World Applied Sciences Journal 33 (7): 1079-1088, 2015 ISSN 1818-4952 © IDOSI Publications, 2015 DOI: 10.5829/idosi.wasj.2015.33.07.94254

Genetic Population Structure of the Aburahaya (Rhynchocypris lagowskii) Based on Mitochondrial DNA Sequence

C.M.M. Hassan, Takanori Ishikawa, Singo SEKI and A. Mahmuda

Laboratory of Aquatic Ecology, Faculty of Agriculture, Kochi University, B-200 Nankoku, Kochi 783-8502,

Abstract: Analyses of partial mitochondrial DNA (mtDNA) sequences support the classification of Aburahaya (Rhynchocypris lagowskii) from the and Pacific Ocean. To investigate genetic population structure, we examine nucleotide sequence of the cytochrome b region. In this study we found three major geographical groups. Molecular phylogenetic analysis revealed that the population of the group 3 differentiation is 0.8759 ± 0.0333. The group 2 differentiation is 0.5333 ± 0.1801 which is collected from the Kamishyou River (Toyama Prefecture) and Hakui River (Ishikawa Prefecture) populations. The neighbor-joining tree of the mitochondrial DNA haplotypes for all specimens constructed from the Kimura’s two parameter. Among these 24 localities being clustered into 3 major geographic groups in NJ tree mtDNA segment and NJ

tree mtDNA haplotype. No significant difference for the population pair wise FST was detected among these localities (P>0.05). The most parsimonious network of mtDNA haplotype of aburahaya 24 localities, estimated using the TCS algorithm. In this network showed three geographical groups. Halpotype 1-29 is one group, haplotype 30-33 is group 2 and haplotype 34-54 is group 3.

Key words: Population genetic mtDNA Cyprinide Aburahaya Geographic groups Haplotype network

INTRODUCTION entire range have been fragmentary. Their relations to geological patterns and processes have not been Aburahaya (Rhynchocypris lagowskii) is a small elucidated sufficiently. Phylogeographic assessments of cyprinid and common fresh water fish that is endemic to the populations structure and historical dynamics of Japan also distributed widely in East Japan. Aburahaya species using molecular genetic markers play an essential prefers to rather higher water temperature and is found in role in the study of historical biogeography, especially the middle and lower course of the rivers. Adults are over relatively short geological time scales (the Neogene- found usually in pools and stagnant waters of river and Quaternary) [1]. Such approaches have been previously are very active in summer. They have a tendency to applied to several fresh-water fishes distributed in entire become quiescent in winter and are hiding among the Japan. Due to habitat degradation (e.g., urbanization and aquatic plants and under the stones on the shore. The river improvement) and the introduction of predatory intestinal contents of this fish are omnivorous and the fishes, any species can be declined throughout its range most important food is aquatic insect. [18, 19]. Previous studies have revealed that cyprinid fish Aburahaya is not an important commercial fish but populations have maintained their genetic diversity and this fish will not be affected by released. In this concept, that the species shows clear genetic population structure there is no comprehensive study on population genetic associated with watersheds in the eastern part of western diversity of aburahaya. So population’s genetic diversity Japan [20, 21]. remains largely unknown. Our fundamental question is This study was conducted primarily to clarify the about aburahaya population’s structure and to population’s structure of Rhynchocypris lagowskii using investigate genetic population structure of aburahaya mtDNA sequence data from specimens collected across samples collected from different locations. the entire range of the species. The populations are Phylogeographic studies of freshwater fish aburahaya the divided into geographic groups are discussed from the

Corresponding Author: C.M.M. Hassan, Laboratory of Aquatic Ecology, Faculty of Agriculture, Kochi University, B-200 Nankoku, Kochi 783-8502, Japan. Tel: +81 9013287572. 1079 World Appl. Sci. J., 33 (7): 1079-1088, 2015 viewpoint of the genetic structure among the geographic groups. Furthermore, estimating the divergence time among the detected major geographical groups, distribution and isolation processes of this species are discussed with reference to potentially associated geological events.

MATERIALS AND METHODS

Study Area and Sample Collections: Aburahaya were collected from the Sea of Japan and Pacific Ocean. After catching the fish we were preserved with 99% ethyl alcohol for DNA extraction. Fig. 1 shows the location of Fig. 1: Sampling localities of Rhynchocypris lagowskii. the sampling sites. Numbers of localities correspond to those in Table 1 Mitochondrial DNA Extraction: One hundred and ninety-one individuals from all population’s samples were The haplotype diversity (h) and nucleotide diversity ( ) used for mtDNA analysis. We were preserved aburahaya of twenty four localities and FST value of population individuals with 99% ethyl alcohol after clipping their fins. structure examined by AMOVA [6] using ARLEQUIN ver. Total DNA was isolated from a piece of fin or muscle by 3.01 [7] A distance matrix was calculated based on standard methods [2]. Kimura’s two-parameter method [8] and clustered by the neighbor-joining method [9] using MEGA ver. 4.0 [4]. Polymerase Chain Reaction Amplification: We amplified The robustness of the phylogenies was assessed by the mitochondrial DNA cytochrome b region by bootstrap analyses consisting of 1000 replicates [10]. polymerase chain reaction (PCR) using a pair of To investigate the relationship between sequence types, oligonucleotide primers: L14391 (5'- the most parsimonious network of mtDNA haplotype of ATGGCAAGCCTACGAAAAAC-3') and H15551 (5'- aburahaya 24 locations, estimated using the TCS GATTACAAGACCGATGCTTT-3') originated [3]. The algorithm [11]. Black dots represent missing haplotypes. PCR condition was initially 1 minute at 94°C for denaturation; then 15 seconds at 94°C, 15 seconds at 50°C RESULT and 30 seconds at 72°C following 30 cycles each; after that 5 minute at 72°C for final elongation. Geographic Grouping: Haplotype diversity among the 24 localities ranged from about (0.0000±0.0000 to 1.0000 ± Sequence Method: The PCR products were purified by 0.0625) (Table 3), the localities being clustered in to 3 filtration with a (EXSO-SAP-IT). These purified products major geographic groups in NJ tree mtDNA segment were used as a template DNA for cycle sequencing (Fig. 2) and NJ tree mtDNA haplotype (Fig. 4) based on reactions performed using Big Dye Terminator Cycle Kimura’s two-parameter distances and most parsimonious Sequencing Kits 3.1 standard protocol in 10-µl volumes network mtDNA haplotypes (Fig. 5). Group 1 haplotype consisting of PCR products 0.5 µl. Sequence buffer 1.75µl. diversity is 0.7748 ± 0.0330, Group 2 haplotype diversity Premix 0.5 µl. The forward primer H15551 (5pM) 0.5 µl. and is 0.5333 ± 0.1801 Group 3 haplotype diversity is 0.8759 ± were ran on an ABI 310 automated DNA Sequencer 0.0333 (Table 4). system Oshikiri river and (Applied Biosystems). Nezugaseki river system Nezugaseki river representatives of two different populations belonging to different Sequence Analysis: We have gotten the 460 bp region geographic groups. of mtDNA cytochrome b region. Phylogenetic and molecular evolutionary analyses were conducted using Relationship among Geographic Groups: We constructed MEGA version 4.0 [4] and the nucleotide sequence phylogenetic tree based on the nucleotide sequence were preliminarily aligned by the program CLASTER W obtained from cytochrome b (cyt-b) region. The [5] the sites showing nucleotide polymorphisms sequence of 460 sites of the cyt-b region was determined were re-examined by human eyes for further alignment. for 191 specimens of Aburahaya with outgroup.

