) after linearizing the treeH furcatus is synonymizedwithH baramensis

from eastern Peninsular Malaysia populations suggestinga recent introgression event Similarly H capitulum in theKapuas River is characterized by sequences of both theSundaic and the Indochinese clades As both sequences arebroadly sympatric it is not possible to distinguish betweenincomplete lineage sorting and introgression of these closelyrelated intraspecific lineages

A Possible Case of Morphological Stasis Clade 6 of thepresent analysis defines a well-supported monophyletic cladefound in Sabah Sarawak and the Endau River of easternpeninsula Malaysia that has no equivalent taxonomic statusin the revision of Ng and Kottelat [7] In Borneo they occursympatrically with the H baramensis species group and areconfounded as either H baramensis or H semotus These

two clades are genetically highly divergent having divergedbetween 8 and 12Ma ago It thus seems highly unlikely thatthey represent retained polymorphisms due to incompletelineage sorting Sequences previously identified as character-izing H chrysops [11] are part of clade 6 and extend to thesouth (Rajang and Sadong Rivers of Sarawak West Borneo)with one closely related genotype found in eastern peninsularMalaysia (Endau River) where it may well be confoundedwith H capitulum H chrysops was synonymized with Hcapitulum by Ng and Kottelat [7] based on their analysis ofspecimens ofH chrysops from the Rajang RiverThus despitea long and apparently independent phylogenetic historythe unspecified species group appears largely cryptic andconfounded principally with members of the H baramensisspecies group

18 Advances in Evolutionary Biology

We suggest that this situation is an example of mor-phological stasis involving the coexistence of a number ofgenetically divergent yet morphologically cryptic species thatare lumped together [24] A striking example of morpho-logical and ecological stasis among fishes is provided by thecoastal marine bonefishes (Albula spp) Distinct evolution-ary lineages (separated by sequence divergence between 5 and30 [25]) of bonefish coexist in several sample locations yetthey show little morphological or ecological differentiationin sympatry [25] Similarly a proportion of the grey mulletspecies (Mugilidae) also appears to consist of cryptic species[26] For exampleMugil cephalus in Taiwan is characterizedby three divergent lineages that have been shown to charac-terize 3 reproductively isolated but morphologically crypticspecies [26 27]

Here we have discovered 2 highly distinct and structuredclades (each composed of 3 mtDNA subclades) that aremorphologically confounded within different species Forexample H spp C of the unspecified species group isidentified as H baramensis in the Baram River and as Hsemotus in the Padas River We speculate that North Borneoharbors a morphologically cryptic species complex possiblycomposed of several species pairs inhabiting different riversystems The reconstruction of this phylogenetic history andthe taxonomic status of its components remains a majorchallenge to the revision of the Hemibagrus genus

42 Phylogeographic Hypotheses The phylogeography ofthe genus appears to have been principally influenced bytwo geological histories one encompassing the formationof North Borneo the second the emergence of the SundaIslands and their connectivity with the Indochinesemainlandassociated with recurrent sea-level changes North Borneoappears to have acted as a species sink promoting speciationand locally high biodiversity within the genus but with littleevidence of subsequent dispersal out of North Borneo Incontrast the geological events influencing the Indochinesemainland and the Sunda Islands have permitted bothspeciation and differential dispersal depending on thebiology of the species For example the H planiceps speciesgroup is found throughout the Greater Sunda Islands andtheThai-Malay Peninsula as is theH nemurus species groupWhereas the H nemurus species group generally occupiesthe low altitude lower reaches of large rivers the speciescomposing the H planiceps group are found in the upperreaches of large rivers The H nemurus species group iscomposed of a number of widely distributed closely relatedspecies whereas theH planiceps group is composed of a largenumber of species that are highly localized geographicallyIt thus appears that sea-level changes have had little impacton the dispersal history of the H planiceps species group buthave deeply influenced that of the H nemurus species group

The Case of North Borneo From 15Ma to 5Ma greater areasof Borneo became emergent and the central mountainson the Sarawak-Kalimantan border extending into Sabahbecame wider and higher which may have represented newbarriers to dispersal [28 29] Borneo gradually developedinto the present large island by emergence of land in the

north and east The H baramensis species group is restrictedto North Borneo as is the unspecified species group of theHnemurus species group H fortis is also distributed in NorthBorneo The isolation of the freshwater fish communities ofNorth Borneo (particularly in the Labuk-Segama watershed)has long been recognized based on the number of endemicspecies and the concentration of relict species [30] The latePliocene-early Pleistocene diversification of H fortis theunspecified species group and the H baramensis speciesgroup may thus be associated with their dispersal to andisolation within the watersheds of North Borneo Only onesequence of the unspecified species group is found outsideof North Borneo and H fortis appears to be distributedthroughout central and east Borneo [7]

ThePhylogeography of the H nemurus Species Group Periodicmarine transgressions on the Sundaland platform during theearly and middle Miocene [2 29] may have been sufficientto isolate populations of freshwater organisms and promotecladogenesis [31] This may have been responsible for theinitial mid to late Miocene divergence of the H nemurusspecies group The majority of sequences assigned to thisspecies group belong to 3 species (H spilopterus H capitulum(including H nemurus) and H hoevenii) all of which havebeen influenced by the more recent geological history of theSunda Islands and their connectivity with the Indochinesemainland H spilopterus is known from river drainages inIndochina including the Mekong Dong Nai Ban PakongChao Phraya and Mae Klong where it is found in a widevariety of lotic and lentic habitats [7] H spilopterus divergedin the late Pliocene-early Pleistocene possibly isolated tothe north of the Kra Peninsula during the late Pliocenemarine transgression that is purported to have created amajor biogeographic transition zone between the Sundaicand Indochinese biotas ([32] but see [33] for a critique ofthis biogeographic scenario) The existence of 2 subclades(A and B) in the Mekong River separated by 2 sequencedivergence is consistent with intraspecific divergence Thetwo subclades possibly represent 2 historical mtDNA lineagesthat are retained within the species reflecting a Pleistoceneisolation event followed by secondary contact within thecontemporary Mekong River Subclade C from the ChaoPhraya and Mae Khlong Rivers exhibits a level of geneticdivergence consistent with sibling species-level divergenceshowing a 5 sequence divergence relative to Mekong RiverH spilopterus

H capitulum may be considered as part of the Sundaicbiota located principally to the south of the Kra marinetransgression zone that may have isolated H spilopterus tothe north on the Indochinese mainlandThe biogeographicalevent leading to the subsequent formation of two intraspecificgroups within H capitulum is not at all clear The onlyarea where the two clades are clearly geographically separateis on the Thai-Malay Peninsula with the ldquoSundaicrdquo cladeon the west coast and the ldquoIndochineserdquo clade on theeast coast separated by the central Titiwangsa mountainrange The genetic distinctiveness of east and west coastpopulations of H capitulum has also been documented fornuclear DNA [34] A similar pattern of separation has been

Advances in Evolutionary Biology 19

reported for 5 additional freshwater fishes within this region[35] However as the divergence of the two H capitulumclades postdates the formation of the mountain range byapproximately 200Ma the geographic separation of the twoclades most likely represents differential dispersal rather thanancient vicariance The presence of the Sundaic clade on thewest coast of the Thai-Malay Peninsula as well as on theSundaic Islands reflects the historical connections betweeneast Sumatra and west Peninsular Malaysia across the Straitof Malacca This area encompasses the Northern CentralSumatra-Western Malaysia freshwater ecoregion [36] TheMuar River forms its southern border coincident with thesouthernmost distribution of the Sundaic clade on the Thai-Malay Peninsula It is important to note however that mostof Sumatra and Java emerged to achieve their present size andelevation only 5Ma ago and much of East Java continuedto be the site of marine deposition until late Pliocene-earlyPleistocene [29] We may speculate that the Sundaic clade ofH capitulum resulted from the relatively recent colonizationof these islands and Borneo The radiation within the cladeonly dates back to approximately 1Ma ago H nemurus fromWest Java may thus best be considered an isolated regionalpopulation of very recent origin

