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Research article

JOURNALOF TROPICAL BIOLOGY AND CONSERVATION 5 : 1 - 13, 2009 1

ABSTRACT. A study on the genetic variationofBarbonymus schwanenfeldii (Bleeker)populations from Malaysia was done usingpartial sequencing of cytochrome b mtDNAgene. Samples were collected from variouslocalities in Peninsular Malaysia, Sarawak andSabah, including the Indonesian (Kalimantan)type ofB. schwanenfeldii from Kapit, Sarawak.Phylogenetic relationship inferred usingneighbour joining, maximum likelihood andmaximum parsimony methods generally dividedsamples into two major groups, consistent withtheir geographic origin: one widespread groupconsisted ofB. schwanenfeldii (locally knownas Lampam sungai) from Peninsular Malaysiawhile another particular group clustered B.

schwanenfeldii (locally known as Tengadak)from Sarawak. Within the Peninsular Malaysia,our current data supported the validation of twomain divisions: central and southern division(CS; consisting of Serting River, Muar River andPadang Piol, Jerantut, Pahang) and north westand north east division (NWE; consisting ofPulau Banding, Perak and Tasik Timah Tasoh,Perlis). The low distance values betweenhaplotype sequences (0.00 to 1.01) and low

Phylogeny and phylogeography ofBarbonymus schwanenfeldii (Cyprinidae)from Malaysia inferred using partialcytochrome b mtDNA gene

Kamarul Rahim KAMARUDIN1,2and Yuzine ESA3

1School of Biological Sciences, The University of Auckland, Private Bag 92019, Auckland

1142, New Zealand.

2Department of Biotechnology, Kulliyyah of Science, International Islamic University Malaysia

(IIUM), Jalan Istana, Bandar Indera Mahkota, 25200 Kuantan, Pahang, Malaysia.

3Molecular Ecology Laboratory, Department of Zoology, Faculty of Resource Science and

Technology, Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia.

nucleotide diversity, Pi (0.005) showed a veryclose genetic relationship between samples,enforcing their taxonomic validation as belongingto a single taxon. However, the low level of geneflow (Nm = 0.07) and high population structuring(Fst = 0.88) between Peninsular Malaysia andSarawak could be correlated with the separationof Borneo Island from mainland Peninsular duringlast Pleistocene Epoch.

INTRODUCTION

The greatest diversity of freshwater fish is foundin tropical South America, Africa and South EastAsia (Helfman et al., 1997). The fish diversity ofSouth East Asia area was once affected during

the Pleistocene Epoch of the Neogene Period.The Pleistocene was characterized by multipleepisodes of glaciation, which caused ice toadvance and retreat approximately every 100,000years beginning about 2 million years ago (Dott& Prothero, 1994). Biogeographically, Malaysiais situated in the Oriental region. PeninsularMalaysia, southern Thailand, southern Indo-China, Sumatra, Java and Borneo were once partof the submerged Sunda Shelf (Mohsin &Ambak, 1983). In recent times, PeninsularMalaysia is separated from Borneo by the SouthKeywords: Barbonymus schwanenfeldii, partial

cytochrome b, population genetics.


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PHYLOGENYAND PHYLOGEOGRAPHYOF BARBONYMUSSCHWANENFELDII (CYPRINIDAE)2

China Sea. Malaysian Borneo is made up ofSarawak and Sabah. The relatively highgeographic distance between mainland of

Peninsular Malaysia and both Sabah andSarawak could be related to the biogeographytheory of the Pleistocene events. Borneo Islandis biogeographically located near the Wallacearegion, the area between Wallace's line andWeber's line. In this case, we predict that geneticdrift might have led to the genetic differentiationin mtDNA lineages between freshwater fish fromPeninsular Malaysia and Borneo Island, afterthe Shelf submerged. According to Halliday

(1993), genetic drift may become the majorsource of genetic variation between somepopulations.

