Plankton Benthos Res 6(2): , 2011 Plankton & Benthos Research The Japanese Association of Benthology

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1 Plankton Benthos Res 6(2): , 2011 Plankton & Benthos Research The Japanese Association of Benthology Genetic characterization of the northwestern Pacific population of a deep-sea demersal fish, Bothrocara hollandi SHIGEAKI KOJIMA 1,*, REINA MAEDA 1, KEI SAKUMA 1, YUTAKA KOKUBU 1, SEISHI HAGIHARA 1 & MASAKI ITOH 2 1 Atmosphere and Ocean Research Institute, the University of Tokyo, Kashiwanoha, Kashiwa, Chiba , Japan 2 Hachinohe Station, Tohoku National Fisheries Research Institute, Fisheries Research Agency, Shimomekurakubo, Same, Hachinohe, Aomori , Japan Received 3 October 2010; Accepted 13 February 2011 Abstract: We determined the nucleotide sequences of the 3 region ( 400 base pairs) of the mitochondrial control region for 97 individuals of the deep-sea demersal fish species Bothrocara hollandi, which were collected at five sites in the northwestern Pacific off Tohoku District, the northeastern part of the Japanese mainland. Phylogenetic analysis based on the sequences showed that these fish form a monophyletic group with individuals of the Okhotsk Sea, which have completely deviated from those fish of the Japan Sea. Furthermore, genetic diversity of fish in the northwestern Pacific was higher than that in the Okhotsk Sea. The population of the northwestern Pacific was shown to have experienced a recent population expansion. Nine of 97 individuals had only one non-coding unit, and the remaining individuals had two units between mitochondrial genes for trna Thr and trna Pro ; however, neither of these groups of individuals formed a monophyletic group in the Okhotsk Sea or the northwestern Pacific, while monophyly of individuals with more than one unit was shown in the Japan Sea. The differences between the populations of the Japan Sea and neighboring sea areas might be attributed to the occurrence of repeated environmental changes and corresponding population bottleneck events in the Japan Sea. Key words: Bothrocara hollandi, mitochondrial DNA, northwestern Pacific, population structure Introduction The Japan Sea is a semi-closed marginal sea area situated between the Japanese Islands and the Asian continent. During the glacial and interglacial cycles of the Pleistocene epoch, dramatic environmental changes repeatedly occurred in the Japan Sea (Tada et al. 1999, Itaki et al. 2004). In particular, during the last glacial maximum (LGM; 27,000 to 17,000 years ago), the water column in the Japan Sea was strongly stratified because of a massive influx of freshwater from the Asian continent, which caused most of the deep-sea layer to become anoxic (Tada et al. 1999, Itaki et al. 2004). In order to reveal the effects of such large environmental changes on the population structures of deep-sea animals in the Japan Sea, we previously analyzed populations of the Japan Sea and the Okhotsk Sea of Bothrocara hollandi * Corresponding author: Shigeaki Kojima; , kojima@aori.utokyo.ac.jp (Jordan & Hubbs), which is the most dominant deep-sea demersal fish species in the Japan Sea (Kojima et al. 2001, Kodama & Kojima 2009, Kodama et al. 2008). These studies revealed the distinct genetic deviation between populations of these two sea areas, which was attributed to the shallowness and narrowness of the straits connecting these two sea areas. The Japan Sea was connected to the Okhotsk Sea through the Mamiya Strait (15 m depth) and the Soya Strait (55 m depth), and to the Pacific Ocean through the Tsugaru Strait (130 m depth), while B. hollandi inhabits depths between 150 m and 1980 m in the Japan Sea (Okiyama 2004). Our studies also showed the Japan Sea population of B. hollandi consists of two genetically distinct groups named Group A and Group B (Kojima et al. 2001). The former is a monophyletic group with a relatively high genetic diversity and the latter is paraphyletic with a relatively low genetic diversity (Kojima et al. 2001, Kodama & Kojima 2009). For a long time, the systematic classification of the genus Bothrocara has been unclear. Recently, Anderson et al.

