Population Structure of Marble Goby Oxyeleotris Marmorata (Bleeker) in Southeast Asia Inferred from Mitochondrial DNA

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 mainland, 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 compared 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 structure 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 version 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. tions using the DTCS Quick Start Kit (Beckman For between population differences, if all haplo- Coulter, CA, USA) according to the manufac- types were identical, then FST equaled 0; if they turer’s instructions. The nucleotide sequences were all different, then FST equaled 1. Thus, were determined for both ends of the PCR prod- pairwise comparisons of FST values among ucts using a BECKMAN COULTER CEQ8000 populations can be considered as standardized automated sequencer (Beckman Coulter). distances between populations (Excoffier et al. 2005). A neighbor-joining tree of the haplotypes Data analysis was constructed under the model of the Kimura Nucleotide composition and number of vari- 2-parameter using MEGA version 4, and evalu- able sites were assessed using MEGA version ated with 1,000 bootstrap replicates. 386 H. C. Ha, S. Senoo, K. Tsunemoto, Y. Nakagawa, S. Miyashita, O. Murata and K. Kato

Ban Cambodia Vietnam Vt

Thailand South China Sea Sulu Sea

BP SK KT Ipoh Sabah Nia West Malaysia PM Btg Rat Sa Sarawak Mek

Sumantera Kalimantan Sam

500 km Java Sea

Suk Java

Fig. 1. Fourteen sampling locations of the marble goby in three regions of Southeast Asia; mainland, peninsula, and islands, which are represented by circles, triangles, and square dots, respectively. Abbreviations of the sampling locations are given in Table 1.

Ayutthaya contained two variable sites, eigh- Results teen variable sites were found in West Malaysia, eleven variable sites were found in Sabah, thir- Sequence variation teen variable sites were found in Sarawak, and A total of eighty-five 394 bp fragments were seven variable sites were found in Indonesia. sequenced successfully from the six populations No variable sites were found in the samples (Table 2). Among the examined sequences, the from Dong Nai (Table 2). A/T base contents were significantly higher than the C/G base content (mean: A=35.4%, Population structure and genetic diversity T=27.7%, C=21.3%, G=15.6%), which is con- Among all the samples, fourteen haplotypes sistent with the results of Brown et al. (1986), were detected (GenBank accession numbers who found that the control region is an A-T JF300363-JF300376); four haplotypes were rich region of the mtDNA. The sequences from found in the mainland, nine haplotypes in the

Table 2. Nucleotide sequence data of six populations based on partial fragments of mtDNA control region, haplotype and nucleotide diversity (mean±SD), and neutrality tests Number of Sample Number of Haplotypic Nucleotide Tajima’s D Fu’s F Regions Populations polymorphic Tajima’s D Fu’s F S size haplotypes diversity diversity P-value S P-value sites Mainland Ayutthaya 10 3 0.6222±0.1383 2 0.0018±0.0017 0.0189 0.662 -0.1561 0.313 Dong Nai 7 1 0.0000±0.0000 0 0.0000±0.0000 0.0000 N.A. 0.0000 N.A. West Peninsula 31 9 0.8021 0.0474 18 0.0125 0.0069 0.3053 0.687 1.1834 0.739 Malaysia ± ± Islands Sabah 18 4 0.6993±0.0840 11 0.0099±0.0058 0.8183 0.827 4.0135 0.955 Sarawak 12 5 0.8333±0.0691 13 0.0115±0.0069 0.2368 0.657 1.9006 0.831 Indonesia 7 3 0.5238±0.2086 7 0.0080±0.0054 0.5177 0.675 2.3136 0.894 Total 6 85 N.A., not available. No significant values of Tajima’s D and Fu’s FS were observed in the neutrality test, showing that all populations were in genetic equilibrium. Population Structure of Marble Goby 387 peninsula, and eight haplotypes in the islands in the sequence data. No significant deviations (Table 3). Six haplotypes were shared between from neutrality were detected in any of the popu- more than one population. Among the shared lations using both of neutrality tests (P>0.1). haplotypes, only Hap-5 was shared in four populations. Hap-5 comprised 29.4% of all samples. Geographic differentiation It was clear that Hap-5 was the most widespread AMOVA produces estimates of variance haplotype among the populations and the most components reflecting the correlation of hap- common among the control region sequences. lotypic diversity at different levels of hierar- Eight distinct haplotypes were found in one chical divisions. AMOVA assessment of the single population only. Ayutthaya, Sarawak, difference among the regions generated a Sabah, and Indonesia each contained a distinct negative variance component (- 0.4070) indicat- haplotype, while West Malaysia population had ing that genetic structure was absent at this four distinct haplotypes. level (Table 4). F statistics from AMOVA also The genetic diversity of all populations’ revealed significant differences among and sequences is shown in Table 2. West Malaysia within populations. 67.32% of the variance was had the highest nucleotide diversity (0.0125). The attributable to individual variation (FST =0.3268, highest haplotype diversity, 0.8333, was found P<0.01), 47.66% was attributable to populations in Sarawak. The zero values for haplotype and (FSC =0.4145, P<0.01), and no variance was due nucleotide diversity in Dong Nai resulted from no to regional site (FCT =- 0.1498). variable sites being detected in the sequences. The genetic differentiation among the six Tajima’s D and Fu’s FS neutrality tests were per- populations was significant, except for the pair- formed to determine departures from neutrality wise FST values between Sarawak-Indonesia