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Table 1: Sampling locations, river names, sampling dates and sample sizes of Rhynchocypris lagowskii River no River System River name Abbreviation Location Sampling Date N 1 Yunosawa River YU Japan Sea 2013.07.14 8 2 Nikkou River Mi River M Japan Sea 2012.09.15 6 3 Mogami River Tachiyazawa River TA Japan Sea 2012.08.26 30 4 Mogami River Oshikiri River OSH Japan Sea 2012.08.15 6 5 Mogami River Ootaru River OT Japan Sea 2012.09.14 8 6 Aka River Kakuda River KAK Japan Sea 2012.09.08 6 7 Nezugaseki River Nezugaseki River NE Japan Sea 2012.09.15 5 8 Daimonn River DA Japan Sea 2012.07,22 8 9 Sinano River Chikuma River CH Japan Sea 2012.05.03 10 10 Kamisyou River Kamisyou River KAM Japan Sea 2012.09.15 6 11 Hakui River Hakui River HA Japan Sea 2012.09.15 5 12 Nanakitada River Nishitanaka River NI Pacific Ocean 2013.05.04 6 13 Harase River HAR Pacific Ocean 2013.04.20 8 14 Abukuma River Kumato River KU Pacific Ocean 2012.06.03 4 15 Same River Yamada River YAMA Pacific Ocean 2013.04.13 7 16 Oshi River OS Pacific Ocean 2012.05.20 11 17 Naka River Kuro River KUR Pacific Ocean 2013.04.06 7 18 Yamada River YAM Pacific Ocean 2012.04.29 8 19 Tone River KA Pacific Ocean 2012.04.15 3 20 Ara River Toki River TO Pacific Ocean 2012.03.31 8 21 Kushi River KUS Pacific Ocean 2013.02.09 8 22 Ooba River OB Pacific Ocean 2012.12.31 7 23 Tenryu River MI Pacific Ocean 2012.11.24 8 24 Amano River AM Pacific Ocean 2013.08.16 8

Table 2: Pair wise Fst of Rhynchocypris lagowskii Population YU M TA OSH OT KAK NE DA CH KAM HA NI YU 0.00000 M 0.46067 0.00000 TA -0.02274 0.57143 0.00000 OSH 0.42295 0.37000 0.64043 0.00000 OT 0.22857 0.23623 0.40657 0.34900 0.00000 KAK 0.09908 0.17647 0.26667 0.27179 -0.00186 0.00000 NE 0.35420 0.30295 0.60002 -0.11211 0.27132 0.20278 0.00000 DA 0.82857 0.62500 0.75706 0.50345 0.42236 0.44484 0.30875 0.00000 CH 0.16331 0.33468 0.27920 0.40415 0.20186 0.13136 0.26673 0.28571 0.00000 KAM 0.97301 0.89577 0.96405 0.61754 0.84079 0.85070 0.62317 0.95438 0.89691 0.00000 HA 0.78514 0.69561 0.89152 0.35505 0.66531 0.64349 0.37075 0.78551 0.74714 0.05973 0.00000 NI 0.00000 0.40000 -0.04847 0.36129 0.17714 0.04444 0.28488 0.80408 0.11964 0.96774 0.74615 0.00000 HAR 1.00000 0.68627 0.79672 0.52304 0.44898 0.48644 0.32496 0.14286 0.30620 0.97540 0.80279 1.00000 KU 0.93133 0.65923 0.85933 0.45365 0.48079 0.49702 0.27192 0.69811 0.50868 0.95204 0.72150 0.91519 YAMA 0.61066 0.54148 0.77619 -0.06583 0.51921 0.47097 -0.00033 0.61556 0.57050 0.61221 0.34130 0.56250 OS 0.45034 0.48934 0.43563 0.48185 0.32968 0.24720 0.42505 0.72427 0.31892 0.94607 0.80044 0.40807 KUR 0.43645 0.40264 0.57939 0.39035 0.28125 0.25107 0.23274 0.23713 0.18105 0.87763 0.70390 0.38037 YAM 0.63265 0.53398 0.69762 0.46919 0.41353 0.35804 0.38611 0.65714 0.44056 0.91521 0.75326 0.58974 KA 1.00000 0.89024 0.97070 0.25000 0.81772 0.82207 0.32750 0.97055 0.89425 0.94340 0.49924 1.00000 TO 0.86111 0.79053 0.91221 0.21107 0.75205 0.73957 0.32315 0.85714 0.80643 0.80033 0.51676 0.84065 KUS 0.97297 0.93317 0.97550 0.75819 0.90447 0.90927 0.75738 0.96383 0.93497 0.94891 0.81041 0.96857 OB 1.00000 0.95563 0.98470 0.75581 0.91784 0.92790 0.75591 0.98736 0.95134 0.98021 0.81852 1.00000 MI 0.81237 0.74973 0.90273 0.51956 0.74680 0.73302 0.52560 0.81349 0.79302 0.74633 0.57461 0.78597 AM 0.86356 0.79752 0.91876 0.30804 0.76694 0.75225 0.39946 0.86003 0.82082 0.78384 0.50122 0.84342

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Table 2: Continued Population HAR KU YAMA OS KUR YAM KA TO KUS OB MI AM HAR 0.00000 KU 0.87335 0.00000 YAMA 0.63279 0.56386 0.00000 OS 0.79741 0.80920 0.64508 0.00000 KUR 0.25956 0.43106 0.53115 0.44293 0.00000 YAM 0.71429 0.70859 0.59479 0.62101 0.47167 0.00000 KA 1.00000 0.97556 0.07353 0.95734 0.86501 0.91638 0.00000 TO 0.87500 0.83496 0.06067 0.85437 0.78236 0.82392 -0.17483 0.00000 KUS 0.97436 0.95917 0.73307 0.96147 0.92376 0.94356 0.91273 0.82445 0.00000 OB 1.00000 0.99132 0.72368 0.98063 0.94192 0.96315 1.00000 0.83824 0.73460 0.00000 MI 0.78597 0.76973 0.49652 0.82779 0.76526 0.79537 0.51201 0.55934 0.54464 0.49342 0.00000 AM 0.84342 0.83404 0.15022 0.86335 0.79659 0.83218 -0.11536 0.01587 0.80551 0.81238 0.54942 0.00000

Table 3: Haplotype and nucleotide diversity of the local samples of Rhynchocypris lagowskii River no River System River name Halpotype Diversity Nucleotide Diversity 1 Omono River Yunosawa River 0.0000 +/- 0.0000 0.000000 +/- 0.000000 2 Nikkou River Mi River 0.6000 +/- 0.1291 0.003913 +/- 0.003012 3 Mogami River Tachiyazawa River 0.1908 +/- 0.0928 0.000685 +/- 0.000801 4 Mogami River Oshikiri River 0.8000 +/- 0.1721 0.014348 +/- 0.009120 5 Mogami River Ootaru River 1.0000 +/- 0.0625 0.006289 +/- 0.004199 6 Aka River Kakuda River 0.9333 +/- 0.1217 0.006232 +/- 0.004389 7 Nezugaseki River Nezugaseki River 0.9000 +/- 0.1610 0.017826 +/- 0.011657 8 Agano River Daimonn River 0.4286 +/- 0.1687 0.000932 +/- 0.001061 9 Sinano River Chikuma River 0.8667 +/- 0.0714 0.003140 +/- 0.002352 10 Kamisyou River Kamisyou River 0.3333 +/- 0.2152 0.001449 +/- 0.001473 11 Hakui River Hakui River 0.9000 +/- 0.1610 0.014348 +/- 0.009543 12 Nanakitada river Nishitanaka River 0.0000 +/- 0.0000 0.000000 +/- 0.000000 13 Abukuma River Harase River 0.0000 +/- 0.0000 0.000000 +/- 0.000000 14 Abukuma River Kumato River 0.5000 +/- 0.2652 0.001087 +/- 0.001348 15 Same River Yamada River 0.8095 +/- 0.1298 0.013043 +/- 0.008106 16 Kuji River Oshi River 0.5455 +/- 0.0722 0.001186 +/- 0.001189 17 Naka River Kuro River 0.7143 +/- 0.1809 0.004762 +/- 0.003418 18 Tone River Yamada River 0.4286 +/- 0.1687 0.002795 +/- 0.002220 19 Tone River Karasu River 0.0000 +/- 0.0000 0.000000 +/- 0.000000 20 Ara River Toki River 0.2500 +/- 0.1802 0.005435 +/- 0.003722 21 Sagami River Kushi River 0.2500 +/- 0.1802 0.002174 +/- 0.001853 22 Kano River Ooba River 0.0000 +/- 0.0000 0.000000 +/- 0.000000 23 Tenryu River Miya River 0.9643 +/- 0.0772 0.013665 +/- 0.008265 24 Yodo River Amano River 0.7857 +/- 0.1508 0.006599 +/- 0.004372

Table 4: Haplotype and nucleotide diversity of the geographical area and group of Rhynchocypris lagowskii Location/Group Haplotype diversity Nucleotide diversity Pacific Ocean 0.9278 ± 0.0113 0.020688 ± 0.010608 Japan sea 0.7646± 0.0436 0.009368 ± 0.005181 Group 1 0.7748± 0.0330 0.004165± 0.002642 Group 2 0.5333 ± 0.1801 0.002077 ± 0.001745 Group 3 0.8759 ± 0.0333 0.013492 ± 0.007234 Here, Group 1: Tohoku, North Kanto, East Chube; Group 2: Ishikawa, Toyama and Group 3: Chube

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Table 5: Pair wise Fst of the geographical area and group of Rhynchocypris lagowskii Population Group 1 Group 2 Group 3 Japan Sea Pacific Ocean Group 1 0.00000 Group 2 0.84107 0.00000 Group 3 0.76975 0.60949 0.00000 Japan Sea 0.00000 Pacific Ocean 0.19147 0.00000 Here, Group 1: Tohoku, North Kanto, East Chube; Group 2: Ishikawa, Toyama and Group 3: Chube

Fig. 2: Unrooted neighbor joining tree of Rhynchocypris lagowskii species of the cyt b region of mtDNA segment with out group based on Kimura’s two-parameter distances. Numbers above branches are bootstrap values for 1000 replications. YU- Yunosawa River, M- Mi River, TA- Tachiyazawa River OSH- Oshikiri River, OT- Ootaru River, KAK- Kakuda River, NE- Kakuda River, DA- Daimonn River, CH - Chikuma River, KAM - Kamisyou River, HA- Hakui River, NI- Nishitanaka River, HAR- Harase River, KU- Kumato River, YAMA- Yamada River, OS- Oshi River, KUR- Kuro River, YAM- Yamada River, KA- Karasu River, TO- Toki River, KUS- Kushi River, OB- Ooba River,MI- Miya River, AM- Amano River

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Fig. 3: Morphological localities of Rhynchocypris lagowskii. Numbers of localities correspond to those in Table 1. The circles represent the sequence types. Blue pie-chart represent Group 1, red pie-chart represent Group 2 and yellow pie-chart represent Group 3. Here, Group 1: Tohoku, North Kanto, East Chube; Group 2: Ishikawa, Toyama and Group 3: Chube

According to these variable sites, a total of 54 haplotypes DISCUSSION were obtained from the species of Rhynchocypris lagowskii (Fig. 4). Haplotype diversity (h) ranged from The present study successfully revealed using (0.0000±0.0000 to 1.0000 ± 0.0625) and nucleotide diversity mtDNA divergence, the major groups within (ð) ranged from (0.0000±0.0000 to 0.006289 ±0.004199) Rhynchocypris lagowskii based on specimens collected (Table 3). To see their gene genealogy, we constructed a from the entire distribution range of the species. phylogenetic network based on mtDNA cyt-b sequences Specifically, the populations in group 2 and group 3 (Fig. 5). The most parsimonious network of mtDNA showed remarkable differentiation from the group 1 haplotype of aburahaya 24 location, estimated using the population which is geographically separated from the TCS algorithm [11]. Total 54 haplotype are occurs in group 1 population by a geographical gap in distribution 24 location. This network showed three geographical from group 2 and group 3 populations. The result of this groups. Halpotype 1-29 is one group, haplotype 30-33 is study agreed with the findings of Matsuda et al. [12] group 2 and haplotype 34-54 is group 3. The neighbor- and Takehana et al. [13] who studied on Oryzias latipes. joining tree (NJ) of mtDNA haplotypes also showed They made a phylogenetic tree and found that similar topologies and consistently revealed three deeply population of one group was geographically separated diverged groups (Group 1-3) of Rhynchocypris lagowskii from others. (Fig. 2). To compare genetic diversity between all However the differentiation of the group 3 (0.8759 ± populations we calculated population genetic statistics. 0.0333) YAMA, TO, KUS, OB, MI and AM population No significant differentiation is found these samples from the other is much larger than this, indicating that the

(P< 0.05) (Table 2). And overall FST between three groups isolation of the species, populations between group 3 and ranged from (0.00000±0.84107); and between two group 1 has been sustained over a long period during geographical areas is (0.00000±0.19147) (Table 5). The which other lowland fish species maintained gene flowed Pacific Ocean area genetic diversity is (0.9278 ± 0.0113); between those regions. while the Japan Sea area is (0.7646± 0.0436) (Table 4). The group 2 (0.5333 ± 0.1801) is collected from the The nucleotide diversity highest the Pacific Ocean area KAM and HA populations. The distributions of group 2 (0.020688 ± 0.010608); and group 3 (0.013492 ± 0.007234); haplotype (Toyama Prefecture and Ishikawa Prefecture) the Japan Sea, group 1 and group 2 showed slightly lower suggest the phenomenon in which one of two closely nucleotide diversity than the Pacific Ocean and group located rivers taken the other river’s course as a result of 3 (0.009368 ± 0.005181, 0.004165± 0.002642 and 0.002077 ± river scramble. The group 1 halpotypes, on the other 0.001745respectively) (Table 4). As a result, three single hand, would have originated from regional populations geographic groups are formed in this study. different from those with the group 2 and 3.

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Fig. 4: Unrooted neighbor joining tree of Rhynchocypris lagowskii species of the cyt b region of mtDNA haplotypes with outgroup based on Kimura’s two-parameter distances. Numbers above branches are bootstrap values for 1000 replications. Blue pie-chart represent Group 1, red pie-chart represent Group 2 and yellow pie-chart represent Group 3 Here, Group 1: Tohoku, North Kanto, East Chube; Group 2: Ishikawa, Toyama and Group 3: Chube

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Fig. 5: The most parsimonious network of mtDNA haplotypes of Rhynchocypris lagowskii. Blue boxes represent Group 1, red boxes represent Group 2 and yellow boxes represent Group 3. Here, Group 1: Tohoku, North Kanto, East Chube; Group 2: Ishikawa, Toyama and Group 3: Chube

Some river containing two different populations The populations occurring in Pacific Ocean area were belonging to different geographic groups that reflect found to be high polyphyletic, some haplotypes in population access from one geographic group to another. populations in Kamishyou River (Toyama Prefecture) For example Mogami River system OSH and Nezugaseki and Hakui River (Ishikawa Prefecture) being closely River System NE shows two populations are same related to those found in continental populations. location. Aburahaya was released many years ago in Lake This might be evidence that two or more largely Biwa. The OSH and NE populations may have colonized differing genetic types had originally been through Lake Biwa. distributed in Japan Sea and Pacific Ocean area Genetic structure of populations of the species before the isolation of the former, or that the Rhynchocypris lagowskii should provide useful populations in the two area had been genetically information for quantifying isolation periods between intermingled with each other in two different geological various areas, such as Japan Sea and Pacific Ocean area. periods at least [14].