The diversification of the Indochinese clade dates fromat least 12Ma ago coincident with the mid-Pleistocenetransition at which time low-frequency high-amplitude andquasiperiodic (100 ka) glacial variability emerged It began125Ma ago and was complete by 07Ma ago [37] The tran-sition marked the beginning of major changes in sea levelsduring which the Sunda Shelf periodically emerged and wastraversed by enormous fluvial systems [3] The contributionof these events to the diversification of this clade cannotbe satisfactorily reconstructed One possible explanation isthat high sea levels produced barriers to gene flow and thepopulations of West Borneo (subclades B C and D) wereisolated from those of east Peninsular Malaysia This couldbe due to one or more dispersal events from Borneo via theGreat Sunda River (Figure 1) to east Peninsular Malaysia orlineage sorting whereby sequences of subclades A and E havebeen lost from West Borneo As in the case of the sympatricoccurrence of H spilopterus and H capitulum subclade E(see above) in the Mekong River we are unable to clearlydefine the relative roles of incomplete lineage sorting andintrogression followingmigration Given the recent origins ofthe Indochinese H capitulum sequences incomplete lineagesorting most probably blurs patterns of recent isolation

The last species of the H nemurus species group is Hhoevenii The species is widespread throughout PeninsularMalaysia and the Sundaic Islands It is found primarily inthe lower reaches of rivers and is apparently capable ofwithstanding low salt concentration [7] H hoevenii thusappears to be the most liable of all members of the Hnemurus species group to be impacted by Pleistocene sea-level changes This is clearly reflected in the widespreaddistribution of the sequences of this species Two lineages areevident having diverged no more than 03Ma ago suggest-ing a late Pleistocene isolation event followed by extensivedispersal and admixture in recent times This represents theclearest example of the dispersal opportunities afforded by

Pleistocene sea level changes on the Sunda Shelf In additionthe relatively low genetic diversity detected (only 6 sequencesin 42 fish) suggests a population bottleneck in the recent pastpossibly related to population declines in the lower reaches ofrivers flooded by increasing sea levels

43 Future Directions We have provided a global picture ofthe evolutionary taxonomy of the genus in Southeast AsiaClearly a more rigorous phylogenetic reconstruction of thegenus Hemibagrus requiring the construction of multiplegene trees based on the analysis of both mitochondrial andnuclear DNA markers from a wider taxonomic array ofhemibagrids will involve an even greater effort of samplingthroughout the region In particular we require samplingat smaller spatial and temporal scales For example smallerscale sampling in the Padas River and the Segama Riverof North Borneo using both mitochondrial and nucleargenetic markers would contribute to clarifying the natureof the cryptic species complex believed to inhabit theserivers The demonstration of reproductive isolation andormicroallopatric distribution among morphologically crypticspecies may result in the description of greater speciesrichness within the genus than presently described Thespecies status of H chrysops merits reconsideration givenits highly divergent evolutionary history Nuclear geneticmarkers in conjunction with mtDNA markers could revealcases of historical introgression particularly in the KapuasRiver and the Mekong River The hemibagrid catfishes con-tinue to provide multiple opportunities for the study of theevolutionary taxonomy and biogeography of a major faunalcomponent of the fluvial ecosystems of Southeast Asia

Disclosure

Ethanol-preserved tissue samples of many of the specimensidentified in Table S1 are freely available upon request fromJJD

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Goh Yan Yih for assistance in collectingspecimens and molecular analyses Mrs Francoise Colom-bani for molecular analyses and Dr Heok Hee Ng forspecies identification This work was funded by the NationalUniversity of Singapore grant to P K L Ng and a CanadianNatural Sciences and Engineering Research Council grant toJ J Dodson

References

[1] D J Lohman M de Bruyn T Page et al ldquoBiogeography ofthe Indo-Australian archipelagordquo Annual Review of EcologyEvolution and Systematics vol 42 pp 205ndash226 2011

20 Advances in Evolutionary Biology

[2] D S Woodruff ldquoNeogene marine transgressions palaeogeog-raphy and biogeographic transitions on the Thai-Malay Penin-sulardquo Journal of Biogeography vol 30 no 4 pp 551ndash567 2003

[3] H K Voris ldquoMaps of Pleistocene sea levels in SoutheastAsia shorelines river systems and time durationsrdquo Journal ofBiogeography vol 27 no 5 pp 1153ndash1167 2000

[4] S Y Yap ldquoOn the distributional patterns of Southeast-EastAsian freshwater fish and their historyrdquo Journal of Biogeographyvol 29 no 9 pp 1187ndash1199 2002

[5] S K J McConnell ldquoMapping aquatic faunal exchanges acrossthe Sunda shelf South-East Asia using distributional andgenetic data sets from the cyprinid fish Barbodes gonionotus(Bleeker 1850)rdquo Journal of Natural History vol 38 no 5 pp651ndash670 2004

[6] T Mo Anatomy Relationships and Systematics of the Bagridae(Teleostei Siluroidei) with a Hypothesis of Siluroid Phylogenyvol 17 ofTheses Zoologicae Koeltz Scientific Books 1991

[7] H H Ng and M Kottelat ldquoRevision of the Asian catfish genusHemibagrus bleeker 1862 (Teleostei Siluriformes Bagridae)rdquoRaffles Bulletin of Zoology vol 61 no 1 pp 205ndash291 2013

[8] P K L Ng and H H Ng ldquoHemibagrus gracilis a new speciesof large riverine catfish (Teleostei Bagridae) from PeninsularMalaysiardquo Raffles Bulletin of Zoology vol 43 pp 133ndash142 1995

[9] H H Tan and M Kottelat ldquoRedescription of Betta picta(Teleostei Osphronemidae) and description of Betta falx sp nfrom central Sumatrardquo Revue Suisse de Zoologie vol 105 no 3pp 557ndash568 1998

[10] J J Dodson F Colombani and P K Ng ldquoPhylogeographicstructure in mitochondrial DNA of a South-east Asian fresh-water fish Hemibagrus nemurus (Siluroidei Bagridae) andPleistocene sea-level changes on the Sunda shelfrdquo MolecularEcology vol 4 no 3 pp 331ndash346 1995

[11] H H Ng and J J Dodson ldquoMorphological and genetic descrip-tions of a new species of catfish Hemibagrus chrysops fromSarawak East Malaysia with an assessment of phylogeneticrelationships (Teleostei Bagridae)rdquo Raffles Bulletin of Zoologyvol 47 no 1 pp 45ndash57 1999

[12] P D N Hebert A Cywinska S L Ball and J R DeWaardldquoBiological identifications through DNA barcodesrdquo Proceedingsof the Royal Society B Biological Sciences vol 270 no 1512 pp313ndash321 2003

[13] J C Carolan T EMurray U Fitzpatrick et al ldquoColour patternsdo not diagnose species quantitative evaluation of a DNAbarcoded cryptic bumblebee complexrdquo PLoS ONE vol 7 no 1Article ID e29251 2012

[14] M Kekkonen and P D N Hebert ldquoDNA barcode-baseddelineation of putative species efficient start for taxonomicworkflowsrdquoMolecular Ecology Resources vol 14 no 4 pp 706ndash715 2014

[15] DM Hillis CMoritz and B KMarbleMolecular SystematicsSinauer Sunderland Mass USA 2nd edition 1996

[16] T D Kocher W K Thomas A Meyer et al ldquoDynamics ofmitochondrial DNA evolution in animals amplification andsequencingwith conserved primersrdquoProceedings of theNationalAcademy of Sciences of the United States of America vol 86 no16 pp 6196ndash6200 1989

[17] T A Hall ldquoBioEdit a user-friendly biological sequence align-ment editor and analysis program for Windows 9598NTrdquoNucleic Acids Symposium Series vol 41 pp 95ndash98 1999

[18] D A Benson I Karsch-Mizrachi K Clark D J Lipman JOstell and EW Sayers ldquoGenBankrdquoNucleic Acids Research vol40 no D1 pp D48ndashD53 2012