Table 3. Number of fish from six populations of marble goby according to the haplotype distribution Haplotypes Ayutthaya Dong Nai West Malaysia Sabah Sarawak Indonesia Hap-1 6 1 Hap-2 2 1 1 Hap-3＊ 2 Hap-4 9 1 Hap-5 7 11 3 4 Hap-6＊ 2 Hap-7 8 3 5 Hap-8 4 2 2 Hap-9＊ 1 Hap-10＊ 3 Hap-11＊ 1 Hap-12＊ 1 Hap-13＊ 1 Hap-14＊ 4 Total 10 7 31 18 12 7 ＊Distinct haplotype. Sample sizes of each population are shown at the bottom of the table.

Table 4. AMOVA of mtDNA control region nucleotide sequence data in six marble goby populations from three regions Variance Percentage of Source of variation df Sum of squares F statistics components variation

Among regions (mainland, peninsula, islands) 2 26.40 -0.4070 -14.98 FCT = - 0.1498 ＊ Among populations, within regions 3 45.97 1.2950 47.66 FSC = 0.4145 ＊ Within populations 79 144.52 1.8294 67.32 FST = 0.3268 Total 84 216.89 2.7174 ＊ The significant level (P < 0.05) of the F statistics, with 1,023 permutations. AMOVA results showing the most molecular variance were observed among and within populations, but no variance was observed among the regions. 388 H. C. Ha, S. Senoo, K. Tsunemoto, Y. Nakagawa, S. Miyashita, O. Murata and K. Kato

Table 5. Pairwise comparison FST value of population differentiation (below diagonal), genetic distance within population (diagonal) and between populations (above diagonal) Ayutthaya Dong Nai West Malaysia Sabah Sarawak Indonesia Ayutthaya 0.0018 0.0207 0.0186 0.0174 0.0179 0.0177 Dong Nai 0.9470＊ 0.0000 0.0089 0.0157 0.0082 0.0074 West Malaysia 0.5168＊ 0.1495＊ 0.0127 0.0151 0.0120 0.0104 Sabah 0.6071＊ 0.5753＊ 0.2390＊ 0.0101 0.0139 0.0121 Sarawak 0.6023＊ 0.2033＊ -0.0204 0.2178＊ 0.0117 0.0097 Indonesia 0.7475＊ 0.4500＊ -0.0227 0.2352＊ -0.0322 0.0081 ＊ Significant level (P<0.05) of FST value, with the sequential Bonferroni correction (Rice 1989).

