Aquaculture Sci. 59（3），383－391（2011） Population Structure of Marble Goby Oxyeleotris marmorata (Bleeker) in Southeast Asia Inferred from Mitochondrial DNA 1 2 1 1 Hou Chew HA , Shigeharu SENOO , Kazunobu TSUNEMOTO , Yoshizumi NAKAGAWA , 1 1 1,＊ Shigeru MIYASHITA , Osamu MURATA and Keitaro KATO Abstract: To investigate the population structure of the marble goby Oxyeleotris marmorata in Southeast Asia, a total of eighty-five samples were collected from three regions (the main- land, the peninsula, and the islands) for mitochondrial DNA analysis. Sampling locations that were geographically close were pooled and treated as a single population. Fourteen haplotypes were detected among all the samples. Hap-5 was the most widespread haplotype among the six populations, comprising of 29.4% of all samples. Both the non-significant values of Tajima’s D and Fu’s FS suggested that all populations were at equilibrium. Analysis of molecular variance (AMOVA) revealed significant differences among and within populations, and no variance was due to regional site (FCT = - 0.1498, P > 0.05). In pairwise comparisons of FST, Ayutthaya, Dong Nai and Sabah showed significant values between the all populations. The negative values of FST showed that Sarawak, Indonesia, and West Malaysia are less genetically different. This suggests that the marble goby in Ayutthaya, Dong Nai and Sabah may be genetically differentiated populations com- pared to the other populations in Southeast Asia. Key words: Oxyeleotris marmorata; Mitochondrial DNA; Control region; Population structure Malaysia is separated by the South China and Luong et al. (2005), the fish can cost 18 to Sea into two regions, Peninsular Malaysia 20 USD/kg. and Malaysian Borneo, which are also known Although many fish farmers have started as West Malaysia and East Malaysia, respec- to culture the marble goby, the seed supply tively. In Malaysia, the marble goby Oxyeleotris is still mainly dependent on natural sources marmorata (Bleeker) is found in freshwater (Ikenoue 1991; Senoo et al. 1992, 2008). Natural rivers, brackish water near estuaries, inland marble goby stocks have decreased significantly ponds, and lakes. The marble goby is one of because of overfishing (Senoo et al. 2008). the most popular freshwater fish in Southeast Therefore, development of mass production Asia, prized for its fine texture and delicious technology is necessary, not only to increase the taste, especially in Malaysia, Singapore, yield, but also to conserve the natural marble Thailand, and Indonesia (Roberts 1989; Senoo goby resource. et al. 1993; Cheah et al. 1994; Amornsakun et al. Mass production of marble goby has not been 2002). Market demand has regularly exceeded widely established because of the high mortal- supply, and it is considered to be one of the ity at the early larval to juvenile stages (Tan most expensive freshwater fish (Senoo et al. and Lam 1973; Tavarutmaneegul and Lin 1988; 1993). According to Amornsakun et al. (2002) Cheah et al. 1991; Senoo et al. 1994a, 1994b, Received 20 December 2010; Accepted 26 April 2011. 1 Fisheries Laboratory, Kinki University, 3153, Shirahama, Wakayama 649-2211, Japan. 2 Borneo Marine Research Institute, Universiti Malaysia Sabah, 88400 Kota Kinabalu, Sabah, Malaysia. ＊Corresponding author: Tel, (+81) 739-42-2625; Fax, (+81) 739-42-2634; E-mail, [email protected] (K. Kato). 384 H. C. Ha, S. Senoo, K. Tsunemoto, Y. Nakagawa, S. Miyashita, O. Murata and K. Kato 2008). Furthermore, newly hatched larvae take The goal of this study was to determine the more than 40 days to grow into juveniles in cap- population structure of the marble goby in tivity. Darwis et al. (2008) reported that marble Southeast Asia using partial sequence analy- goby juveniles are only 0.3 g in body weight and sis of the control region of the mtDNA. Study 3.5 cm in total length after 100 days of rearing of the marble goby population structure will in captivity. The slow growth from the juvenile enhance the broodstock management for stage to a commercial size is also another con- aquaculture seed production. Marble goby straint on marble goby culture. fins were sampled from three regions: the Studies on artificial seed production for the mainland (Thailand and Vietnam), the penin- marble goby have been ongoing since the sula (West Malaysia), and the islands (Sabah 1970’s, however, the life cycle of the marble and Sarawak in East Malaysia, and Indonesia). goby in nature is not clear (Senoo et al. 1992; Partial mtDNA control regions were amplified, Cheah et al. 1994). This species is widely dis- sequenced, and analyzed to determine the pop- tributed in Southeast Asia regions. Thus, a ulation structures among the regions. better understanding of its population struc- ture would aid more effective and sustainable Materials and Methods fisheries management. Molecular genetics has become a powerful tool to determine the Fin samples levels of differentiation among populations. In this study, the term of“ population” is used Examination of mitochondrial DNA (mtDNA) to indicate a pool of close sampling locations of markers is now an established technique for marble goby, according to their geographical elucidating population genetic structure. The distribution. Due to the difficulties of collecting genetic diversity present in a species is hierar- samples, the small sample numbers from sam- chically structured. In addition to differences pling locations that were geographically close among individuals within any one population, were pooled and treated as a single population there may be differences among populations for the comparison between populations. within a given geographical region, differences Eighty-five samples were collected from six among populations from different geographi- populations from August 2009 until February cal regions, and differences among entire geo- 2010, among which two were from the mainland, graphical regions. one was from the peninsula, and three were Table 1. List of sample sizes and sample abbreviations for mtDNA analysis according to the regions, populations and sampling locations SEA Regions Populations Sampling locations Abbreviations Sample size Mainland Ayutthaya, Thailand Ayutthaya Ban 10 Dong Nai, Vietnam Dong Nai Vt 7 Peninsula West Malaysia Ipoh Ipoh 9 Melaka Mek 5 Rantau Rat 6 Kuala Selangor PM 9 Kuala Terengganu KT 2 Islands Sabah, East Malaysia Penampang BP 9 Kimanis SK 9 Sarawak, East Malaysia Bintangor Btg 1 Kuching Sa 9 Niah Nia 2 Indonesia Samarinda, Kalimantan Sam 2 Sukamandi, Java Suk 5 Total 6 14 85 In total eighty-five samples were collected from fourteen sampling locations in six populations. Population Structure of Marble Goby 385 from the islands (Table 1). A small portion of the 4 (Tamura et al. 2007). The aligned sequences pectoral fin was cut from each individual and pre- were used to analyze the population structure served in 90% ethanol for DNA examination. and genetic variation using ARLEQUIN ver- sion 3.5 (CMPG, University of Berne; Excoffier DNA extraction, amplification, and sequencing et al. 2005). Genetic diversity in each popula- Fin samples were digested using proteinase tion was measured as haplotypic diversity (Nei K, followed by standard phenol-chloroform 1987) and nucleotide diversity (Tajima 1983). extraction. 3 M sodium acetate (pH5.2) was Heterozygous nucleotide sites can be estimated added, and the DNA was precipitated with 70% sufficiently with a sample size of ten (or even ethanol. The DNA samples were resuspended five) (Tajima 1983). Tajima’s D (Tajima 1989) in 200μl of TE buffer (10 mM Tris-HCl, 1 mM and Fu’s FS (Fu 1997) tests, as implemented in EDTA, pH 8.0) and stored in a freezer at -20℃. ARLEQUIN version 3.5, were used to evaluate A pair of primers (forward primer 5’-CGGA the neutrality of the investigated sequences. To GGTTAAAATCCTCCCT-3’, reverse primers perform the test, homologous DNA sequences 5’-TAGGAACCAAATGCCAGGAATA-3’) was from at least three individuals were used for designed to amplify the partial mtDNA con- Tajima’s D test to compute a standardized mea- trol region using polymerase chain reaction sure of the total number of segregating sites (PCR). The forward primer is located inside the and the average number of mutations between threonine tRNA gene, and the reverse primer pairs. Fu’s FS test detects an excess of mutation is located inside the control region. PCR was and it is more powerful in cases of population performed using a thermal cycler, GENEAMP expansion. The level of genetic population dif- PCR SYSTEM9700 (Applied Biosystems, CA, ferentiation was tested using analysis of molecu- USA) in 20μl reaction volumes containing lar variance (AMOVA) as implemented in approximately 20 ng of DNA, with 0.5 unit Ex ARLEQUIN version 3.5 (Excoffier et al. 2005), Taq (TaKaRa Bio Inc, Otsu, Japan), 1×Ex Taq using the genetic distance matrix to estimate Buffer, 200μM each dNTP, and 0.5μM of prim- the components of variance that are attributable ers. The PCR cycling conditions were 94℃ for to differences among populations and among 5 min; 35 cycles at 94℃ for 30 s, 52℃ for 30 s, individuals within populations. Populations were and 72℃ for 90 s; followed by a final extension combined into three regions, as defined by geo- for 10 min at 72℃. graphical features (Fig. 1). Significance of vari- The PCR products were electrophoresed on ance components was tested by a nonparametric a 1.5% agarose gel in 1×TAE buffer to check permutation procedure with 1,000 permutations the yield. The amplified DNA was excised (Excoffier et al. 1992). The pairwise fixation from the gel under irradiation UV rays, and index (FST) was employed to check the genetic extracted using a QIAQUICK Gel Extraction differentiation between populations. The cor- Kit (QIAGEN, Hilden, Germany), following the relation of genes of different individuals in the manufacturer’s instructions. The same PCR same population and the genetic differences primers were also used for sequencing reac- among populations were also tested by the FST.