2 NW Pacific population of Bothrocara hollandi 109 Table 1. List of sampling sites. No. Area Location Depth (m) Cruise N 1 off Hachinohe N, E * 2 off Shiogama N, E 557 WK ** 3 off Soma N, E 510 WK ** 4 off Onahama N, E 362 WK ** 5 off Onahama N, E 558 WK ** * Collected by the training ship Tanshu-maru of Hyogo Prefectural Kasumi High School. ** Collected by the research vessel Wakataka-maru of Tohoku National Fisheries Research Institute, Fisheries Research Agency (2009) reviewed various Bothrocara species and reported that B. hollandi is distributed in an area of the Pacific Ocean near northern and central Japan, the Bering Sea, the Japan Sea, and the Okhotsk Sea. However, phylogeographic analysis has not yet been carried out for the Pacific population of this species. Since the Okhotsk Sea is connected to the Pacific Ocean by deep straits, it is expected that little genetic deviation exists between the local populations of these two sea areas. However, some genetic cline of this species along the coast-line may be detected. In this study, we obtained B. hollandi specimens from the northwestern Pacific Ocean and examined their genetic characteristics on the basis of nucleotide sequences of two regions of mitochondrial DNA, namely, the control region and the non-coding region between mitochondrial genes for trna Thr and trna Pro. Based on the results, we discussed processes and factors of formation of the genetic characteristics as well as population structure of this species. Materials and Methods Ninety-seven individuals of the Bothrocara hollandi species were collected at five sampling sites in the northwestern Pacific off Tohoku District, the northeastern part of the Japanese mainland. We collected the specimens during cruises of both the training ship Tanshu-maru of Hyogo Prefectural Kasumi High School and the research vessel Wakataka-maru of Tohoku National Fisheries Research Institute, Fisheries Research Agency (Table 1; Fig. 1). We stored all specimens in a freezer ( 30 C) until use. Total DNA was extracted from a small piece (approximately 0.03 cm 3 ) of muscle tissue from each specimen with a DNeasy Tissue Extraction Kit (Qiagen, Valencia, CA) according to the manufacturer s instructions. Mitochondrial DNA fragments, including the 3 region of the trna Thr gene, the non-coding region, the trna Pro gene, and the 5 region of CR, were amplified in a polymerase chain reaction (PCR) using the primers ZThr-L (5 - AGAGCGCCGGTCTTGTAARCCG-3 ) and CR3-H (5 - GAAACCCCTTGCACCCGTGG-3 ), as described previously (Kodama et al. 2008). The steps used to perform PCR were as follows: incubation at 95 C for 120 s, followed by 30 to 40 cycles at 95 C for 40 s, 66 C for 60 s, and 72 C for 90 s. To degrade remaining primers and nucleotide, 5 µl of Fig. 1. Location of the sites where Bothrocara hollandi specimens were collected; details are listed in Table 1. the PCR products were mixed with 1 ml of ExoSAP-IT (United States Biochemical, Cleveland, OH), incubated at 37 C for 15 min and 80 C for 15 min. Each purified PCR product was used in cycle sequence reactions with the following primers: CR-2 (5 -CRAATACTTGTCCCTCACC- CTC-3 ) (Kojima et al. 2001) and Pro-L (5 -CTACCTC- CAACTCCCAAAGC-3 ) (Palumbi et al. 1991), using the BigDye Terminator Cycle Sequencing Kit, version 3.0 (Applied Biosystems, Foster City, CA). The sequences were obtained using an ABI 3100 automated DNA sequencer (Applied Biosystems). The nucleotide sequences reported in the present study have been added to the GSDB, DDBJ, EMBL, and NCBI nucleotide sequence databases under accession numbers AB (control region) and AB (non-coding unit). The sequences were aligned using the computer program

3 110 S. KOJIMA et al. Clustal W (Thompson et al. 1994, Jeanmougin et al. 1998) in the MEGA, version 5.0 Beta software package (Tamura et al. 2007), using the default settings. We also checked the alignments visually. In the following analyses, we used transitions, transversions, and insertions/deletions, all of which were equally weighted. We constructed a phylogenetic network by the median-joining method, using the computer program Network, version (Bandelt et al. 1999), based on differences in nucleotide sequences. We estimated the genetic diversity of specimens based on both haplotype (gene) diversity (h), which is the probability that two randomly chosen haplotypes are different (Nei 1987), and nucleotide diversity (P ), which is the probability that two randomly chosen homologous nucleotides are different (Tajima 1983, Nei 1987). Furthermore we examined the differences in the frequencies of