(Vt/7, Mek/5, Rat/1, PM/5, BP/3, Sa/2, Nia/2) (Ipoh/7, PM/1, Sa/3, Suk/5) (Rat/1, PM/2) (Ipo h/2, KT/2, S a /2,SK/2) SK/2) (PM/1) (Sam/1) (BP/6, Sam/1, SK/3) (SK/4) (Btg/1, Sa/1) (Rat/1) (Ban/2) (Rat/1) (Ban/6, Rat/1) (Ban/2, Rat/1, Sa/1)

Fig. 2. The evolutionary history of marble goby haplotypes was inferred using the Neighbor-Joining method (1,000 bootstraps). Numbers at nodes indicate the bootstrap values, and only values >50% are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Kimura 2-parameter method, and the units are the number of base substitutions per site. The abbreviations (refer to Table 1) and numbers in the brackets show the sampling locations and the number of samples, respectively.

(-0.0322), Sarawak-West Malaysia (-0.0204), and Phylogenetic relationship Indonesia-West Malaysia (-0.0227) (Table 5). The A neighbor-joining tree was constructed using negative values of pairwise FST showed that the the Kimura 2-parameter model. The tree indi- average number of pairwise difference within a cated no obvious genealogy among the marble population was more than between populations. goby (Fig. 2). The topology of the neighbor- Thus, Sarawak, Indonesia and West Malaysia joining tree of marble goby haplotypes was shal- are less genetically different. The significant FST low, and there were no significant genealogical values between populations showed Ayutthaya, branches or clusters of samples corresponding Dong Nai and Sabah were genetically differenti- to sampling locations. Typically, haplotypes in a ated from the other populations (Table 5). population were scattered throughout the tree, The genetic distances in Table 5 show that except for the Dong Nai population. the highest value between populations was between Ayutthaya and Dong Nai (0.0207), and Discussion lowest value was between Indonesia and Dong Nai (0.0074). Within populations, the genetic Genetic diversity and structure distances of more than 0.0100 occurred within In this study, the genetic diversity, as West Malaysia, Sabah, and Sarawak. expressed by haplotype and nucleotide diversities, is higher in the West Malaysia, Sarawak, Population Structure of Marble Goby 389 and Sabah populations compared to other popu- provide cogent evidence of strong evolutionary lations (Table 2). This might be explained by forces at work (Reinaldo et al. 2002). In future the smaller sample size in the other populations studies, the demography of marble goby popula- (Tajima 1983; Nei 1987): the seven samples tion may be revealed by increasing the sample from Dong Nai (Vietnam) consisted of one number in each population, and sampling from haplotype only with no polymorphic sites in smaller scale regions. the sequences. To further elucidate the genetic In this study, we obtained negative variance diversities of the marble goby, more samples components in AMOVA at the region hierar- per site should be collected from different popu- chical level. The AMOVA methodology relies lations (Rand et al. 1994). on estimates of relationships between haplo- mtDNA is maternally inherited; samples with types in the same populations relative to hap- distinct haplotypes in Ayutthaya, West Malaysia, lotypes of different populations (Weir 1996). Sarawak, Sabah, and Indonesia probably repre- Negative variance components can result from sent the indigenous populations in those regions very small, but positive estimates of genetic (Table 3). These distinct haplotypes have no structure indices from data (Weir 1996). This introgression stemming from other popula- implies that haplotypes are more related among tions, which could be explained by the natural than within regions, and most of the variability geographic boundaries and different maternal occurs among and within populations (Weir and ancestors (Weiss et al. 2001). These distinct Cockerham 1984; Weir 1996). haplotypes can be used as indicators or DNA markers to identify the indigenous populations Phylogenetic