Family Cyprinidae is currently the largest familyof freshwater fish in terms of its abundant generaand species throughout Malaysia. Accordingto Mohsin & Ambak (1983), the Malaysianregion is considered as the southern centre fordistribution of primary freshwater fish.Generally, freshwater fish from the genusPuntius are the most abundant species inMalaysia and can be found in almost everywater body (Inger & Chin, 1962; Mohsin &Ambak, 1983; Zakaria Ismail, 1990). In this study,Puntius schwanenfeldii (Bleeker), Barbodesschwanenfeldii (Bleeker) or recently known as

Barbonymus schwanenfeldii (Bleeker) waschosen to test the glacial effects on thefreshwater fish diversity during the Pleistocenelow sea levels. Barbonymus schwanenfeldii is

known as lampam sungai in PeninsularMalaysia and tengadak in Sarawak. It is apretty river-dwelling fish and morphologicallyquite similar to Puntius gonionotus (Java borbfish). This fish is widely distributed in all riversand lakes in Peninsular Malaysia particularly inPahang, Perak, Kelantan, Terengganu andSelangor (Mohsin & Ambak, 1991) andindigenous to upper and mid-zone of RejangRiver system, as well as Limbang and Batang Airivers (Litis et al., 1997).

The sequence evolution of mitochondrial DNAof freshwater fish has been relatively wellstudied in the phylogeographic structure and

in clarifying the phenomena of postglacialcolonization (Dodson et al., 1995; Wang et al.,2000). Fish mitochondrial genomes areeffectively inherited maternally, haploid,apparently non-recombining and thereforecorrespond exactly to the model of bifurcatingevolutionary tree (Stepien & Kocher, 1997).Overall mtDNA substitution rates are estimated5-10 times greater than in 'single-copy' nuclearDNA, and commonly occur in transition or

transversion form (Hartl & Clark, 1989; Hoelzel& Dover, 1991).

In general, molecular analyses ofcytochrome bpartial sequence were done in this study as anadditional approach to study the Pleistoceneimpacts by comparing the genetic differentiationbetween B. schwanenfeldii from PeninsularMalaysia region with Sarawak and Sabah as theBorneo representatives. The cytochrome b isprobably the best-studied mtDNA gene in fishes(Zhu et al., 1998; Jansson & st, 1997) and thisregion is used to analyze relationships amongspecies, phylogeographic questions andpopulation genetic structuring. Wang et al.(2000) used cytochrome b gene to assess thegenetic and phylogeographic structure of

Acrossocheilus paradoxus populations fromTaiwan. In addition, a number of B.schwanenfeldii from Kalimantan might havebeen transferred to Kapit, Sarawak and had been

put together into the population of local B.schwanenfeldii. Thus, the phylogenetic studywas applied to test the taxonomic assumptionof the Indonesian type B. schwanenfeldii asdistinct from other B. schwanenfeldiipopulations from Sarawak, due to the minordifference in colour observed between the twotypes ofB. schwanenfeldii (Stephen M. Sungan,personal comment).


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KAMARUL RAHIM KAMARUDIN & YUZINE ESA 3

MATERIALS AND METHODS

Sample Description and Location

Samples ofB. schwanenfeldii were collectedfrom several populations in Peninsular Malaysia,Sarawak and Sabah (Table 1, Figure 1).

Barbonymus schwanenfeldii fish from Kapit,Sarawak were identified as Indonesian type fishas they are believed to have originated fromKalimantan. Fish were sampled using a varietyof fishing methods, including seine net, gill nets,cast nets and fishing rod. Fresh samples of local

typeB. schwanenfeldii fish and pure IndonesiantypeB. schwanenfeldii fish from Kapit, Sarawakwere stored at -80oC freezer for long-termstorage. The other samples were preserved in95% ethanol and stored at -20oC until geneticanalyses were performed.

DNA Extraction

Total DNA was extracted from muscle tissueusing modified Cetyl trimethylammoniumbromide (CTAB) method (Grewe et al., 1993) withthe presence of Proteinase K. Pelleted DNA wasre-dissolved in 100L of sterilized distilled water.Quality and approximate yield were determinedby electrophoresis in a 1% agarose gelcontaining ethidium bromide at 90 V for 30 min.Isolated genomic DNA were used for mtDNAanalysis.