haplotypes between populations by using the exact test of population differentiation (Raymond & Rousset 1995) performed with the Arlequin software package, version (Excoffier et al. 2010). We estimated the unbiased fixation indices F ST (Weir & Cockerham 1984) and F ST (Excoffier et al. 1992), and tested the significance of the indices by using a nonparametric permutation approach (10,000 permutations) performed with Arlequin, version (Excoffier et al. 2010). We analyzed the demographic population history based on the mismatch distributions of pairwise differences between all individual pairs. Furthermore, we examined the probable population expansion by performing mismatch distribution analysis (Rogers & Harpending 1992), using Arlequin, version (Excoffier et al. 2010). Results On the basis of nucleotide sequences of the 3 part ( 400 base pairs) of the mitochondrial control region, we identified 36 kinds of sequences (haplotypes) from 97 individuals of Bothrocara hollandi that were collected in the northwestern Pacific (Table 2). Twenty-five of 398 sites were variable, 4 sites contained insertions/deletions, and transversion/transition bias was Specifically, we found all five haplotypes that were obtained from the Okhotsk Sea (haplotypes in Table 2) in a previous study (Kodama et al. 2008). Three of these five haplotypes were found to dominate the Pacific population. In contrast, the remaining 31 haplotypes that we obtained were rare. None of these rare haplotypes were observed in any of the individuals sampled from the Japan Sea whose sequences were reported in a previous study (Kodama et al. 2008). Figure 2 shows the median-joining network based on 38 variable sites among all haplotypes of B. hollandi that have been obtained to date. The haplotypes from the Japan Sea and those from the remaining sea areas formed distinct groups. We determined that genetic diversity was greater in the group from the northwestern Pacific than the group from the Okhotsk Sea (Table 3). Specifically, the diversity value of the former was found to be almost the same as that Table 2. Haplotype composition of individuals collected at each sampling site. Number of sampling sites is the same as in that in Table 1. Number of haplotypes follow those in Kodama et al. (2008). Haplotypes were previously reported to be present in the Okhotsk Sea (Kodama et al. 2008). The numbers in parentheses denote the number of individuals with a single non-coding unit between mitochondrial genes for trna Thr and trna Pro. Sampling site Haplotype Total (1) 2(1) 3(2) (1) 2(2) 3(1) 3(1) 10(5) (1) 1(1) (1) 1(1) Total 21(1) 18(4) 22 20(2) 16(2) 97(9) of the group from the Japan Sea. The test we performed to examine the unbiased fixation indices F ST and F ST showed a significant genetic differentiation between the populations of the Okhotsk Sea and the northwestern Pacific (F ST 0.073, p 0.000; F ST 0.072, p 0.000), while the exact test of population differentiation showed no genetic differentiation between the populations (p 0.219). Furthermore, mismatch distribution analysis

4 NW Pacific population of Bothrocara hollandi 111 Fig. 2. Median-joining network of the haplotypes of Bothrocara hollandi. Closed triangles, open circles, and closed circles denote haplotypes from the Japan Sea, from both the Okhotsk Sea and the northwestern Pacific, and from only the northwestern Pacific, respectively. The abbreviations used for the haplotypes from the Okhotsk Sea and the northwestern Pacific are the same as those used in Table 2. Sequences of hyplotypes 1 56 (AB AB262646) are from Kodama et al. (2008). Table 3. Genetic diversity of the populations of Bothrocara hollandi. Number of sampling sites is the same as in that in Table 1. Sampling site No. of No.of speci- haplomens types Haplotype diversity Diversity index Nucleoyide diversity NW Pacific Japan Sea (Group A)* Japan Sea (Group B)* Japan Sea (all)* Okhotsk Sea* * Calculated based on data in Kodama et al. (2008) showed that a population of the northwestern Pacific has experienced an expansion in population size (mismatch observed mean 2.082, t 2.109, q , q , SSD , p 0.760). We also determined the number of non-coding units between mitochondrial genes for trna Thr and trna Pro for the 97 B. hollandi individuals that we collected in the northwestern Pacific. However, the nucleotide sequences of this region could not be unambiguously determined for 11 individuals, likely due to heteroplasmy (Kodama & Kojima 2009). For the remaining 86 Pacific individuals, we successfully determined nucleotide sequences of the non-coding region, and obtained 10 