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Our analysis on cyt-b based populations reveals the ACKNOWLEDGEMENTS aburahaya’s demographic history. Each of the local populations had relatively high within–genetic diversity. We are indebted to the following persons who helped Similarly, largest divergence was found in easternmost us in collecting specimens: Natsuo Okabe, Hiroshi Aiki, populations (Isa Bay area and the east) of some cyprinids, Seiichi Muraki and Osamu Inaba. e.g. Biwia zezera [15] and Sarcocheilichthys variegates [16]. We found fifty-four haplotype distinct mtDNA REFERENCES sequences. The FST value the statistic representing between-population genetic diversity being consistent 1. Avise, J.C., 2000. Phylogeography: the history and with high population differentiation and low gene formation of species. Harvard University Press, flow [17]. Because the sampling sites of the local Cambrige. populations are in different river systems, it is unlikely 2. Asahida, T., T. Kobayashi, K. Saitoh and that these populations are frequently interbreeding. I. Nakayama, 1996. Tissue preservation and total In Japan irrigation channels make a huge network DNA extraction from fish stored at ambient beyond the river systems and inter population mating temperature using buffers containing high would be more frequently. However, the recent reduction concentration of urea. Fisheries, Sci., 62: 727-730. in the area covered by rice fields across Japan has 3. Sasaki, T., P. Yuri, Kartavtsev, N. Satoru, Chiba, probably isolated the local populations and limited Takayuki Uematsu, V. Valadimir, Sviridov and N. inter-population matching, further reducing gene flow Hanzawa, 2007. Genetic divergence and phylogenetic and leading to loss genetic variability by genetic drift in independence of Far Eastern species in subfamily each population. From the findings of the present study Leuciscinae (Pisces: Cyprinide) inferred from discontinuous genetic variation resulting in the mitochondrial DNA analyses. Genes Genet. Syst., occurrence of several different forms or types of 82: 329-340. individuals among the members of a single species 4. Tamura, K., J. Dudley, M. Nei and S. Kumar, 2007. was found and this discontinuous genetic variation MEGA4: Molecular Evolutionary Genetics divides the individuals of a population into two or Analysis (MEGA) software version 4.0 Mo. Biol., more sharply distinct forms. So the population of 24: 1596-1599. Aburahaya (Rhynchocypris lagowskii) was in highly 5. Thompson, J.D., D.G. Higgins and T.J. Gibson, 1994. polymorphism. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through CONCLUSIONS sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res., We clarified the unique aspects of the 22: 4673-4680. phylogeographic pattern of Rhynchocypris lagowskii 6. Excoffier, L., PE. Smouse and M. Quattro, 1992. compared with several group, regional populations and Analysis of molecular variance inferred from metric previously studied freshwater fish in Japan. The distances among DNA haplotypes: application to distribution processes of freshwater fishes are generally human mitochondril DNA restriction data. Genetics, thought to be affected by geographical factors, such as 131: 479-491. isolation by mountain uplift, range expansion following 7. Excoffier, L., G. Laval and S. Schneider, 2006. sea regression and isolation and reduction following ARLEQUIN ver. 3.01: An integrated Software sea-level rise. Simultaneously, the effects of such Package for population Genetics Data analysis. geographical factors are, at least partly, dependent on Computational and Molecular Population Genetics ecological traits of the species. In order to elucidate the Lab (CMPG), Institute of Zoology, University of population structure and development of the geographic Berne, Bern, Switzerland. distribution of Rhynchocypris lagowskii, further study is 8. Kimura, M., 1980. A simple method for estimating needed based on more extensive materials from evolutionary rates of base substitutions through populations throughout its overall distribution range, as comparative studies of nucleotide sequence. J. Mol. well as of other related species. Evol., 16: 111-120.

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