Polymerase Chain Reaction (PCR)

Approximately 450 base pairs (bp) section ofthe mtDNA genome from the cytochrome b genewas successfully amplified using standardpolymerase chain reaction (PCR) procedures. Twouniversal primers ofcytochrome b were used:GludG-L (5'-TGACTTGAARAACCAYCGTTG-3',forward) and CB2-H (5'-CCCTCAGAATGATATTTGTCCTCA-3',reverse) (Palumbi et al., 1991). Each thermal cycleamplification was performed in 50 L reactionvolume containing 31.75 L sterilized distilled

water, 0.25 L ofTaq DNA polymerase, 5.0 Lof 10X reaction buffer, 1.0L of dNTP (10 mM),3.0L of Magnesium Chloride (25 mM ) and 2.5L of each primer (10 M). Approximately 4.0L of the DNA was added to each PCR cocktails.Cycle parameters were 5 min at 96oC for initialdenaturation, 45 sec at 95oC for denaturation, 1min 30 sec at 47oC for annealing, 1 min 30 sec at72oC (30 cycles) for elongation, and 7 min at72oC for final elongation. Master mix was usedfor large quantities of PCR reactions. Theamplified products were visualized on 1%agarose gel containing ethidium bromide, run

for approximately 30 min at 90 V andphotographed under UV light. A digestedlambda DNA (GeneRulerTM 1 kb DNA Ladder)was used as a standard size marker (Fermentas).PCR products were purified prior to sequencingusing purification kit (Promega).

DNA Sequencing

Purified PCR products were pelleted and air-dried before sending for sequencing.Sequencing was done using the BigDyeTerminator v3.0 Cycle Sequencing kit (ACGT).Cycle sequencing reaction was done in aprogrammable cycler (Tpersonal CombiThermocycler). Cycle sequencing reaction wasdone for 35 cycles of 96oC: 10 sec, 55oC: 5 sec,60oC: 4 min hold and proceeded to Ethanol/Sodium Acetate precipitation. Rapid thermalramp was 1oC/sec. Sequencing was carried outon ABI 377 automated sequencer (PE Applied

Biosystem).

Statistical Analysis

CHROMAS (version 1.45; Technelysium Pty.Ltd., Tewantin Qld 4565, Australia) program wasused to display fluorescence-based DNAsequence analysis results. Multiple sequencealignment for forward reactions ofcytochromeb partial sequences ofB. schwanenfeldii wasdone by using CLUSTAL X program (version1.81; Thompson et al., 1997), and subsequently


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Table 1. Description of Barbonymus schwanenfeldiisamples from several populations in Malaysia.

No. Region Study site n Abbreviation Gen Bank no.

1 Sabah Tenom (BSFT) 1 BSFT1 -2 Sarawak (S) Kapit (BSFKS) 14

(a) Local type (BSFK) 4 BSFK11 AY243354BSFK12 AY355431BSFK14 AY355432BSFK15 AY462409

(b) Indonesian type (BSFI) - 10 BSF11 AY355434originated from BSFI4 AY462413Kalimantan, Indonesia BSFI9 AY355436

BSFI13 AY462412BSFI14 AY355433

BSFI15 AY462414BSFI16 AY462410BSFI20 AY462411BSFI21 AY462408BSFI22 AY355435

3 Paku River, Betong, Sri Aman (BSFB) 2 BSFB1 AY355418BSFB2 AY355437BSFB3 AY355438

4 Bau (BSFBP) 1 BSFBP1 AY4624155 Peninsular Serting River

Malaysia (PM)

Jempol Pump Station, 2 BSFJ1 AY243355Serting Hilir (BSFJ) BSFJ2 AY355425Pulapah Lama Road, 4 BSFPL1 AY355428Negeri Sembilan (BSFPL) BSFPL2 AY355423

BSFPL4 AY355429BSFPL5 AY355424

Triang Selatan 2, Serting Hilir (BSFTS) 3 BSFTS1 AY355430BSFTS2 AY355421BSFTS3 AY355422

Ayer Hitam (BSFAH) 1 BSFAH1 AY462416

6 Padang Piol, Jerantut, 4 BSFPP1 AY355419Pahang (BSFPP) BSFPP2 AY355426

BSFPP3 AY355427BSFPP4 AY355420

7 Pulau Banding, Perak (BSFPB) 3 BSFPB1 AY462404BSFPB2 AY462405BSFPB3 AY462406

8 Tasik Timah Tasoh, Perlis (BSFTT) 1 BSFTT1 AY4624079 Muar River, Johor (BSFM) 2 BSFM1 AY576455

BSFM3 AY576456

TOTAL 39

* Location and individual abbreviations are used in figures, tables and text.


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Figure 1. Small figure on the upper right shows the location of Malaysia region (filled) in South East Asia. Theleft figure shows the location map ofBarbonymus schwanenfeldii study areas in the mainland of PeninsularMalaysia while the lower figure shows the location map of B. schwanenfeldii study areas in Sabah and Sarawak.Each study area is detailed in Table 1.


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aligned by eye. A sequence ofBarbodesschwanenfeldii from GenBank (GenBankaccession number: AF180823) was chosen as

corresponding sequence. PhylogeneticAnalysis Using Parsimony (PAUP*) programversion 4.0b10 (Swofford, 1998) was used toreconstruct neighbour joining tree (Saitou &Nei, 1987), maximum parsimony and maximumlikelihood trees. This program was also utilizedto examine other relevant analyses such as basefrequencies. Kimura 2-parameter distance (1980)was selected, base on equal base frequenciesand unequal ratio of transition to transversion

(Ti:Tv). Phylogenetic confidence was estimatedby bootstrapping (Felsenstein, 1985) with 1000replicate data sets. In addition, MolecularEvolutionary Genetics Analysis (MEGA version2.1; Kumar et al., 2001) was used as comparison.The level of genetic diversity between, withinand among populations and geographicalregions were quantified by pairwise estimatesof nucleotide diversity (Pi; Jukes & Cantor,1969); level of gene flow (Nm) and F-statisticanalysis (Fst) from Hudson et al. (1992). Theparameters were analyzed using DNA SequencePolymorphism (DNA SP) version 3.53 (Rozas &Rozas, 1999).

RESULTS

Genetic Diversity and Population Structure

Partial sequences of 450 base pairs (bp) ofcytochrome b mtDNA gene were obtained from

38 individuals ofB. schwanenfeldii and afterexcluding alignment gaps, 399 bp were alignedprior to DNA sequence analyses. All sequenceswere registered with GenBank (Table 1).However, as a precautionary step to minimizebias, one sequence ofB. schwanenfeldii fromTenom, Sabah was excluded due to the lownumber of sample from Sabah population, i.e.one sample. Alignment was made against asequence of Helostoma temminckii fromBakong, Sarawak, as an outgroup. Out of 399sites, seven sites were considered as variable

sites (1.75%), including five parsimony-informative characters (1.25%). Three hundredand ninety two out of 399 sites were conserved

(98.25%). There were six haplotypes (NHap); onlyone haplotype within the Sarawak region andfive haplotypes within the Peninsular region,with haplotype (gene) diversity 0% and 70%respectively. Within the Peninsular region, fourhaplotypes originated from central and southerndivision (CS; consisting of Serting River, MuarRiver and Padang Piol, Jerantut, Pahang) and asingle haplotype from north west and north eastdivision (NWE; consisting of Pulau Banding,

Perak and Tasik Timah Tasoh, Perlis). Thecurrent divisions were modified from and basedon regional partition by Mohsin & Ambak (1983,1991; Figure 2).

The Kimura 2-parameter distance values (resultswere not shown) between sequences wereremarkably low, with a range between 0% and1.0%. The distance values within the PeninsularMalaysia samples ranged from 0% to 1.0%.Within the Borneo region, the average distancevalue was 0%, suggesting identical mtDNAlineage. Moreover, the distance values betweenthe Peninsular region and Sarawak populationas the sub-Borneo representative rangedbetween 0.8% and 1.0%. The standard deviationvalues likely supported the Kimura 2-parameterdistance values between samples of

B. schwanenfeldii.

Likewise, low nucleotide diversity, Pi was

calculated within regions, ranging from 0% to0.3% (Table 2). BSFTS has the highestnucleotide diversity (0.3%) with threehaplotypes (haplotype diversity=1.00). Withinthe Peninsular region, the average Pi was 0.2%,relatively higher than within the Sarawak region(0%). However, the nucleotide diversitybetween Peninsular and Sarawak regions maybe considered as similar, due to the smalldifference in percentage (0.5%). Most samplesfrom central and southern division (CS) shareda single and identical haplotype. The average Pi


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Figure 2. Three main divisions of Peninsular Malaysia. Adapted from Mohsin & Ambak (1983, 1991)

Table 2. Calculation of nucleotide diversity, Pi (JC) (Jukes & Cantor, 1969) within populations and geographicalregions ofBarbonymus schwanenfeldii in Malaysia. Each study area is detailed in Table 1.

Study site Nucleotide diversity, Pi (JC) %

Sarawak 0.0Kapit 0.0Bau 0.0

Peninsular Malaysia 0.2Central and Southern Division (CS) 0.2

North West and North East Division (NWE) 0.0

Jempol Pump Station, Serting Hilir (CS) 0.0Pulapah Lama Road, Negeri Sembilan (CS) 0.1Triang Selatan 2, Serting Hilir (CS) 0.3Padang Piol, Jerantut, Pahang (CS) 0.0Muar River (CS) 0.0Pulau Banding, Perak (NWE) 0.0

Malaysia 0.5


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of CS and NWE were 0.2% and 0% respectively,with 0.2% in difference. Overall, the nucleotidediversity ofB. schwanenfeldii throughout

Malaysia was 0.5%, featuring a very closesimilarity among haplotypes.

The extent of gene flow level between Peninsularand Sarawak regions was low (Nm = 0.07, Table3). The outcome was expected based on therecent separation of Borneo Island fromPeninsular Malaysia by South China Sea.Consequently, the population structuringbetween both regions, which has been

estimated from F-statistic analysis was high (Fst= 0.88). These two types of data gave valuableclarification towards the Pleistocene events.Within the Peninsular region, the level of geneflow (Table 3) between NWE and CS was low(Nm= 0.15) with high population structuring (Fst=0.76), supporting parts of the regional partitionby Mohsin & Ambak (1983, 1991).

Phylogenetic Analysis

A total of 39 partial sequences (399 bp) ofcytochrome b mtDNA gene were aligned;including one sequence of Helostomatemminckii as an outgroup to root thephylogenetic trees. The neighbour joining treeusing bootstrap test (Figure 3) of B.schwanenfeldii populations revealed a majorclade of B. schwanenfeldii samples fromSarawak (96% bootstrap value) while B.schwanenfeldii samples from Peninsular

Malaysia region were basal to the Sarawakclade. Within the Peninsular Malaysia, samplesfrom NWE formed a particular cluster, with 65%bootstrap value. Likewise, the maximumparsimony and maximum likelihood tree usingbootstrap test (Figure 3) ofB. schwanenfeldiipopulations revealed almost the same pattern,with 96% and 93% bootstrap value for Sarawakclade, respectively; 59% and 64% bootstrapvalue for NWE cluster, respectively. In view ofthe level of genetic diversity, the clusteringshowed by both neighbour joining and

maximum parsimony trees suggested thepossibilities of genetic drift impacts on Borneopopulations that subsequently led to the

genetic variation inB. schwanenfeldii mtDNAgenome. These data were corresponding to thelow level of gene flow (Nm = 0.07) and highpopulation structuring (Fst = 0.88) betweenmainland of Peninsular Malaysia and Sarawakas the Borneo representative.

In addition, the individuals of the IndonesiantypeB. schwanenfeldii mixed with the local type

B. schwanenfeldii samples from Sarawak (Figure

3). Regarding the results of genetic distance andnucleotide diversity (Pi), the phylogeneticpattern apparently showed that the IndonesiantypeB. schwanenfeldii shared the same mtDNAlineages with the local typeB. schwanenfeldiifrom Sarawak. In other words, the identicalgenetic lineage betweenB. schwanenfeldii fromKalimantan andB. schwanenfeldii from Sarawaktotally unraveled the taxonomic similaritybetween them.

Generally, the phylogenetic relationship inferredusing the neighbour joining, maximumlikelihood and maximum parsimony methodsrevealed two major groups: the widespreadgroup of B. schwanenfeldii samples fromPeninsular Malaysia, which is basal, and aparticular group ofB. schwanenfeldii samplesfrom Sarawak support the historical theory ofPleistocene events. Within the PeninsularMalaysia there are currently two main valid

divisions: central and southern division (CS;consisting of Serting River, Muar River andPadang Piol, Jerantut) and north west and northeast division (NWE; consisting of PulauBanding, Perak and Tasik Timah Tasoh, Perlis).The B. schwanenfeldii sequences of NWEdiffered from CS populations with one transition(Ti=1). Furthermore, the phylogeneticrelationship did not support the taxonomicassumption of the pure Indonesian (Kalimantan)typeB. schwanenfeldii as a distinct from other

B. schwanenfeldii populations from Sarawak.


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Figure 3. Phylogenetic tree (consensus tree) of the populations ofBarbonymus schwanenfeldii inferred from cytochrome b mtDNAgene. Abbreviations refer to individuals in populations identified in Table 1. The tree was rooted with a sequence ofHelostomatemminckii from Bakong, Sarawak. Kimura 2-parameter distance (1980) with 1000 replications was used. Indonesian (Kalimantan)

type B. schwanenfeldii (BSFI) is labelled with asterisk symbol (*). Numbers at nodes indicate the bootstrap values in percentage(Bold neighbour joining method; underline- maximum likelihood; italic- maximum parsimony method). NWE north west and north

east division.

Table 3. Calculation of nucleotide diversity, Pi (JC) (Jukes & Cantor, 1969), level of gene flow, Nm (Hudson et al., 1992) and populationstructuring, Fst (Hudson et al., 1992) within and between geographical regions of Barbonymus schwanenfeldii in Malaysia. NWE -North West and North East Division, Peninsular Malaysia; CS - Central and Southern Division, Peninsular Malaysia.

Nucleotide Level of gene flow, Pop ulat iondiversity, Pi (JC) Nm (Hudson et al., structuring, Fst

(Jukes & Cantor, 1969) 1992) (Hudson et al.,1992)

Peninsular Malaysia 0.2 - -

NWE vs CS 0.2 0.15 0.76

Sarawak 0.0 - -

Peninsular Malaysia vs 0.5 0.07 0.88

Sarawak


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DISCUSSION

Haplotype Diversity and Population Structure

Geographical isolation of allopatric populationsis the major way of preventing gene flowbetween two populations, which allows anevolution of a genome adapted to localcondition (Hall, 1993). Likewise, the low level ofgene flow of B. schwanenfeldii betweenPeninsular Malaysia and Sarawak may beassociated with the isolation of Borneo; thesubmersion of the Sunda River and the rise of

the sea level after the Pleistocene Epoch havecaused the rise in the sea level between 40 to100 meters. This gradual separation of Borneofrom the mainland of Peninsular Malaysia wassuspected to have caused accumulative geneticdrift. Genetic drift is likely to occur, particularlyin small populations that are isolated from themain population of a species (Halliday, 1993).

The relatively high similarity in B.schwanenfeldii sequences between PeninsularMalaysia and Sarawak samples was incongruence with the Pleistocene glaciation(about 10,000 years ago; see Halliday, 1993). Theresults may reflect the formation of the SundaRiver during the Pleistocene, which allowed theflow and migration ofB. schwanenfeldii betweenthe eastern part of the Peninsular Malaysia andthe western parts of Borneo, until thesubmersion of the great river system (Mohsin& Ambak, 1983; Dodson et al., 1995). During

this period of Pleistocene land bridge, there weregreat potential for the movement of freshwaterfish between both regions. Furthermore, the highsimilarity in B. schwanenfeldii sequencesbetween Peninsular Malaysia and Sarawaksamples presumably may indicate remnants ofidentical haplotypes from both lineages, andthey were essentially similar at one time beforethe separation (Inger & Chin, 1962, 2002).Dodson et al. (1995) found that Hemibagrusnemurus colonizing the Kapuas river of westBorneo, east Sumatra and south PeninsularMalaysia; tentatively identified asH. hoevenii ,

have formed the most morphologically andgenetically distinct Sundaic clade suggestingthe occurrence of gene flow between the areas.

However, there was no report on B.schwanenfeldii in North Borneo (Inger & Chin,1962, 2002). Interestingly, we managed to getone sample ofB. schwanenfeldii from Tenom,Sabah and thus giving the possibility of recentintroduction of the species into the northernpart of Borneo. In terms of biogeography, Sabahrivers were never connected to Sunda Riverduring the last Pleistocene (Figure 4; Voris, 2000).

All individuals of B. schwanenfeldii fromSarawak were represented by one haplotype andthere was no genetic differentiation amongthem, suggesting an identical geneticinheritance between the Indonesian typeindividuals and individuals from Sarawak rivers.Even though relatively low values of geneticdistances were calculated between individualsfrom mainland of Peninsular Malaysia andSarawak, the status of Peninsular MalaysiaB.schwanenfeldii as basal to the Sarawak clade inall the phylogenetic trees (Figure 3) and thepresence of five different haplotypes inPeninsular Malaysia B. schwanenfeldiisuggested the occurrence of nucleotidesubstitutions resulted from the isolationphenomenon of Borneo. Accordingly, we alsosuggest that the direction of gene flow was fromPeninsular Malaysia to Borneo through theSunda River during the last Pleistocene. Thereconstructed phylogenetic trees also showed

population structure within Peninsular Malaysiabut the findings were not fully supported bythe zoogeographic reconstruction from Dodsonet al. (1995) and not entirely in congruence withthe main divisions suggested by Mohsin &Ambak (1983, 1991). More detailed studies willbe required to clarify such population structure.

Phylogeography

According to Mohsin & Ambak (1983), duringthe middle Pleistocene, maximum lowering of sealevel had occurred and the eastern slopes of


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Peninsular Malaysia, West Borneo andSumatera were drained by Sunda River. SundaRiver was rich in freshwater fish and has been

dominated by cyprinids. Meanwhile, the faunasof Sunda shelf may have mingled in theselowlands. The formation of Pleistocene landbridges had permitted free migration of faunaand flora between mainland and islands. Theinterconnection between some rivers inPeninsular Malaysia and Sarawak during the lastPleistocene (Figure 4) may have led to theintermixing ofcytochrome b gene of cyprinidsbetween the two regions, by which low genetic

differentiation was calculated among samplesofH. macrolepidota from southern PeninsularMalaysia, and southern and central Sarawak(Ryan & Esa, 2006). The sharing of samehaplotype between Tor tambroides from BatangAi, Sarawak and Perak, northern PeninsularMalaysia (Esa et al., 2008) further supportedthe historical connection of drainages. Whenthis great river system submerged and the sealevel increased, Borneo was formed andseparated from Peninsular Malaysia by theSouth China Sea (Mohsin & Ambak, 1983;Dodson et al., 1995).

Two major groupings consisted of a widespreadbasal group of Peninsular Malaysia and anotherdistinct group of Sarawak region, as revealedby the phylogenetic trees (Figure 3), correspondto the low level of gene flow observed betweenthe two geographical regions. In this case, theSouth China Sea is considered as the

geographic barrier, which leads to the restrictionof gene flow between the two isolated regions.Likewise, Dodson et al. (1995) has suggested agenetically distinct 'Sarawak' group of

Hemibagrus nemurus Valenciennes (1839) inWest Borneo, based on mtDNA test andmorphological analysis. Clearly, the identicalmtDNA lineage observed among B.schwanenfeldii from Sarawak as well asKalimantan indicated as if Borneo was one bigpopulation instead of a region with multiplepopulations ofB. schwanenfeldii. Within thePeninsular Malaysia, our current data support

the validation of two main divisions central andsouthern divisions: CS and NWE. The presenceof a single haplotype representing NWE and

four different haplotypes for CS further supportthe division validation.

Based on our current findings, we suggest thatthere was no colonization ofB. schwanenfeldiiin Borneo, particularly in Sarawak, before theperiod of the Pleistocene land bridge. When theislands of the Sunda shelf and the mainland wereconnected by lowlands traversed by the SundaRiver, the faunas of Peninsular Malaysia,

Sumatera, Borneo and Java may have mingled(Dodson et al., 1995. In the meantime, it wasbelieved that founders ofB. schwanenfeldiimigrated into Borneo and colonized the riversduring the last Pleistocene. Consequently, afterthe recent isolation period, genetic drift hasslowly caused allopatric speciation betweenPeninsular Malaysia and Borneo B.schwanenfeldii, in this caseB. schwanenfeldiifrom Sarawak and Kalimantan. In an isolatedpopulation, gradual adaptation to a newcondition could allow a genome to evolvegradually. The small number of transitionsbetween B. schwanenfeldii from PeninsularMalaysia and Sarawak (Ti= 3) correspond withthe recent isolation of Borneo from the mainlandof Peninsular Malaysia.

The proposition of the Kalimantan type B.schwanenfeldii as a distinct species from anyother B. schwanenfeldii from Sarawak

populations was not discerned by thephylogenetic trees (Figure 3). All individuals ofIndonesian typeB. schwanenfeldii (BSFI) mixedwith local individuals ofB. schwanenfeldii fromSarawak. Morphologically, both types ofB.schwanenfeldii shared similar physicalappearance. Furthermore, the identical mtDNAlineage between local type and pure Indonesiantype B. schwanenfeldii from Kapit, suggestedsimilar genetic relationship between the two typesofB. schwanenfeldii. These corresponding datafrom the morphological characteristics andmtDNA information strongly rejected the


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proposition of Indonesian typeB. schwanenfeldiias a distinct species from any other B.schwanenfeldii from Sarawak populations. Inother words, bothB. schwanenfeldii from Sarawakand Kalimantan are morphologically andgenetically identical.

ACKNOWLEDGEMENTS

We wish to thank Field Museum of NaturalHistory, Chicago, Illinois, USA for the onlinemap of Pleistocene sea level for tropicalSoutheast Asia and Austral-Asia. Thanks alsogo to all the lecturers of Department ofBiotechnology, Kulliyyah of Science,International Islamic University Malaysia(IIUM) and Faculty of Resource Science andTechnology (FRST) UNIMAS for theircontinuous support, encouragement and

valuable discussion (Prof. Dr. Mohd. TajuddinAbdullah, Mrs. Ramlah Zainudin, Dr. Edmund

Sim Ui Hang, Dr. Awang Ahmad Sallehin AwangHussaini and Mr. Khairul Adha A. Rahim). Wewould also like to express our gratitude to allthe lab assistants, technicians, undergraduate,postgraduate students (especially Mr. JeffrineRovie Ryan Japning) at the laboratories of FRSTUNIMAS for assistance in lab assays andreviewers of this manuscript. Many thanks tothe Tarat Indigenous Fisheries Production and

Research Station in Serian, Sarawak, Malaysiafor providing the fresh samples ofBarbonymusschwanenfeldii. This research was supportedby ARCBC L18403 F07 research grant award.

REFERENCES

Dodson, J.J., F. Colombani & P.K.L Ng. 1995 .Phylogeographic structure in mitochondrial DNAof a South-east Asian freshwater fish, Hemibagrusnemurus (Siluroidei; Bagridae) and Pleistocene sea-level changes on the Sunda shelf. Molecular Ecology4: 331-346.

Figure 4. Map of Pleistocene sea level for tropical Southeast Asia and Austral-Asia based on depth contour of120 m below present level. Modified from Voris (2000).


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