units with new sequences (Fig. 3; Table 4). Nine of the 97 individuals had only one noncoding unit, and the remaining individuals had two units between mitochondrial genes for trna Thr and trna Pro. Individuals with a single unit were obtained from four haplotypes based on the control region (haplotypes 53, 56, 77, and 82 in Table 2). Furthermore, individuals with a single unit and those with two units shared two dominant haplotypes (haplotypes 53 and 56). We could not obtain a reliable phylogenetic relationship among haplotypes, likely due to the finding that most haplotypes exhibited little difference from each other (data not shown). Regardless, the present study suggests that neither individuals with a single unit nor those with two units form a monophyletic group in the Okhotsk Sea or the northwestern Pacific. Discussion Among three distributional areas of Bothrocara hollandi, which is the most dominant deep-sea demersal fish of the Japan Sea, genetic characteristics have been reported for two areas, namely, the Japan Sea and the Okhotsk Sea. Information on a remaining area, the northwestern Pacific provided by the present study enables us to reveal population structure of this interesting species as a whole. The

5 112 S. KOJIMA et al. Fig. 3. Nucleotide sequences of the units of the non-coding region of Bothrocara hollandi. Dashes denote alignment gaps. Sequences of units 0 17 (AB AB ) are from Kodama & Kojima (2009). haplotypes from the Japan Sea and those from the remaining sea areas formed distinct groups. Of note, while the Japan Sea is connected with neighboring sea areas only by narrow and shallow straits, the straits that connect the Okhotsk Sea and the Pacific Ocean are wide and deep enough for deep-sea demersal fishes to readily migrate between these two areas. Nevertheless, the present study suggested some degree of genetic deviation between the Okhotsk Sea and the Pacific Ocean. It might show the existence of some kind of dispersal barrier between these two sea areas. The present results showed genetic diversity of the population of B. hollandi in the Okhotsk Sea is much lower than of that in the Japan Sea or of that in the Pacific Ocean (Table 3). In a previous study (Kodama et al. 2008), we reported significant deviation of mismatch distribution of B. hollandi in the Okhotsk Sea from the sudden expansion model, and concluded that they have not experienced recent population expansion. The present study showed recent population expansion of the Pacific population to be similar to that of the Japan Sea. One possible explanation of the low genetic diversity of the Okhotsk population is its moderate isolation from the Pacific population and incomplete recovery following the reduction of population size due to a shortage of food supply during the last glacial period (Kojima et al. 2009). Nine of the 97 individuals had only one non-coding unit between mitochondrial genes for trna Thr and trna Pro (Table 4), while all individuals from the Okhotsk Sea examined to date have been shown to have two units (Kodama & Kojima 2009). The present study suggests that neither individuals with a single unit nor those with two units form a monophyletic group in the Okhotsk Sea or the northwestern Pacific. In contrast, individuals with a single unit are domi-

6 NW Pacific population of Bothrocara hollandi 113 Table 4. Composition of haplotypes of the non-coding region between the genes for trna Thr and trna Pro of Bothrocara hollandi. Units are the same as those in Fig. 2, and the permutation is indicated in from the 5 end to the 3 end. Number of sampling sites is the same as that in Table 1. Permutation of units Sampling site Total Total nant in the Japan Sea population (Table 5) and those with more than two units formed a monophyletic group (Kodama & Kojima 2009). Since its sister species, B. tanakae, exhibits a single unit, the common ancestor of B. hollandi was thought to also have only a single unit (Kodama & Kojima 2009). When the present population of the Japan Sea was isolated from the neighboring sea areas, all or most of the B. hollandi individuals may have exhibited a single unit, and repeated severe environmental changes may have prevented individuals with two units from dominating in the Japan Sea population. However, the almost complete replacement by individuals with two units may have occurred in the Okhotsk Sea and the northwestern Pacific, where environmental conditions during the glacial period were not as severe as those in the Japan Sea (Gorbarenko et al. 2004, Minoshima et al. 2007). In addition to revealing a complete