[19] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[20] X Ku Z Peng R Diogo and S He ldquoMtDNA phylogenyprovides evidence of generic polyphyleticism for East Asianbagrid catfishesrdquoHydrobiologia vol 579 no 1 pp 147ndash159 2007

[21] L Yang and S He ldquoPhylogeography of the freshwater catfishHemibagrus guttatus (Siluriformes Bagridae) implications forSouth China biogeography and influence of sea-level changesrdquoMolecular Phylogenetics and Evolution vol 49 no 1 pp 393ndash398 2008

[22] B W Bowen A L Bass L A Rocha W S Grant and DR Robertson ldquoPhylogeography of the trumpetfishes (Aulosto-mus) ring species complex on a global scalerdquo Evolution vol 55no 5 pp 1029ndash1039 2001

[23] Y P Kartavtsev ldquoDivergence at Cyt-b and Co-1 mtDNA geneson different taxonomic levels and genetics of speciation inanimalsrdquoMitochondrial DNA vol 22 no 3 pp 55ndash65 2011

[24] SWada Y Kameda and S Chiba ldquoLong-term stasis and short-term divergence in the phenotypes of microsnails on oceanicislandsrdquoMolecular Ecology vol 22 no 18 pp 4801ndash4810 2013

[25] J Colborn R E Crabtree J B Shaklee E Pfeiler and B WBowen ldquoThe evolutionary enigma of bonefishes (Albula spp)cryptic species and ancient separations in a globally distributedshorefishrdquo Evolution vol 55 no 4 pp 807ndash820 2001

[26] J-D Durand K-N Shen W-J Chen et al ldquoSystematics of thegrey mullets (Teleostei Mugiliformes Mugilidae) molecularphylogenetic evidence challenges two centuries ofmorphology-based taxonomyrdquo Molecular Phylogenetics and Evolution vol64 no 1 pp 73ndash92 2012

[27] K-N Shen B W Jamandre C-C Hsu W-N Tzeng and J-D Durand ldquoPlio-Pleistocene sea level and temperature fluc-tuations in the northwestern Pacific promoted speciation inthe globally-distributed flathead mullet Mugil cephalusrdquo BMCEvolutionary Biology vol 11 no 1 article 83 2011

[28] R Hall ldquoThe plate tectonics of Cenozoic SE Asia and thedistribution of land and seardquo in Biogeography and GeologicalEvolution of SE Asia R Hall and J D Holloway Eds pp 99ndash131Backhuys Publishers Leiden The Netherlands 1998

[29] R Hall ldquoSoutheast Asiarsquos changing palaeogeographyrdquo Blumeavol 54 no 1ndash3 pp 148ndash161 2009

[30] R F Inger and P K Chen ldquoThe freshwater fishes of NorthBorneordquo Fieldiana Zoology vol 45 pp 1ndash268 1962

[31] J Bohlen V Slechtova H H Tan and R Britz ldquoPhylogenyof the Southeast Asian freshwater fish genus Pangio (Cyprini-formes Cobitidae)rdquoMolecular Phylogenetics and Evolution vol61 no 3 pp 854ndash865 2011

[32] M de Bruyn E Nugroho M M Hossain J C Wilson andP B Mather ldquoPhylogeographic evidence for the existence ofan ancient biogeographic barrier the Isthmus of Kra SeawayrdquoHeredity vol 94 no 3 pp 370ndash378 2005

[33] J Parnell ldquoThe biogeography of the Isthmus of Kra region areviewrdquoNordic Journal of Botany vol 31 no 1 pp 001ndash015 2013

[34] S Usmani S G Tan S S Siraj and K Yusoff ldquoPopulationstructure of the Southeast Asian river catfishMystus nemurusrdquoAnimal Genetics vol 34 no 6 pp 462ndash464 2003

[35] M P Tan A F J Jamsari and M N Siti Azizah ldquoPhylo-geographic pattern of the striped snakehead Channa striatain Sundaland ancient river connectivity geographical and

Advances in Evolutionary Biology 21

anthropogenic singnaturesrdquo PLoS ONE vol 7 no 12 Article IDe52089 2012

[36] R Abell M L Thieme C Revenga et al ldquoFreshwater ecore-gions of the world a new map of biogeographic units forfreshwater biodiversity conservationrdquo BioScience vol 58 no 5pp 403ndash414 2008

[37] P U Clark D Archer D Pollard et al ldquoThemiddle Pleistocenetransition characteristics mechanisms and implications forlong-term changes in atmospheric pCO

2rdquo Quaternary Science

Reviews vol 25 no 23-24 pp 3150ndash3184 2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 18: ResearchArticle - Université Laval Julian... · ResearchArticle A DNA Barcode-Based Evaluation of the Southeast Asian Catfish Genus Hemibagrus Bleeker, 1862 (Teleostei: Siluriformes;

18 Advances in Evolutionary Biology

We suggest that this situation is an example of mor-phological stasis involving the coexistence of a number ofgenetically divergent yet morphologically cryptic species thatare lumped together [24] A striking example of morpho-logical and ecological stasis among fishes is provided by thecoastal marine bonefishes (Albula spp) Distinct evolution-ary lineages (separated by sequence divergence between 5 and30 [25]) of bonefish coexist in several sample locations yetthey show little morphological or ecological differentiationin sympatry [25] Similarly a proportion of the grey mulletspecies (Mugilidae) also appears to consist of cryptic species[26] For exampleMugil cephalus in Taiwan is characterizedby three divergent lineages that have been shown to charac-terize 3 reproductively isolated but morphologically crypticspecies [26 27]

Here we have discovered 2 highly distinct and structuredclades (each composed of 3 mtDNA subclades) that aremorphologically confounded within different species Forexample H spp C of the unspecified species group isidentified as H baramensis in the Baram River and as Hsemotus in the Padas River We speculate that North Borneoharbors a morphologically cryptic species complex possiblycomposed of several species pairs inhabiting different riversystems The reconstruction of this phylogenetic history andthe taxonomic status of its components remains a majorchallenge to the revision of the Hemibagrus genus

42 Phylogeographic Hypotheses The phylogeography ofthe genus appears to have been principally influenced bytwo geological histories one encompassing the formationof North Borneo the second the emergence of the SundaIslands and their connectivity with the Indochinesemainlandassociated with recurrent sea-level changes North Borneoappears to have acted as a species sink promoting speciationand locally high biodiversity within the genus but with littleevidence of subsequent dispersal out of North Borneo Incontrast the geological events influencing the Indochinesemainland and the Sunda Islands have permitted bothspeciation and differential dispersal depending on thebiology of the species For example the H planiceps speciesgroup is found throughout the Greater Sunda Islands andtheThai-Malay Peninsula as is theH nemurus species groupWhereas the H nemurus species group generally occupiesthe low altitude lower reaches of large rivers the speciescomposing the H planiceps group are found in the upperreaches of large rivers The H nemurus species group iscomposed of a number of widely distributed closely relatedspecies whereas theH planiceps group is composed of a largenumber of species that are highly localized geographicallyIt thus appears that sea-level changes have had little impacton the dispersal history of the H planiceps species group buthave deeply influenced that of the H nemurus species group

The Case of North Borneo From 15Ma to 5Ma greater areasof Borneo became emergent and the central mountainson the Sarawak-Kalimantan border extending into Sabahbecame wider and higher which may have represented newbarriers to dispersal [28 29] Borneo gradually developedinto the present large island by emergence of land in the

north and east The H baramensis species group is restrictedto North Borneo as is the unspecified species group of theHnemurus species group H fortis is also distributed in NorthBorneo The isolation of the freshwater fish communities ofNorth Borneo (particularly in the Labuk-Segama watershed)has long been recognized based on the number of endemicspecies and the concentration of relict species [30] The latePliocene-early Pleistocene diversification of H fortis theunspecified species group and the H baramensis speciesgroup may thus be associated with their dispersal to andisolation within the watersheds of North Borneo Only onesequence of the unspecified species group is found outsideof North Borneo and H fortis appears to be distributedthroughout central and east Borneo [7]

ThePhylogeography of the H nemurus Species Group Periodicmarine transgressions on the Sundaland platform during theearly and middle Miocene [2 29] may have been sufficientto isolate populations of freshwater organisms and promotecladogenesis [31] This may have been responsible for theinitial mid to late Miocene divergence of the H nemurusspecies group The majority of sequences assigned to thisspecies group belong to 3 species (H spilopterus H capitulum(including H nemurus) and H hoevenii) all of which havebeen influenced by the more recent geological history of theSunda Islands and their connectivity with the Indochinesemainland H spilopterus is known from river drainages inIndochina including the Mekong Dong Nai Ban PakongChao Phraya and Mae Klong where it is found in a widevariety of lotic and lentic habitats [7] H spilopterus divergedin the late Pliocene-early Pleistocene possibly isolated tothe north of the Kra Peninsula during the late Pliocenemarine transgression that is purported to have created amajor biogeographic transition zone between the Sundaicand Indochinese biotas ([32] but see [33] for a critique ofthis biogeographic scenario) The existence of 2 subclades(A and B) in the Mekong River separated by 2 sequencedivergence is consistent with intraspecific divergence Thetwo subclades possibly represent 2 historical mtDNA lineagesthat are retained within the species reflecting a Pleistoceneisolation event followed by secondary contact within thecontemporary Mekong River Subclade C from the ChaoPhraya and Mae Khlong Rivers exhibits a level of geneticdivergence consistent with sibling species-level divergenceshowing a 5 sequence divergence relative to Mekong RiverH spilopterus

H capitulum may be considered as part of the Sundaicbiota located principally to the south of the Kra marinetransgression zone that may have isolated H spilopterus tothe north on the Indochinese mainlandThe biogeographicalevent leading to the subsequent formation of two intraspecificgroups within H capitulum is not at all clear The onlyarea where the two clades are clearly geographically separateis on the Thai-Malay Peninsula with the ldquoSundaicrdquo cladeon the west coast and the ldquoIndochineserdquo clade on theeast coast separated by the central Titiwangsa mountainrange The genetic distinctiveness of east and west coastpopulations of H capitulum has also been documented fornuclear DNA [34] A similar pattern of separation has been

Advances in Evolutionary Biology 19

reported for 5 additional freshwater fishes within this region[35] However as the divergence of the two H capitulumclades postdates the formation of the mountain range byapproximately 200Ma the geographic separation of the twoclades most likely represents differential dispersal rather thanancient vicariance The presence of the Sundaic clade on thewest coast of the Thai-Malay Peninsula as well as on theSundaic Islands reflects the historical connections betweeneast Sumatra and west Peninsular Malaysia across the Straitof Malacca This area encompasses the Northern CentralSumatra-Western Malaysia freshwater ecoregion [36] TheMuar River forms its southern border coincident with thesouthernmost distribution of the Sundaic clade on the Thai-Malay Peninsula It is important to note however that mostof Sumatra and Java emerged to achieve their present size andelevation only 5Ma ago and much of East Java continuedto be the site of marine deposition until late Pliocene-earlyPleistocene [29] We may speculate that the Sundaic clade ofH capitulum resulted from the relatively recent colonizationof these islands and Borneo The radiation within the cladeonly dates back to approximately 1Ma ago H nemurus fromWest Java may thus best be considered an isolated regionalpopulation of very recent origin

The diversification of the Indochinese clade dates fromat least 12Ma ago coincident with the mid-Pleistocenetransition at which time low-frequency high-amplitude andquasiperiodic (100 ka) glacial variability emerged It began125Ma ago and was complete by 07Ma ago [37] The tran-sition marked the beginning of major changes in sea levelsduring which the Sunda Shelf periodically emerged and wastraversed by enormous fluvial systems [3] The contributionof these events to the diversification of this clade cannotbe satisfactorily reconstructed One possible explanation isthat high sea levels produced barriers to gene flow and thepopulations of West Borneo (subclades B C and D) wereisolated from those of east Peninsular Malaysia This couldbe due to one or more dispersal events from Borneo via theGreat Sunda River (Figure 1) to east Peninsular Malaysia orlineage sorting whereby sequences of subclades A and E havebeen lost from West Borneo As in the case of the sympatricoccurrence of H spilopterus and H capitulum subclade E(see above) in the Mekong River we are unable to clearlydefine the relative roles of incomplete lineage sorting andintrogression followingmigration Given the recent origins ofthe Indochinese H capitulum sequences incomplete lineagesorting most probably blurs patterns of recent isolation

The last species of the H nemurus species group is Hhoevenii The species is widespread throughout PeninsularMalaysia and the Sundaic Islands It is found primarily inthe lower reaches of rivers and is apparently capable ofwithstanding low salt concentration [7] H hoevenii thusappears to be the most liable of all members of the Hnemurus species group to be impacted by Pleistocene sea-level changes This is clearly reflected in the widespreaddistribution of the sequences of this species Two lineages areevident having diverged no more than 03Ma ago suggest-ing a late Pleistocene isolation event followed by extensivedispersal and admixture in recent times This represents theclearest example of the dispersal opportunities afforded by

Pleistocene sea level changes on the Sunda Shelf In additionthe relatively low genetic diversity detected (only 6 sequencesin 42 fish) suggests a population bottleneck in the recent pastpossibly related to population declines in the lower reaches ofrivers flooded by increasing sea levels

43 Future Directions We have provided a global picture ofthe evolutionary taxonomy of the genus in Southeast AsiaClearly a more rigorous phylogenetic reconstruction of thegenus Hemibagrus requiring the construction of multiplegene trees based on the analysis of both mitochondrial andnuclear DNA markers from a wider taxonomic array ofhemibagrids will involve an even greater effort of samplingthroughout the region In particular we require samplingat smaller spatial and temporal scales For example smallerscale sampling in the Padas River and the Segama Riverof North Borneo using both mitochondrial and nucleargenetic markers would contribute to clarifying the natureof the cryptic species complex believed to inhabit theserivers The demonstration of reproductive isolation andormicroallopatric distribution among morphologically crypticspecies may result in the description of greater speciesrichness within the genus than presently described Thespecies status of H chrysops merits reconsideration givenits highly divergent evolutionary history Nuclear geneticmarkers in conjunction with mtDNA markers could revealcases of historical introgression particularly in the KapuasRiver and the Mekong River The hemibagrid catfishes con-tinue to provide multiple opportunities for the study of theevolutionary taxonomy and biogeography of a major faunalcomponent of the fluvial ecosystems of Southeast Asia

Disclosure

Ethanol-preserved tissue samples of many of the specimensidentified in Table S1 are freely available upon request fromJJD

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Goh Yan Yih for assistance in collectingspecimens and molecular analyses Mrs Francoise Colom-bani for molecular analyses and Dr Heok Hee Ng forspecies identification This work was funded by the NationalUniversity of Singapore grant to P K L Ng and a CanadianNatural Sciences and Engineering Research Council grant toJ J Dodson

References

[1] D J Lohman M de Bruyn T Page et al ldquoBiogeography ofthe Indo-Australian archipelagordquo Annual Review of EcologyEvolution and Systematics vol 42 pp 205ndash226 2011

20 Advances in Evolutionary Biology

[2] D S Woodruff ldquoNeogene marine transgressions palaeogeog-raphy and biogeographic transitions on the Thai-Malay Penin-sulardquo Journal of Biogeography vol 30 no 4 pp 551ndash567 2003

[3] H K Voris ldquoMaps of Pleistocene sea levels in SoutheastAsia shorelines river systems and time durationsrdquo Journal ofBiogeography vol 27 no 5 pp 1153ndash1167 2000

[4] S Y Yap ldquoOn the distributional patterns of Southeast-EastAsian freshwater fish and their historyrdquo Journal of Biogeographyvol 29 no 9 pp 1187ndash1199 2002

[5] S K J McConnell ldquoMapping aquatic faunal exchanges acrossthe Sunda shelf South-East Asia using distributional andgenetic data sets from the cyprinid fish Barbodes gonionotus(Bleeker 1850)rdquo Journal of Natural History vol 38 no 5 pp651ndash670 2004

[6] T Mo Anatomy Relationships and Systematics of the Bagridae(Teleostei Siluroidei) with a Hypothesis of Siluroid Phylogenyvol 17 ofTheses Zoologicae Koeltz Scientific Books 1991

[7] H H Ng and M Kottelat ldquoRevision of the Asian catfish genusHemibagrus bleeker 1862 (Teleostei Siluriformes Bagridae)rdquoRaffles Bulletin of Zoology vol 61 no 1 pp 205ndash291 2013

[8] P K L Ng and H H Ng ldquoHemibagrus gracilis a new speciesof large riverine catfish (Teleostei Bagridae) from PeninsularMalaysiardquo Raffles Bulletin of Zoology vol 43 pp 133ndash142 1995

[9] H H Tan and M Kottelat ldquoRedescription of Betta picta(Teleostei Osphronemidae) and description of Betta falx sp nfrom central Sumatrardquo Revue Suisse de Zoologie vol 105 no 3pp 557ndash568 1998

[10] J J Dodson F Colombani and P K Ng ldquoPhylogeographicstructure in mitochondrial DNA of a South-east Asian fresh-water fish Hemibagrus nemurus (Siluroidei Bagridae) andPleistocene sea-level changes on the Sunda shelfrdquo MolecularEcology vol 4 no 3 pp 331ndash346 1995

[11] H H Ng and J J Dodson ldquoMorphological and genetic descrip-tions of a new species of catfish Hemibagrus chrysops fromSarawak East Malaysia with an assessment of phylogeneticrelationships (Teleostei Bagridae)rdquo Raffles Bulletin of Zoologyvol 47 no 1 pp 45ndash57 1999

[12] P D N Hebert A Cywinska S L Ball and J R DeWaardldquoBiological identifications through DNA barcodesrdquo Proceedingsof the Royal Society B Biological Sciences vol 270 no 1512 pp313ndash321 2003

[13] J C Carolan T EMurray U Fitzpatrick et al ldquoColour patternsdo not diagnose species quantitative evaluation of a DNAbarcoded cryptic bumblebee complexrdquo PLoS ONE vol 7 no 1Article ID e29251 2012

[14] M Kekkonen and P D N Hebert ldquoDNA barcode-baseddelineation of putative species efficient start for taxonomicworkflowsrdquoMolecular Ecology Resources vol 14 no 4 pp 706ndash715 2014

[15] DM Hillis CMoritz and B KMarbleMolecular SystematicsSinauer Sunderland Mass USA 2nd edition 1996

[16] T D Kocher W K Thomas A Meyer et al ldquoDynamics ofmitochondrial DNA evolution in animals amplification andsequencingwith conserved primersrdquoProceedings of theNationalAcademy of Sciences of the United States of America vol 86 no16 pp 6196ndash6200 1989

[17] T A Hall ldquoBioEdit a user-friendly biological sequence align-ment editor and analysis program for Windows 9598NTrdquoNucleic Acids Symposium Series vol 41 pp 95ndash98 1999

[18] D A Benson I Karsch-Mizrachi K Clark D J Lipman JOstell and EW Sayers ldquoGenBankrdquoNucleic Acids Research vol40 no D1 pp D48ndashD53 2012

[19] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[20] X Ku Z Peng R Diogo and S He ldquoMtDNA phylogenyprovides evidence of generic polyphyleticism for East Asianbagrid catfishesrdquoHydrobiologia vol 579 no 1 pp 147ndash159 2007

[21] L Yang and S He ldquoPhylogeography of the freshwater catfishHemibagrus guttatus (Siluriformes Bagridae) implications forSouth China biogeography and influence of sea-level changesrdquoMolecular Phylogenetics and Evolution vol 49 no 1 pp 393ndash398 2008

[22] B W Bowen A L Bass L A Rocha W S Grant and DR Robertson ldquoPhylogeography of the trumpetfishes (Aulosto-mus) ring species complex on a global scalerdquo Evolution vol 55no 5 pp 1029ndash1039 2001

[23] Y P Kartavtsev ldquoDivergence at Cyt-b and Co-1 mtDNA geneson different taxonomic levels and genetics of speciation inanimalsrdquoMitochondrial DNA vol 22 no 3 pp 55ndash65 2011

[24] SWada Y Kameda and S Chiba ldquoLong-term stasis and short-term divergence in the phenotypes of microsnails on oceanicislandsrdquoMolecular Ecology vol 22 no 18 pp 4801ndash4810 2013

[25] J Colborn R E Crabtree J B Shaklee E Pfeiler and B WBowen ldquoThe evolutionary enigma of bonefishes (Albula spp)cryptic species and ancient separations in a globally distributedshorefishrdquo Evolution vol 55 no 4 pp 807ndash820 2001

[26] J-D Durand K-N Shen W-J Chen et al ldquoSystematics of thegrey mullets (Teleostei Mugiliformes Mugilidae) molecularphylogenetic evidence challenges two centuries ofmorphology-based taxonomyrdquo Molecular Phylogenetics and Evolution vol64 no 1 pp 73ndash92 2012

[27] K-N Shen B W Jamandre C-C Hsu W-N Tzeng and J-D Durand ldquoPlio-Pleistocene sea level and temperature fluc-tuations in the northwestern Pacific promoted speciation inthe globally-distributed flathead mullet Mugil cephalusrdquo BMCEvolutionary Biology vol 11 no 1 article 83 2011

[28] R Hall ldquoThe plate tectonics of Cenozoic SE Asia and thedistribution of land and seardquo in Biogeography and GeologicalEvolution of SE Asia R Hall and J D Holloway Eds pp 99ndash131Backhuys Publishers Leiden The Netherlands 1998

[29] R Hall ldquoSoutheast Asiarsquos changing palaeogeographyrdquo Blumeavol 54 no 1ndash3 pp 148ndash161 2009

[30] R F Inger and P K Chen ldquoThe freshwater fishes of NorthBorneordquo Fieldiana Zoology vol 45 pp 1ndash268 1962

[31] J Bohlen V Slechtova H H Tan and R Britz ldquoPhylogenyof the Southeast Asian freshwater fish genus Pangio (Cyprini-formes Cobitidae)rdquoMolecular Phylogenetics and Evolution vol61 no 3 pp 854ndash865 2011

[32] M de Bruyn E Nugroho M M Hossain J C Wilson andP B Mather ldquoPhylogeographic evidence for the existence ofan ancient biogeographic barrier the Isthmus of Kra SeawayrdquoHeredity vol 94 no 3 pp 370ndash378 2005

[33] J Parnell ldquoThe biogeography of the Isthmus of Kra region areviewrdquoNordic Journal of Botany vol 31 no 1 pp 001ndash015 2013

[34] S Usmani S G Tan S S Siraj and K Yusoff ldquoPopulationstructure of the Southeast Asian river catfishMystus nemurusrdquoAnimal Genetics vol 34 no 6 pp 462ndash464 2003

[35] M P Tan A F J Jamsari and M N Siti Azizah ldquoPhylo-geographic pattern of the striped snakehead Channa striatain Sundaland ancient river connectivity geographical and

Advances in Evolutionary Biology 21

anthropogenic singnaturesrdquo PLoS ONE vol 7 no 12 Article IDe52089 2012

[36] R Abell M L Thieme C Revenga et al ldquoFreshwater ecore-gions of the world a new map of biogeographic units forfreshwater biodiversity conservationrdquo BioScience vol 58 no 5pp 403ndash414 2008

[37] P U Clark D Archer D Pollard et al ldquoThemiddle Pleistocenetransition characteristics mechanisms and implications forlong-term changes in atmospheric pCO

2rdquo Quaternary Science

Reviews vol 25 no 23-24 pp 3150ndash3184 2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 19: ResearchArticle - Université Laval Julian... · ResearchArticle A DNA Barcode-Based Evaluation of the Southeast Asian Catfish Genus Hemibagrus Bleeker, 1862 (Teleostei: Siluriformes;

Advances in Evolutionary Biology 19

reported for 5 additional freshwater fishes within this region[35] However as the divergence of the two H capitulumclades postdates the formation of the mountain range byapproximately 200Ma the geographic separation of the twoclades most likely represents differential dispersal rather thanancient vicariance The presence of the Sundaic clade on thewest coast of the Thai-Malay Peninsula as well as on theSundaic Islands reflects the historical connections betweeneast Sumatra and west Peninsular Malaysia across the Straitof Malacca This area encompasses the Northern CentralSumatra-Western Malaysia freshwater ecoregion [36] TheMuar River forms its southern border coincident with thesouthernmost distribution of the Sundaic clade on the Thai-Malay Peninsula It is important to note however that mostof Sumatra and Java emerged to achieve their present size andelevation only 5Ma ago and much of East Java continuedto be the site of marine deposition until late Pliocene-earlyPleistocene [29] We may speculate that the Sundaic clade ofH capitulum resulted from the relatively recent colonizationof these islands and Borneo The radiation within the cladeonly dates back to approximately 1Ma ago H nemurus fromWest Java may thus best be considered an isolated regionalpopulation of very recent origin

The diversification of the Indochinese clade dates fromat least 12Ma ago coincident with the mid-Pleistocenetransition at which time low-frequency high-amplitude andquasiperiodic (100 ka) glacial variability emerged It began125Ma ago and was complete by 07Ma ago [37] The tran-sition marked the beginning of major changes in sea levelsduring which the Sunda Shelf periodically emerged and wastraversed by enormous fluvial systems [3] The contributionof these events to the diversification of this clade cannotbe satisfactorily reconstructed One possible explanation isthat high sea levels produced barriers to gene flow and thepopulations of West Borneo (subclades B C and D) wereisolated from those of east Peninsular Malaysia This couldbe due to one or more dispersal events from Borneo via theGreat Sunda River (Figure 1) to east Peninsular Malaysia orlineage sorting whereby sequences of subclades A and E havebeen lost from West Borneo As in the case of the sympatricoccurrence of H spilopterus and H capitulum subclade E(see above) in the Mekong River we are unable to clearlydefine the relative roles of incomplete lineage sorting andintrogression followingmigration Given the recent origins ofthe Indochinese H capitulum sequences incomplete lineagesorting most probably blurs patterns of recent isolation

The last species of the H nemurus species group is Hhoevenii The species is widespread throughout PeninsularMalaysia and the Sundaic Islands It is found primarily inthe lower reaches of rivers and is apparently capable ofwithstanding low salt concentration [7] H hoevenii thusappears to be the most liable of all members of the Hnemurus species group to be impacted by Pleistocene sea-level changes This is clearly reflected in the widespreaddistribution of the sequences of this species Two lineages areevident having diverged no more than 03Ma ago suggest-ing a late Pleistocene isolation event followed by extensivedispersal and admixture in recent times This represents theclearest example of the dispersal opportunities afforded by

Pleistocene sea level changes on the Sunda Shelf In additionthe relatively low genetic diversity detected (only 6 sequencesin 42 fish) suggests a population bottleneck in the recent pastpossibly related to population declines in the lower reaches ofrivers flooded by increasing sea levels

43 Future Directions We have provided a global picture ofthe evolutionary taxonomy of the genus in Southeast AsiaClearly a more rigorous phylogenetic reconstruction of thegenus Hemibagrus requiring the construction of multiplegene trees based on the analysis of both mitochondrial andnuclear DNA markers from a wider taxonomic array ofhemibagrids will involve an even greater effort of samplingthroughout the region In particular we require samplingat smaller spatial and temporal scales For example smallerscale sampling in the Padas River and the Segama Riverof North Borneo using both mitochondrial and nucleargenetic markers would contribute to clarifying the natureof the cryptic species complex believed to inhabit theserivers The demonstration of reproductive isolation andormicroallopatric distribution among morphologically crypticspecies may result in the description of greater speciesrichness within the genus than presently described Thespecies status of H chrysops merits reconsideration givenits highly divergent evolutionary history Nuclear geneticmarkers in conjunction with mtDNA markers could revealcases of historical introgression particularly in the KapuasRiver and the Mekong River The hemibagrid catfishes con-tinue to provide multiple opportunities for the study of theevolutionary taxonomy and biogeography of a major faunalcomponent of the fluvial ecosystems of Southeast Asia

Disclosure

Ethanol-preserved tissue samples of many of the specimensidentified in Table S1 are freely available upon request fromJJD

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Goh Yan Yih for assistance in collectingspecimens and molecular analyses Mrs Francoise Colom-bani for molecular analyses and Dr Heok Hee Ng forspecies identification This work was funded by the NationalUniversity of Singapore grant to P K L Ng and a CanadianNatural Sciences and Engineering Research Council grant toJ J Dodson

References

[1] D J Lohman M de Bruyn T Page et al ldquoBiogeography ofthe Indo-Australian archipelagordquo Annual Review of EcologyEvolution and Systematics vol 42 pp 205ndash226 2011

20 Advances in Evolutionary Biology

[2] D S Woodruff ldquoNeogene marine transgressions palaeogeog-raphy and biogeographic transitions on the Thai-Malay Penin-sulardquo Journal of Biogeography vol 30 no 4 pp 551ndash567 2003

[3] H K Voris ldquoMaps of Pleistocene sea levels in SoutheastAsia shorelines river systems and time durationsrdquo Journal ofBiogeography vol 27 no 5 pp 1153ndash1167 2000

[4] S Y Yap ldquoOn the distributional patterns of Southeast-EastAsian freshwater fish and their historyrdquo Journal of Biogeographyvol 29 no 9 pp 1187ndash1199 2002

[5] S K J McConnell ldquoMapping aquatic faunal exchanges acrossthe Sunda shelf South-East Asia using distributional andgenetic data sets from the cyprinid fish Barbodes gonionotus(Bleeker 1850)rdquo Journal of Natural History vol 38 no 5 pp651ndash670 2004

[6] T Mo Anatomy Relationships and Systematics of the Bagridae(Teleostei Siluroidei) with a Hypothesis of Siluroid Phylogenyvol 17 ofTheses Zoologicae Koeltz Scientific Books 1991

[7] H H Ng and M Kottelat ldquoRevision of the Asian catfish genusHemibagrus bleeker 1862 (Teleostei Siluriformes Bagridae)rdquoRaffles Bulletin of Zoology vol 61 no 1 pp 205ndash291 2013

[8] P K L Ng and H H Ng ldquoHemibagrus gracilis a new speciesof large riverine catfish (Teleostei Bagridae) from PeninsularMalaysiardquo Raffles Bulletin of Zoology vol 43 pp 133ndash142 1995

[9] H H Tan and M Kottelat ldquoRedescription of Betta picta(Teleostei Osphronemidae) and description of Betta falx sp nfrom central Sumatrardquo Revue Suisse de Zoologie vol 105 no 3pp 557ndash568 1998

[10] J J Dodson F Colombani and P K Ng ldquoPhylogeographicstructure in mitochondrial DNA of a South-east Asian fresh-water fish Hemibagrus nemurus (Siluroidei Bagridae) andPleistocene sea-level changes on the Sunda shelfrdquo MolecularEcology vol 4 no 3 pp 331ndash346 1995

[11] H H Ng and J J Dodson ldquoMorphological and genetic descrip-tions of a new species of catfish Hemibagrus chrysops fromSarawak East Malaysia with an assessment of phylogeneticrelationships (Teleostei Bagridae)rdquo Raffles Bulletin of Zoologyvol 47 no 1 pp 45ndash57 1999

[12] P D N Hebert A Cywinska S L Ball and J R DeWaardldquoBiological identifications through DNA barcodesrdquo Proceedingsof the Royal Society B Biological Sciences vol 270 no 1512 pp313ndash321 2003

[13] J C Carolan T EMurray U Fitzpatrick et al ldquoColour patternsdo not diagnose species quantitative evaluation of a DNAbarcoded cryptic bumblebee complexrdquo PLoS ONE vol 7 no 1Article ID e29251 2012

[14] M Kekkonen and P D N Hebert ldquoDNA barcode-baseddelineation of putative species efficient start for taxonomicworkflowsrdquoMolecular Ecology Resources vol 14 no 4 pp 706ndash715 2014

[15] DM Hillis CMoritz and B KMarbleMolecular SystematicsSinauer Sunderland Mass USA 2nd edition 1996

[16] T D Kocher W K Thomas A Meyer et al ldquoDynamics ofmitochondrial DNA evolution in animals amplification andsequencingwith conserved primersrdquoProceedings of theNationalAcademy of Sciences of the United States of America vol 86 no16 pp 6196ndash6200 1989

[17] T A Hall ldquoBioEdit a user-friendly biological sequence align-ment editor and analysis program for Windows 9598NTrdquoNucleic Acids Symposium Series vol 41 pp 95ndash98 1999

[18] D A Benson I Karsch-Mizrachi K Clark D J Lipman JOstell and EW Sayers ldquoGenBankrdquoNucleic Acids Research vol40 no D1 pp D48ndashD53 2012

[19] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[20] X Ku Z Peng R Diogo and S He ldquoMtDNA phylogenyprovides evidence of generic polyphyleticism for East Asianbagrid catfishesrdquoHydrobiologia vol 579 no 1 pp 147ndash159 2007

[21] L Yang and S He ldquoPhylogeography of the freshwater catfishHemibagrus guttatus (Siluriformes Bagridae) implications forSouth China biogeography and influence of sea-level changesrdquoMolecular Phylogenetics and Evolution vol 49 no 1 pp 393ndash398 2008

[22] B W Bowen A L Bass L A Rocha W S Grant and DR Robertson ldquoPhylogeography of the trumpetfishes (Aulosto-mus) ring species complex on a global scalerdquo Evolution vol 55no 5 pp 1029ndash1039 2001

[23] Y P Kartavtsev ldquoDivergence at Cyt-b and Co-1 mtDNA geneson different taxonomic levels and genetics of speciation inanimalsrdquoMitochondrial DNA vol 22 no 3 pp 55ndash65 2011

[24] SWada Y Kameda and S Chiba ldquoLong-term stasis and short-term divergence in the phenotypes of microsnails on oceanicislandsrdquoMolecular Ecology vol 22 no 18 pp 4801ndash4810 2013

[25] J Colborn R E Crabtree J B Shaklee E Pfeiler and B WBowen ldquoThe evolutionary enigma of bonefishes (Albula spp)cryptic species and ancient separations in a globally distributedshorefishrdquo Evolution vol 55 no 4 pp 807ndash820 2001

[26] J-D Durand K-N Shen W-J Chen et al ldquoSystematics of thegrey mullets (Teleostei Mugiliformes Mugilidae) molecularphylogenetic evidence challenges two centuries ofmorphology-based taxonomyrdquo Molecular Phylogenetics and Evolution vol64 no 1 pp 73ndash92 2012

[27] K-N Shen B W Jamandre C-C Hsu W-N Tzeng and J-D Durand ldquoPlio-Pleistocene sea level and temperature fluc-tuations in the northwestern Pacific promoted speciation inthe globally-distributed flathead mullet Mugil cephalusrdquo BMCEvolutionary Biology vol 11 no 1 article 83 2011

[28] R Hall ldquoThe plate tectonics of Cenozoic SE Asia and thedistribution of land and seardquo in Biogeography and GeologicalEvolution of SE Asia R Hall and J D Holloway Eds pp 99ndash131Backhuys Publishers Leiden The Netherlands 1998

[29] R Hall ldquoSoutheast Asiarsquos changing palaeogeographyrdquo Blumeavol 54 no 1ndash3 pp 148ndash161 2009

[30] R F Inger and P K Chen ldquoThe freshwater fishes of NorthBorneordquo Fieldiana Zoology vol 45 pp 1ndash268 1962

[31] J Bohlen V Slechtova H H Tan and R Britz ldquoPhylogenyof the Southeast Asian freshwater fish genus Pangio (Cyprini-formes Cobitidae)rdquoMolecular Phylogenetics and Evolution vol61 no 3 pp 854ndash865 2011

[32] M de Bruyn E Nugroho M M Hossain J C Wilson andP B Mather ldquoPhylogeographic evidence for the existence ofan ancient biogeographic barrier the Isthmus of Kra SeawayrdquoHeredity vol 94 no 3 pp 370ndash378 2005

[33] J Parnell ldquoThe biogeography of the Isthmus of Kra region areviewrdquoNordic Journal of Botany vol 31 no 1 pp 001ndash015 2013

[34] S Usmani S G Tan S S Siraj and K Yusoff ldquoPopulationstructure of the Southeast Asian river catfishMystus nemurusrdquoAnimal Genetics vol 34 no 6 pp 462ndash464 2003

[35] M P Tan A F J Jamsari and M N Siti Azizah ldquoPhylo-geographic pattern of the striped snakehead Channa striatain Sundaland ancient river connectivity geographical and

Advances in Evolutionary Biology 21

anthropogenic singnaturesrdquo PLoS ONE vol 7 no 12 Article IDe52089 2012

[36] R Abell M L Thieme C Revenga et al ldquoFreshwater ecore-gions of the world a new map of biogeographic units forfreshwater biodiversity conservationrdquo BioScience vol 58 no 5pp 403ndash414 2008

[37] P U Clark D Archer D Pollard et al ldquoThemiddle Pleistocenetransition characteristics mechanisms and implications forlong-term changes in atmospheric pCO

2rdquo Quaternary Science

Reviews vol 25 no 23-24 pp 3150ndash3184 2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 20: ResearchArticle - Université Laval Julian... · ResearchArticle A DNA Barcode-Based Evaluation of the Southeast Asian Catfish Genus Hemibagrus Bleeker, 1862 (Teleostei: Siluriformes;

20 Advances in Evolutionary Biology

[2] D S Woodruff ldquoNeogene marine transgressions palaeogeog-raphy and biogeographic transitions on the Thai-Malay Penin-sulardquo Journal of Biogeography vol 30 no 4 pp 551ndash567 2003

[3] H K Voris ldquoMaps of Pleistocene sea levels in SoutheastAsia shorelines river systems and time durationsrdquo Journal ofBiogeography vol 27 no 5 pp 1153ndash1167 2000

[4] S Y Yap ldquoOn the distributional patterns of Southeast-EastAsian freshwater fish and their historyrdquo Journal of Biogeographyvol 29 no 9 pp 1187ndash1199 2002

[5] S K J McConnell ldquoMapping aquatic faunal exchanges acrossthe Sunda shelf South-East Asia using distributional andgenetic data sets from the cyprinid fish Barbodes gonionotus(Bleeker 1850)rdquo Journal of Natural History vol 38 no 5 pp651ndash670 2004

[6] T Mo Anatomy Relationships and Systematics of the Bagridae(Teleostei Siluroidei) with a Hypothesis of Siluroid Phylogenyvol 17 ofTheses Zoologicae Koeltz Scientific Books 1991

[7] H H Ng and M Kottelat ldquoRevision of the Asian catfish genusHemibagrus bleeker 1862 (Teleostei Siluriformes Bagridae)rdquoRaffles Bulletin of Zoology vol 61 no 1 pp 205ndash291 2013

[8] P K L Ng and H H Ng ldquoHemibagrus gracilis a new speciesof large riverine catfish (Teleostei Bagridae) from PeninsularMalaysiardquo Raffles Bulletin of Zoology vol 43 pp 133ndash142 1995

[9] H H Tan and M Kottelat ldquoRedescription of Betta picta(Teleostei Osphronemidae) and description of Betta falx sp nfrom central Sumatrardquo Revue Suisse de Zoologie vol 105 no 3pp 557ndash568 1998

[10] J J Dodson F Colombani and P K Ng ldquoPhylogeographicstructure in mitochondrial DNA of a South-east Asian fresh-water fish Hemibagrus nemurus (Siluroidei Bagridae) andPleistocene sea-level changes on the Sunda shelfrdquo MolecularEcology vol 4 no 3 pp 331ndash346 1995

[11] H H Ng and J J Dodson ldquoMorphological and genetic descrip-tions of a new species of catfish Hemibagrus chrysops fromSarawak East Malaysia with an assessment of phylogeneticrelationships (Teleostei Bagridae)rdquo Raffles Bulletin of Zoologyvol 47 no 1 pp 45ndash57 1999

[12] P D N Hebert A Cywinska S L Ball and J R DeWaardldquoBiological identifications through DNA barcodesrdquo Proceedingsof the Royal Society B Biological Sciences vol 270 no 1512 pp313ndash321 2003

[13] J C Carolan T EMurray U Fitzpatrick et al ldquoColour patternsdo not diagnose species quantitative evaluation of a DNAbarcoded cryptic bumblebee complexrdquo PLoS ONE vol 7 no 1Article ID e29251 2012

[14] M Kekkonen and P D N Hebert ldquoDNA barcode-baseddelineation of putative species efficient start for taxonomicworkflowsrdquoMolecular Ecology Resources vol 14 no 4 pp 706ndash715 2014

[15] DM Hillis CMoritz and B KMarbleMolecular SystematicsSinauer Sunderland Mass USA 2nd edition 1996

[16] T D Kocher W K Thomas A Meyer et al ldquoDynamics ofmitochondrial DNA evolution in animals amplification andsequencingwith conserved primersrdquoProceedings of theNationalAcademy of Sciences of the United States of America vol 86 no16 pp 6196ndash6200 1989

[17] T A Hall ldquoBioEdit a user-friendly biological sequence align-ment editor and analysis program for Windows 9598NTrdquoNucleic Acids Symposium Series vol 41 pp 95ndash98 1999

[18] D A Benson I Karsch-Mizrachi K Clark D J Lipman JOstell and EW Sayers ldquoGenBankrdquoNucleic Acids Research vol40 no D1 pp D48ndashD53 2012

[19] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[20] X Ku Z Peng R Diogo and S He ldquoMtDNA phylogenyprovides evidence of generic polyphyleticism for East Asianbagrid catfishesrdquoHydrobiologia vol 579 no 1 pp 147ndash159 2007

[21] L Yang and S He ldquoPhylogeography of the freshwater catfishHemibagrus guttatus (Siluriformes Bagridae) implications forSouth China biogeography and influence of sea-level changesrdquoMolecular Phylogenetics and Evolution vol 49 no 1 pp 393ndash398 2008

[22] B W Bowen A L Bass L A Rocha W S Grant and DR Robertson ldquoPhylogeography of the trumpetfishes (Aulosto-mus) ring species complex on a global scalerdquo Evolution vol 55no 5 pp 1029ndash1039 2001

[23] Y P Kartavtsev ldquoDivergence at Cyt-b and Co-1 mtDNA geneson different taxonomic levels and genetics of speciation inanimalsrdquoMitochondrial DNA vol 22 no 3 pp 55ndash65 2011

[24] SWada Y Kameda and S Chiba ldquoLong-term stasis and short-term divergence in the phenotypes of microsnails on oceanicislandsrdquoMolecular Ecology vol 22 no 18 pp 4801ndash4810 2013

[25] J Colborn R E Crabtree J B Shaklee E Pfeiler and B WBowen ldquoThe evolutionary enigma of bonefishes (Albula spp)cryptic species and ancient separations in a globally distributedshorefishrdquo Evolution vol 55 no 4 pp 807ndash820 2001

[26] J-D Durand K-N Shen W-J Chen et al ldquoSystematics of thegrey mullets (Teleostei Mugiliformes Mugilidae) molecularphylogenetic evidence challenges two centuries ofmorphology-based taxonomyrdquo Molecular Phylogenetics and Evolution vol64 no 1 pp 73ndash92 2012

[27] K-N Shen B W Jamandre C-C Hsu W-N Tzeng and J-D Durand ldquoPlio-Pleistocene sea level and temperature fluc-tuations in the northwestern Pacific promoted speciation inthe globally-distributed flathead mullet Mugil cephalusrdquo BMCEvolutionary Biology vol 11 no 1 article 83 2011

[28] R Hall ldquoThe plate tectonics of Cenozoic SE Asia and thedistribution of land and seardquo in Biogeography and GeologicalEvolution of SE Asia R Hall and J D Holloway Eds pp 99ndash131Backhuys Publishers Leiden The Netherlands 1998

[29] R Hall ldquoSoutheast Asiarsquos changing palaeogeographyrdquo Blumeavol 54 no 1ndash3 pp 148ndash161 2009

[30] R F Inger and P K Chen ldquoThe freshwater fishes of NorthBorneordquo Fieldiana Zoology vol 45 pp 1ndash268 1962

[31] J Bohlen V Slechtova H H Tan and R Britz ldquoPhylogenyof the Southeast Asian freshwater fish genus Pangio (Cyprini-formes Cobitidae)rdquoMolecular Phylogenetics and Evolution vol61 no 3 pp 854ndash865 2011

[32] M de Bruyn E Nugroho M M Hossain J C Wilson andP B Mather ldquoPhylogeographic evidence for the existence ofan ancient biogeographic barrier the Isthmus of Kra SeawayrdquoHeredity vol 94 no 3 pp 370ndash378 2005

[33] J Parnell ldquoThe biogeography of the Isthmus of Kra region areviewrdquoNordic Journal of Botany vol 31 no 1 pp 001ndash015 2013

[34] S Usmani S G Tan S S Siraj and K Yusoff ldquoPopulationstructure of the Southeast Asian river catfishMystus nemurusrdquoAnimal Genetics vol 34 no 6 pp 462ndash464 2003

[35] M P Tan A F J Jamsari and M N Siti Azizah ldquoPhylo-geographic pattern of the striped snakehead Channa striatain Sundaland ancient river connectivity geographical and

Advances in Evolutionary Biology 21

anthropogenic singnaturesrdquo PLoS ONE vol 7 no 12 Article IDe52089 2012

[36] R Abell M L Thieme C Revenga et al ldquoFreshwater ecore-gions of the world a new map of biogeographic units forfreshwater biodiversity conservationrdquo BioScience vol 58 no 5pp 403ndash414 2008

[37] P U Clark D Archer D Pollard et al ldquoThemiddle Pleistocenetransition characteristics mechanisms and implications forlong-term changes in atmospheric pCO

2rdquo Quaternary Science

Reviews vol 25 no 23-24 pp 3150ndash3184 2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 21: ResearchArticle - Université Laval Julian... · ResearchArticle A DNA Barcode-Based Evaluation of the Southeast Asian Catfish Genus Hemibagrus Bleeker, 1862 (Teleostei: Siluriformes;

Advances in Evolutionary Biology 21

anthropogenic singnaturesrdquo PLoS ONE vol 7 no 12 Article IDe52089 2012

[36] R Abell M L Thieme C Revenga et al ldquoFreshwater ecore-gions of the world a new map of biogeographic units forfreshwater biodiversity conservationrdquo BioScience vol 58 no 5pp 403ndash414 2008

[37] P U Clark D Archer D Pollard et al ldquoThemiddle Pleistocenetransition characteristics mechanisms and implications forlong-term changes in atmospheric pCO

2rdquo Quaternary Science

Reviews vol 25 no 23-24 pp 3150ndash3184 2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 22: ResearchArticle - Université Laval Julian... · ResearchArticle A DNA Barcode-Based Evaluation of the Southeast Asian Catfish Genus Hemibagrus Bleeker, 1862 (Teleostei: Siluriformes;

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology