Genetic Relationships of Oshorokoma Charr (Salvelinus malma kraschennikovi), Distributed in Hokkaido, Japan

Genetic diversities and relationships were examined among ‘Oshorokoma’ charr, Salvelinus malma krascheninnikovi, distributed in the rivers flowing into the Sea of Japan, the Okhotsk Sea, Nemuro Strait and the Pacific Ocean, in Hokkaido, Japan, based on DNA sequences of the cytochrome b region of mitochondrial DNA. A total of 6 haplotypes was recognized. There were 5 polymorphic sites in a 500 bp fragment. Oshorokoma was still more distant from S. leucomaenis than from S. alpinus or S. fontinalis in the genetic tree. Haplotype 2, distributed only in the Churui River, was estimated to be the oldest lineage. Haplotype 5 was the most dominant and was not distributed in the rivers flowing into Nemuro Strait. The average genetic distance was 0.003 for the Yubetsu River whereas it was only 0.001 for the Tokachi and Ishikari Rivers. Besides, it was only 0.003 between the rivers originating from the Taisetsu and Shari mountain masses. There was a significant positive correlation between geographical and genetic distances. These results suggest a westward invasion of an ancestral Oshorokoma population, a high genetic diversity in some populations, and a low genetic differentiation among populations. Further studies, including a survey in the whole of Hokkaido, and also including Rishiri and Rebun Islands, are considered to be necessary in the future. et al. (1998) suggested a sister relationship between S. malma and S. alpinus based on analysis of FokIfamily of short interspersed repetitive elements (SINEs). Oleinik et al. (2014) examined 27 Alaskan and Asian populations of S. malma malma based on mtDNA sequence, recorded 76 haplotypes in 436 fish, and reported weak spatial differences and low levels of divergence. Based on a genetic research of Oshorokoma in 11 rivers in Hokkaido, genetic differences are high among the populations whereas it is extremely low within the population (Saito, 2004). If they remain in rivers, there may be some genetic separations among the populations during every interglacial period. However, genetic structures and relationships of Oshorokoma in 8 Koichiro Kawai & Hidetoshi Saito Hokkaido still remain to be clarified. In this study, Oshorokoma samples were collected from some rivers flowing into different seas, determined for the DNA sequences in a mitochondrial region, genetic distance was calculated between individuals, and a genetic tree was constructed. Further, genetic distances and relationships were discussed in relation to geographical viewpoints. Materials and methods Samples Charr samples were collected at 14 sites in 11 rivers flowing into the Sea of Japan, the Ohotsuk Sea, the Nemuro Strait and the Pacific Ocean (Fig. 1). Sampling was performed by fishing using earthworm as a main bait. Samples were transported to the laboratory as a live form using a potable aeration system. After killing by bleeding, samples were measured for body size. Fin was cut, liver was dissected out and both stored in Eppendorf tubes at -30°C until use. PCR Template DNA was prepared from the samples using DNeasy Tissue Kit (Qiagen, Tokyo, Japan), according to the manufacturer’s instruction. The cytochrome b region of mitochondrial DNA was partially amplified by PCR with a mixture of a template DNA (50 ng) and primers H15915 (5’-ACCTCCGATCTYCGGATTACAAGAC-3’; Aoyama et al., 2000) and L15285 (5’-CCCTAACCGGVTTCTTYGC-3’; Inoue et al., 2000) by using the TaKaRa PCR Amplification kit (TaKaRa, Ohtsu, Japan) in a thermal cycler (Mastercycler personal; Eppendorf, Hamburg, Germany) using the following protocol: preheating at 94°C for 11 min, followed by 30 cycles of denaturation at 94°C for 30 s→annealing at 55°C for 30 s→extension at 72°C for 1 min and a final extension at 72°C for 7 min. PCR products was purified using NucleoSpin Gel and PCR cleanup (Takara, Ohtsu, Japan). Sequencing was performed directly with the Genetic Analyzer 3130xl (Applied Biosystem, CA, USA) in the Genetic Research Center of Hiroshima University. Genetic distance Genetic distance was determined as TajimaNei-patameter using MEGA 6. 142°E 143°E 144°E