deviation of the B. hollandi populations between the Japan Sea and the neighboring sea areas, the present study also showed some population structure among the latter areas. Bothrocara hollandi has been thought to inhabit the benthopelagic layer at all stages of life, and to have relatively low dispersal ability (Okiyama 2004). With more detailed analyses of population structure, as well as exhaustive studies of its life history, B. hollandi is expected to be an ideal subject to reveal the history of global marine environmental changes and the reaction of marine organisms. Table 5. Composition of haplotypes of the non-coding region between the genes for trna Thr and trna Pro of Bothrocara hollandi. Data of the Japan Sea and the Okhotsk Sea are from Kodama and Kojima (2009). Permutation Japan Sea Okhotsk NW of units Group A Group B Sea Pacific Acknowledgements The authors would like to thank the captains, officers, and crews of the training ship Tanshu-maru of Hyogo Prefectural Kasumi High School and the research vessel Wakataka-maru of Tohoku National Fisheries Institute for their support in sampling specimens. This study was supported in part by KAKENHI (No ). References Anderson ME, Stevenson DE, Shinohara G (2009) Systematic review of the genus Bothrocara Bean 1890 (Teleostei: Zoarcidae). Ichthyol Res 56: Bandelt H-J, Foster P, Röhl A (1999) Median-joining networks for inferring intraspecific phylogenies. Mol Biol Evol 16: Excoffier L, Lischer HEL (2010) Arlequin suite ver 3.5: A new se-

7 114 S. KOJIMA et al. ries of programs to perform population genetics analyses under Linux and Windows. Mol Ecol Resour 10: Excoffier L, Smouse PE, Quattro JM (1992) Analysis of molecular variance inferred from metric distances among DNA haplotypes: application to human mitochondrial DNA restriction data. Genetics 131: Gorbarenko SA, Southon JR, Keigwin LD, Cherepanova MV, Gvozdeva IG (2004) Last Pleistocene Holocene oceanographic variability in the Okhotsk Sea: geochemical, lithological and paleontological evidence. Palaeogeogr Palaeoclimatol Palaeoecol 209: Itaki T, Ikehara K, Motoyama I, Hasegawa S (2004) Abrupt ventilation changes in the Japan Sea over the last 30 ky: evidence from deep-dwelling radiolarians. Palaeogeogr Palaeoclimatol Palaeoecol 208: Jeanmougin F, Thompson JD, Gouy M, Higgins DG, Gibson TJ (1998) Multiple sequence alignment with Clustal X. Trends Biochem Sci 23: Kodama Y, Kojima S (2009) The population history of the deepsea demersal fish Bothrocara hollandi in the Japan Sea revealed by tandem repeat units in the mitochondrial non-coding region. Plankton Benthos Res 4: Kodama Y, Yanagimoto T, Shinohara G, Hayashi I, Kojima S (2008) Deviation age of a deep-sea demersal fish, Bothrocara hollandi, between the Japan Sea and the Okhotsk Sea. Mol Phylogenet Evol 49: Kojima S, Moku M, Kawaguchi K (2009) Genetic diversity and population structure of three dominant myctophid fishes (Diaphus theta, Stenobrachius leucopsarus, and S. nannochir) in the North Pacific Ocean. J Oceanogr 65: Kojima S, Segawa R, Hayashi I, Okiyama M (2001) Phylogeography of a deep-sea demersal fish, Bothrocara hollandi, in the Japan Sea. Mar Ecol Prog Ser 217: Minoshima K, Kawahata H, Irino T, Ikehara K, Aoki K, Uchida M, Yoneda M, Shibata Y (2007) Deep water ventilation in the northwestern North Pacific during the last deglaciation and the early Holocene (15-5 cal. Kyr B.P.) based on AMS 14 C dating. Nucl Instrum Meth B 259: Nei M (1987) Molecular Evolutionary Genetics. Columbia University Press, New York, 512 pp. Okiyama M (2004) Deepest demersal fish community in the Sea of Japan: a review. Contrib Biol Lab Kyoto Univ 29: Palumbi S, Martin A, Romano S, McMillan WO, Stice L, Grabowski G (1991) The simple fool s guide to PCR, Ver. 2. Department of Zoology and Kewalo marine Laboratory, University of Hawaii, Honolulu, 44 pp. Raymond M, Rousset F (1995) An exact test for population differentiation. Evolution 49: Rogers AR, Harpending H (1992) Population growth makes waves in the distribution of pairwise genetic differences. Mol Biol Evol 9: Tada R, Irino T, Koizumi I (1999) Land-ocean linkages over orbital and millennial timescales recorded in late Quaternary sediments of the Japan Sea. Paleoceanography 14: Tajima F (1983) Evolutionary relationship of DNA sequences in finite populations. Genetics 105: Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24: Thompson JD, Higgins DG, Gibson TJ (1994) Clustal W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22: Weir BS, Cockerham CC (1984) Estimating F-statistics for the analysis of population structure. Evolution 38: