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, Japan Abstract: Analyses of partial mitochondrial DNA (mtDNA) sequences support the classification of Aburahaya (Rhynchocypris lagowskii) from the Sea of Japan 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). Mogami river 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. 1080 World Appl. Sci. J., 33 (7): 1079-1088, 2015 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 Omono River 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 Agano River 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 Abukuma River 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 Kuji River Oshi River OS Pacific Ocean 2012.05.20 11 17 Naka River Kuro River KUR Pacific Ocean 2013.04.06 7 18 Tone River Yamada River YAM Pacific Ocean 2012.04.29 8 19 Tone River Karasu River KA Pacific Ocean 2012.04.15 3 20 Ara River Toki River TO Pacific Ocean 2012.03.31 8 21 Sagami River Kushi River KUS Pacific Ocean 2013.02.09 8 22 Kano River Ooba River OB Pacific Ocean 2012.12.31 7 23 Tenryu River Miya River MI Pacific Ocean 2012.11.24 8 24 Yodo River Amano River AM Pacific Ocean 2013.08.16 8 Table 2: Pair