ISSN: 1511-3701 PertanikaJ. Trap. Agric. Sci. 29 (l & 2): 47-55 (2006) ©Universiti Putra Malaysia Press

Mitochondrial DNA Diversity of douronensis Valenciennes () in Malaysian Borneo

I·ZYUZI E ESA, 2SITI SHAPOR SIRAJ, 2SITI KHALIJAH DAUD, lKHAIRUL ADHAA. RAHIM, IMOHD TAJUDDI ABDULLAH, ~JEFFRINE ROVIE RYAN JAPNING & 2S00 GUAN, TAN

IFaculty ofResource Science and Technology, Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia 2Biology Department, Universiti Putra Malaysia 43400 Serdang, Selangor. Malaysia 3Institute ofBiodiversity, Bukit Rengit, 28500 Lanchang, Pahang, Malaysia E-mail: [email protected]

Keywords: Tor douronensis, freshwater fish, , population structure, COl sequence

ABSTRAK Kajian ini telah dijalankan untuk mengkaji struktur populasi dan taksonomi Tor douronensis, sejenis ikan air tawar tempatan yang penting di Malaysia Borneo, menggunakan analisis penjujukan 466 pasangan bes gen mitokondria sitokrom c oksides I (COl). Sejumlah 62 ekor sampel ikan telah diperoleh dari lima lokasi di Sarawak (N=53) dan Sabah (N=8). Analisis filogenetik menggunakan kaedah "NeighbourJoining" (N]) menyokong status monofiletik di antara'T. douronensis dan ; seterusnya mengukuhkan lagi status taksonomi kedua­ duanya sebagai spesies yang berlainan. Haplotaip T. douronensis seterusnya boleh dibahagikan kepada tiga kumpulan yangutama, dengan ikanPelian dari Sabah membentuk kumpulannya sendiri (Kluster Ill) dengan sokongan (bootstrap) statistik yang kuat. Perbezaan genetik yang tinggi di antara haplotaip-haplotaip dari Sabah dengan Sarawak menunjukkan bahawa ikan Pelian Sabah mungkin merupakan sejenis spesies kriptik. Kajian ini menunjukkan variasi­ variasi intra dan inter-po-pulasi yang tinggi dalam T. douronensis. Variasi dalam kalangan sampel dalam populasi dijumpai di dalam kesemua populasiT. douronensis kecuali di dalam populasi Bario. Kehadiran perbezaan-perbezaan

haplotaip yang tetap (unik) bersamaan dengan nilai FST yang tinggi antara populasi-populasi T. douronensis, menyokong kesimpulan bahawa sedikit atau tiada migrasi berlaku di antara populasi-populasi yang dipisahkan oleh jarak geografi yangjauh atau sistem sungai yang berlainan. Walau bagaimanapun, perkongsian beberapa haplotaip antara populasi-populasi tersebut, contohnya antam Batang Ai dan Bario (HS6) dan antara Batang Ai dan Ulu Limbang/Ba Kelalan (HS2) memberi bukti yang T. douronensis mempunyai taburan yang meluas di kawasan tersebut di masa lalu, terutama semasa zaman Q:.taternary. Keseluruhannya, kajian ini berjaya memberikan maklumat­ makluat yang berguna tentang struktur populasi dan taksonomi ikan T. douronensis di Malaysia Borneo.

ABSTRACT This study examines the population structure and taxonomy ofTor douronensis, an important indigenousfreshwater fish species in Malaysian Borneo, by using sequence analysis of 466 base pairs of the mitochondrial cytochrome c oxidase I (COl) gene. A total of62 fish samples were collectedfrom five locations in Sarawak (N=54) and Sabah (N=8). The phylogenetic analysis using the NeighbourJoining (N]) method supported the monophyletic status between T. douronensis and Tor tambroides, which further reinforced their taxonomic status as distinct species. The T. douronensis haplotypes were further divided into three majorgroups, with the Pelian[uhfrom Sabah forming its own group (Cluster Ill) with strong bootstrap support. The large genetic differences separating the Sabah haplotypesfrom its Sarawak congeners suggested that the Pelianfish might represent a cryptic species. The current study showed high levels of intra and inter-population variations in T. douronensis. Within all population variations, T. douronensis

populations were found, except in Bario. The presence offixed haplotype differences along with high FST values among the populations ofT. douronensis, support the conclusion that little or no migration occurred among the extant YUZINE ESA ET AL. populations separated by large geographic distances or river systems. However, the sharing ofhaplotypes between some such papulations, for example between Batang Ai and Bario (HS6), and between Batang Ai and Ulu Limbang/Ba Kelalan (HS2) provided support that T. douronensis had a historically widespread natural distribution in the region probably during the Quaternary period. Overall, the present study was able to shed light on the taxonomy and population structure ofT. douronensis in Malaysian Borneo.

INTRODUCTION Rainboth, 1996; Zhou and Chu, 1996; g, Freshwater fishes of the genus Tor Gray, 2004). Roberts (1999) classified them to be a commonly known as , belong to the single species, and a junior synonym to T family Cyprinidae (subfamily Cyprininae) tambra. (Mohsin and Ambak, 1983; Roberts, 1989; Thus, the application of molecular Kotellat et al., 1993). They are distributed techniques (such as DNA sequencing) should throughout the Indian subcontinent, offer better insights into the unresolved Southeast Asia and Southern China and taxonomy and population size of T douronensis inhabit the upper streams and headwaters of (Nguyen et al., 2006). Molecular markers can most major river systems (Kottelat et al., 1993; give more reliable and consistent results for Rainboth, 1996). Environmental degradation rapid species identification (Ryan and Esa, such as river pollution, deforestration and 2006), levels of genetic variability, levels of watershed erosion had led to the rapid gene flow and population subdivisions and for destruction of Tor natural habitat. understanding factors contributing to fitness Uncontrolled fish harvest (overfishing) has in freshwater fishes (Vrijenhoek, 1998). also greatly reduced their population size (Ng, Analysis of DNA sequence polymorphism 2004). Their distributions in Malaysian Borneo utilizing the existing "universal primers" for are now limited to the upper streams and mitochondrial DNA (mtD A) (Palumbi et al., protected areas (natural parks) ofSarawak and 1991) provides the highest resolution of Sabah (Litis et al., 1997; yanti et al., 1999; g, genetic variation which had been widely 2004). There are currently three described Tor applied in molecular systematic studies species in Malaysia: Tor tamlJroides Bleeker, Tor (Amason et al., 2002; Liu and Chen, 2003; tambra Valenciennes, and Tor douronensis guyen et al., 2006). Thus, this study was Valenciennes (Kottelat and Whitten, 1996; conducted to clarify aspects of the systematics Roberts, 1989; Rainboth 1996; Ng, 2004). and population structure of T douronensis in T douronensis is presumably the most various populations in Malaysian Borneo by widespread species recorded in East analysing the cytochrome oxidase I (COl) Malaysia, apart from the less abundant T nucleotide sequences of the mitochondrial tamlJroides found in Sarawak, and it is the only DNA. mahseer currently described from Sabah (Inger and Chin, 2002). T douronensis, locally MATERIALS AND METHODS known as "ikan semah" in Sarawak and "ikan Sample Description and Collection Location pelian" in Sabah has been named""the state Samples of T douronensis were collected from fish ofSarawak" due to its importance as a high five locations in Sarawak and three locations value food fish as well as for eco-tourism and in Sabah (Fig. 1). However, samples from recreational fishing (Litis et al., 1997; g, Sabah (Keningau, Liwagu and Tawau) were 2004). pooled together into a single population Nevertheless, the taxonomic differen­ (Sabah population) due to the small number tiation of T douronensis and its related species, ofindividuals obtained for this study. The fish T tamlJroides is still unresolved, with many were sampled using a variety of fishing conflicting descriptions among different methods, including seine, gill and cast nets and researchers (Roberts, 1989; Kottelat et al., 1993; fishing rod. Some samples from Sarawak were

48 PERTANlKAJ. TROP. ACRIC. SCI. VOL. 29 OS. I & 2 (2006) MITOCHO DRIAL DA DIVERSITY OF TOR DOURONENSISVALE CIENNES (CYPRI IOAE)

0 50 100Mi!es , I I i , i 0 100 200 Kilometers i N

South Chinu SEta

Kalimantan

Fig. 1: Map showing sampling locations and sample sizes ofT. douronensis collected for this study. N= sample size. provided by the Indigenous Fish Research and A 500 bp segment ofthe cytochrome c oxidase Production Center (IFRPC), Tarat, Sarawak. I gene was amplified with the oligonucleotide The weight and standard length of the fish primers COIf (5' CCTGCAGGAGGAGGAG samples used in the study ranged from 50-500 AYCC 3', forward) and COle (5' CCAGAGATI g and 5-100 em, respectively. Whole samples AGAGGGAATCAGTG 3', reverse) (Palumbi et were morphologically identified by using the at., 1991). Approximately, 50-100 ng of the keys provided by Inger and Chin (2002), template DA was amplified in a 25 ml Mohsin and Ambak (1983), and Kottelat et all reaction mixture containing 50 mM lOX

(1993). Fresh samples in the form of full buffer, 2 mM MgCI2, 0.2 mM of each dNTP specimens, muscle tissues, scales or fin clips (Promega), 0.1 mM of each primer, and 0.5 were placed in a -80°C freezer for long-term units of Tag DA Polymerase (Promega). The storage. However, in most cases, samples cycle parameters consisted of 35 cycles of collected in the field were preserved in 95% denaturation (95°C, 30 seconds), annealing ethanol and stored at -20°C prior to genetic (45°C, 30 seconds), and extension (72°C, 60 analyses. seconds. The amplified products were visualized on a 1% agarose gel containing DNA Extraction and Polymerase Chain Reaction ethidium bromide for approximately 30 min (PCR) at 90 V and photographed under UV light. A Total DNA was isolated using the modified digested lambda DNA ladder (GeneRuler™ 1 CTAB method (Grewe et at., 1993) in the kb DNA Ladder) was used as a size standard presence of Proteinase K. The pelleted DNA marker (Promega). The PCR products were was redissolved in 100,uL ofsterilized distilled further purified using a D A purification kit water. The DA quality and approximate yield (Invitrogen) according to the manufacturer's were determined by electrophoresis in a 1% instructions. The purified PCR products were agarose gel containing ethidium bromide at then directly sequenced using the'BigDye'" 90 V for 30 min. The isolated genomic DA Terminator v3.0 Cycle Sequencing kit on an was used for the mtDNA analysis.

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ABI 377 automated DNA sequencer (PE RESULTS Applied Biosystem) using only the forward Sixty-two partial sequences of 466 base pairs primer (Calf). (bp) each of the mtD A cal gene were obtained, representing the six populations of Statistical Analysis T. douronensis. We observed 38 (8%) variable/ The CHROMAS (version 1.45) program was polymorphic sites including 33 (7%) used to display the fluorescence-based DA parsimony-informative sites while 430 sites sequencing results. The multiple sequence (92%) were conserved. A total of14 haplotypes alignment for the forward reactions was done were distinguished in the nucleotide data set using the CLUSTAL X program (version 1.81; with 11 haplotypes being unique and three Thompson et al., 1997), and subsequently haplotypes being shared among the six aligned by eye. The pairwise genetic distance populations (Table 1). In total, 41 substitutions between populations was calculated using the were found among the haplotypes, of which Tamura-Nei distance (Tamura and Nei, 1993), there were 35 transitions and 6 transversions. based on unequal base frequencies and The sequences of each of the haplotype have unequal ratios of transition to transversion been deposited in the GenBank (GeneBank (Ti:Tv) implemented in MEGA (version 3.1; Reference Numbers: EFI92444-EFI92457). Kumar et al., 2004). The MEGA program was The mean total nucleotide composition was also used to construct a neighbourjoining A=25.7%, T=32.3%, C=22.7% and G=19.3%. (NJ) tree (Saitou and ei, 1987) using two The T douronensis samples from Sabah indigenous cyprinids, (Barbonymus gonionotus harboured four unique haplotypes (HSIIP to (Genbank accession number: DQ532806) and HSI4P) which were not shared with T. Barbonymus schwanenfeldii (Genbank accession douronensis populations from Sarawak. On the number: DQ532805)) obtained from the other hand, HS6 was the only haplotype found Jempol River, egeri Sembilan as outgroup in the Bano population although it was also species. Four haplotypes of T. tambroides found in the BatangAipopulation. The Layar/ (Genbank accession number: DQ532827, Spak population also had four haplotypes, EF192458, EF192460, EF192461) were also three being unique haplotypes while the included in the analysis to demonstrate the fourth one (HSI0L) was also found in the reciprocally monophyletic status between the Batang Ai population (Table 1). two mahseers. The phylogenetic confidence Overall, the nucleotide diversity was low was estimated by bootstrapping with 1000 with the Sabah population showing the highest replicate data sets (Felsenstein, 1985). value (0.016). The haplotype diversity varied, The levels ofmtDNA calvariation within ranging from 0 to 0.900 (Layar/Spak) (Table

the T. douronensis population were examined 1). The pairwise FST (Hudson et al., 1992) and by computing the nucleotide (with theJukes­ the results of the Chi-square tests for genetic Cantor correction; Jukes and Cantor, 1969) differentiation among the populations are and haplotype diversity indices implemented presented in Table 2. Significant levels of in the DnaSP (version 4.0) program (Rozas et genetic differentiation were found in all aL, 2003). The level ofpopulation subdivision comparisons among the T. douronensis

(FST) (Hudson et al., 1992) between populations except between the Ulu Limbang populations and the Chi-square probability test and the Ba Kelalan populations (F~ 0.075), for population differentiation using 1000 and between the Layar/Spak and the Sabah

permutations of the data sets were also populations, although their pairwise FST value estimated using the DnaSP program. was high (0.726) (Table 2). Phylogenetic analysis of the haplotypes using the IT method strongly supported the reciprocally monophyletic status between T

50 PERTANlKAJ. TROP. AGRIC. SCI. VOL. 29 OS. 1 & 2 (2006) MITOCHONDRIAL D A DIVERSITY OF TOR DOURONENSISVALENCIE NES (CYPRI IDAE)

TABLE 1 Distribution of 14 observed mill A COl haplotypes, nucleotide diversity, number of haplotypes and number of polymorphic sites among populations of T. douronensis

Population

Haplotypes GenBank DIu Ba Bario Sabah Layarl Batang Accession Limbang Kelalan Spak Ai umbers

HS1BA EF192444 1.000 HS2 EF192445 0.350 0.210 0.140 HS3 EF192446 1.000 HS4 EF192447 1.000 HS5 EF192448 1.000 HS6 EF192449 0.530 0.170 HS7L EF192450 1.000 HS8L EF192451 1.000 HS9L EF192452 1.000 HS10L EF192453 0.100 0.600 HSllP EF192454 1.000 HS12P EF192455 1.000 HS13P EF192456 1.000 HS14P EF192457 1.000 Nucleotide diversity (p;JC) 0.001 0.001 0.000 0.016 0.006 0.008 umber of haplotypes 2 3 1 4 4 4 Haplotype diversity (Hd) 0.200 0.607 0.000 0.750 0.900 0.697 Number of polymorphic sites 1 2 0 15 4 13

Numbers under each population indicate the frequencies of individuals with that haplotype in each population.

TABLE 2 Lower diagonal: pairwise Tamura-Nei genetic distances among the six populations of T. douronensis

1. DIu Limbang 2. Ba Kelalan 3. Bario 4.Sabah 5. LayarISpak 6. Batang Ai

1 0.075" 0.947'" 0.811" 0.880' 0.147' 2 0.001 0.857" 0.804' 0.865' 0.175' 3 0.004 0.005 0.817' 0.890' 0.327' n 4 0.046 0.047 0.047 0.726 , 0.724'" 5 0.026 0.027 0.027 0.042 0.706" 6 0.005 0.006 0.006 0.046 0.024

Upper diagonal: population subdivision (Fsr) values and probability test (Chi-square) for population differentiation based on 1000 permutations of the sequence data set, significance levels (ns=not significant, P<0.05=*, P

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HS4 '13 HS1BA 66 HS3 99 HS2

63

T. douronensis HS9L 51 HS7L HSllL HS13P HS14P 100 HS12P m HS11P

69 00532827 '------1 EF192458 IT. tambroides 100 EF192460 53 EF192461 .------00532005 (8. schwanenfeldil) LL.------__ 00532006 (B. QOllionotus)

002

Fig. 2: Neighbour-joining (N]) phylogram showing the relationships among COl haplotypes ofT. douronensis, T. tambroides and two outgroups analysed in the present study. The number at each node rep-resents the bootstrap percentage value based on 1000 pseudoreplications for NJ analysis douronensis and T tambroides (Fig. 2) and Kelalan populations of Sarawak (4.7%) while further divided the former mahseer into three the lowest value was between the Ulu Limbang major groups (Cluster I to III) with strong population and the Ba Kelalan population bootstrap supports. Cluster I grouped (0.1 %). Within the Sarawak populations, the haplotypes from the Ulu Limbang, Ba Kelalan Layar/Spak population had genetic distances and Bario populations (Northern Sarawak) of 2.4% to 2.7% separating it from the other with those from the Batang Ai population four Sarawak populations. Interestingly, the (Southern Sarawak). Cluster II grouped all the Batang Ai population had a closer genetic Southern Sarawak haplotypes consisting of distance (0.5% to 0.6%) with the Northern three unique haplotypes from the Layar/Spak Sarawak populations (Ulu Limbang, Ba Kelalan population and one shared haplotype and Bario) than with the Southern Layar/Spak (HS10L) with the Batang Ai population. population (2.4%). Interestingly, Cluster III grouped all the four unique haplotypes from Sabah (North DISCUSSION Borneo). The results ofthe mtDNA analysis in this study The pairwise genetic distances (number enabled us to shed light on the taxonomic ofnucleotide substitutions per site) calculated status of T douronensis in the Malaysian part using the Tamura- ei model (Tamura and of Borneo Island. The phylogenetic analysis ei, 1993) among the T douronensis of the COl gene confirmed the reciprocally populations are shown in Table 2. The highest monophyletic status between T douronensis and genetic distance was observed between the T tambroides, thus further reinforcing their Sabah population and both the Bario and Ba taxonomic status as distinct species (Roberts,

52 PERT IKAJ. TROP. AGRIC. SCI. VOL. 29 OS. 1 & 2 (2006) MITOCHONDRIAL DNA DIVERSITY OF TORDOURONENSISVALENCIENNES (CYPRINIDAE)

1989; Kottelat et at., 1993; Rainboth, 1996; congeners from Sarawak, and the presence of Zhou and Chu, 1996; Ng, 2004). The current fixed haplotypes supported the hypothesis that mtDNA results also did not show any mixing the North Borneo region was the most isolated of haplotypes between T. douronensis and T. region and probably had no connection with tambroides as was observed by Nguyen et al. the other Borneo regions during the (2006). The major finding of this study is the Pleistocene glaciation periods (Inger and bifurcation of the T. douronensis haplotypes Chin, 2002). Thus, the Pelian fish from Sabah into three highly differentiated groups, with could possibly have evolved through allopatric the Sabah (North Borneo) haplotypes forming speciation and formed new or cryptic mtDNA its own subgroup (Cluster III). The bootstrap lineages. The lack of a clear geographical support among the three clusters was high structuring ofhaplotype distributions between although the consensus positioning ofCluster the Semah fish from the Northern and the I and Cluster II with regards to Cluster III was Southern parts of Sarawak is also moderately supported. A plausible explanation demonstrated in other indigenous freshwater for this is that the Pelianfish from Sabah might fish species with a widespread natural represent a cryptic species. distribution such as in Hampala macrolepidota This study found high levels of intra and (Ryan and Esa, 2006). inter-population variations in T. douronensis. This study demonstrated the usefulness of Within population variations were found in all genetic studies in assessing the taxonomy and the T. douronensis populations except in the population structures ofMalaysia's indigenous Bario population. The large mtDNA freshwater fish taxa for appropriate differences currently found among the T. conservation and management strategies. douronensis populations could be explained by However, further studies are required using one or several factors induding small larger sample sizes per population, samples population sizes, past bottleneck events, or the from other areas of their geographical presence of physical barriers to gene flow distributions, sequence data from other among the populations (Nguyen et al., 2006). mtDNA regions and information based on The presence of fixed haplotype differences nuclear DNA (i.e. single locus microsatellite) among the populations, along with high FST markers. values among populations of T. douronensis, supported the conclusion that little or no ACKNOWLEDGEMENTS migration occurred among the extant We thank Dr Stephen Sungan and officers populations separated by large geographic from the Indigenous Fish Production Research distances, or river systems (Nguyen et at., 2006). Centre (IFPRC), Department ofAgriculture, Nevertheless, the sharing of haplotypes Sarawak, for their assistance in sample between such populations does occur, for collection. We also thank the Fisheries example between Batang Ai and Bario (HS6), Department ofMalaysia for their full support and between Batang Ai and Ulu Limbang/Ba throughout the project, members of the Kelalan (HS2) and this provided support that, Molecular Ecology Laboratory, Universiti in the past, T. douronensis had a widespread Malaysia Sarawak, members of the Genetics natural distribution in the region. Geological Laboratory, Universiti Putra Malaysia, and all evidence suggested that the river systems of the people involved in the sample collection the Northern and the Southern parts of and laboratory work. This projectwas funded Sarawakwere historically interconnected, most by Universiti Malaysia Sarawak through probably during the Tertiary and Quaternary research grant 01 (80)411/2003(148) and periods (Inger and Chin, 2002). National Biotechnology Directorate (MOST!) The large genetic differences between the grant no: 01-02-04-008/BTK/ER/33. T. douronensis population from Sabah with its

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