relationship of those regions. Genetic diversity should be The neighbor-joining tree revealed that hap- higher in the ancestral population compared to lotypes of marble goby were widespread in a derived population (Savolainen et al. 2002). Southeast Asia, except for the eight distinct hap- West Malaysia has the highest number of haplo- lotypes (Fig. 2, Table 3). The complete life cycle types (nine) and the highest nucleotide diversity of the marble goby in nature has not been clearly (0.0125) among the six populations, indicating defined. However, according to Akihito et al. that the West Malaysia population probably rep- (2000), there is a possibility that the worldwide resents ancestral population in Southeast Asia. distribution of the gobioid fishes occurs by the Hap-5 is the most common haplotype among the passive migrations of larvae via currents. Akihito fourteen haplotypes, and was present in 29.4% of et al. (1984) reported that some freshwater gobi- the samples. Even though Hap-5 can be found in oid species could have positively advanced into four different populations, it might derive from a continental inland areas, consequently changing single ancestor according to the theory of mater- their life style from amphidromous to a fluvial nal inheritance of mtDNA. type. It is generally assumed that gobioids arose Tajima’s D neutrality test for all the populations in freshwater, from a marine ancestor, and then indicated that the populations are in genetic equi- returned to marine habitats once or many times librium (Kimura 1983). Fu’s FS test, which is pro- (Allen 1989; Allen et al. 2002). These could be posed to detect population growth, also did not the reasons for the widespread occurrence of show significant values for any of the populations. Hap-5 in Southeast Asia. However, further study In fact, smaller sample sizes were used in this on the life cycle of marble goby in nature is study, and a reduction in the number of sig- necessary to understand their habitat and physi- nificant deviations from neutrality are expected ology to improve artificial seed production of (Simonsen et al. 1995; Wayne and Simonsen marble goby in hatcheries. 1998). Small sample sizes preclude a reasonable There is a probable explanation for the occur- argument for neutrality if neutrality tests fail to be rence of the same haplotypes in different popu- significant. On the other hand, when neutrality is lations. During the last glacial period, the sea significantly rejected, the small sample sizes may level was lower and much of the archipelago 390 H. C. Ha, S. Senoo, K. Tsunemoto, Y. Nakagawa, S. Miyashita, O. Murata and K. Kato of Southeast Asia was terrestrial (Sundaland) Mr. Lim Kai Siang of Aquacliq, Selangor and Mr. (Harold 2000; Marwick 2009). During this Thian Hai Chung of Honey Aquaculture, Kuching period, freshwater fish, including marble goby, for providing the fin samples and the transporta- would have dispersed throughout the Sundaland tion to conduct the sampling trips. Thanks to Mrs. via freshwater currents. In the Holocene, the Chun Kia Huey and Dr. Muhammad Darwis for Sunda Shelf, which includes southern Thailand, their help with the sampling from Vietnam and Peninsular Malaysia, Sumatra, Java, and Borneo, Indonesia, respectively. was flooded when thawing occurred, thus form- ing the current geographical features (Harold References 2000; Marwick 2009). Thus, the marble goby had been separated and isolated on the archipelago, Akihito Prince, M. Hayashi, T. Yoshino, K. Shimada, T. peninsula, and mainland respectively. This could Yamamoto and H. Senou (1984) Suborder Gobioidei. In“ The Fishes of the Japanese Archipelago” (ed. by K. explain the widespread distribution of the same Masuda, K. Amaoka, C. Araga, T. Uyeno and T. haplotypes in these regions. The genetic similarity Yoshino), Tokai University Press, Tokyo, pp. 236-289. between West Malaysia, Sarawak, and Indonesia Akihito, A. Iwata, T. Kobayashi, K. Ikeo, T. Imanishi, H. also supports this hypothesis (Table 5). Fauna Ono, Y. Umehara, C. Hamamatsu, K. Sugiyama, Y. Ikeda, K. Sakamoto, A. Fumihito, S. Ohno and T. from Vietnam and Cambodia include species Gojobori (2000) Evolutionary aspects of gobioid fishes that are widely distributed across the Indochina based upon a phylogenetic analysis of mitochondrial and Sunda provinces, suggesting that taxa from cytochrome b genes. Gene, 259, 5-15. these assemblages would be affected by common Allen, G. R. (1989) Freshwater Fishes of Australia, T. F. H. Publications Inc., Neptune City, New Jersey, 240 pp. Paleo-geographical events (Marwick 2009). Allen, G. R., S. H. Midgley and M. Allen (2002) Field Guide The results of the present study have impor- to the Freshwater Fishes of Australia, Western Australia tant implications for the fisheries management Museum, Perth, 408 pp. of marble goby. This study revealed highly Amornsakun, T., W. Sriwatana and U. Chamnanwech (2002) Some aspects in early life stage of sand goby, Oxyeleotris significant differences in the mtDNA control marmoratus larvae. Songklanakarin J. Sci. Techno., 24, region of Ayutthaya, Dong Nai, and Sabah com- 611-619. pared to other populations (by FST values and Brown, G. G., G. Gadaleta, G. Pepe, C. Saccone and E. Sbica genetic distances). This could be because these (1986) Structural conservation and variation in the D-loop-containing region of vertebrate mitochondrial populations are geographically much farther DNA. J. Mol. Biol., 192, 503-511. from the other populations, and no gene flow Cheah, S. H., S. Y. Lam and K. J. Ang (1991) Preliminary existed among them. Our data showed that the report on induced spawning of the marble goby, populations of West Malaysia, Sarawak, and Oxyeleotris marmoratus in Malaysia. In “Recent Innovations in the Animal and Animal Product Indonesia were closely related. A more rigor- Industry”, Malaysian Society of Animal Product, ous study using larger samples sizes and longer Serdang, pp. 111-114. mtDNA fragments is recommended to help elu- Cheah, S. H., S. Senoo, S. Y. Lam and K. J. Ang (1994) cidate the genetic variation of these populations. Aquaculture of a high value freshwater fish in Malaysia: the marble goby (Oxyeleotris marmoratus, Bleeker). In “NAGA”, The ICLARM Quarterly, Penang, pp. 22-25. Acknowledgments Darwis, M., S. R. Muhd. Saleh and S. Senoo (2008) Effects of different salinity levels on growth and survival of We would like to express our gratitude to the marble goby, Oxyeleotris marmoratus juvenile. Aquaculture, 56, 433-439. Ministry of Education, Culture, Sports, Science Excoffier, L., P. E. Smouse and J. M. Quattro (1992) and Technology, Japan, for funding this study Analysis of molecular variance inferred from metric under the G-COE program of Kinki University, distance among DNA haplotypes: application to Japan. We thank all the staff of the Borneo Marine human mitochondrial DNA restriction data. Genetics, 131, 479-491. Research Institute Fish Hatchery, Universiti Excoffier, L., G. Laval and S. Schneider (2005) Arlequin Malaysia Sabah for their cooperation in the experi- (version 3.0): an integrated software package for pop- ment. We also thank Mr. Yusuke Takeuchi and ulation genetics data analysis. Evol. Bioinformatics Mr. Yusuke Sato of Shimakyu Co. Ltd., Thailand, Online, 1, 47-50. Population Structure of Marble Goby 391

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東南アジアにおけるマーブルゴビ Oxyeleotris marmorata のミトコンドリア DNA による集団構造解析

夏孝洲・瀬尾重治・常本和伸・中川至純・宮下盛・村田修・家戸敬太郎

東南アジアにおけるマーブルゴビ Oxyeleotris marmorata の集団構造を調べるために，ミトコンドリア DNA 解析用に85尾のサンプルを 3 地域（タイおよびベトナム，マレー半島，並びにジャワおよびボルネオ島）から収集し，近隣地から得られたサンプルをプールして 1 集団と仮定して解析した。

全サンプルから14ハプロタイプが得られた。全ての集団において Tajima’s D および Fu’s FS 値に有意差はなかったことから，遺伝的に平衡状態にあることが示唆された。分散分析（AMOVA）の結果，集団間および集団内に有意差がみられた。Ayutthaya，Dong Nai および Sabah は他のすべての集団と

の間で有意な FST 値および高い遺伝的距離を示し，Ayutthaya，Dong Nai および Sabah のマーブルゴビが他の集団と比較してそれぞれ遺伝的に異なる集団であることが推察された。