Genotyping of two congeneric bitterling fish species by nuclear SNP markers and the detection of hybridization in a sympatric region

Bitterling fish species (subfamily: Acheilognathinae) are endangered in Japan due to habitat loss. The decline of freshwater mussels also enhances the decrease of bitterlings because bitterlings lay eggs in the gills of freshwater mussels. Furthermore, loss of unionid mussels causes hybridization between bitterling species. This study aims to analyze the frequency of hybridization between congeneric species of bitterlings-Tanakia lanceolata and Tanakia limbata-at a well-preserved site, where both species occur naturally and compare this with other regions more severely impacted by human intervention. One part of the irrigation streams of the Asahi River System, Okayama, is inhabited by a natural monument species of Japan, a benthic cobitid, Parabotia curtus, and therefore, the natural substratum and the structurally heterogeneous stream banks are maintained. We collected Tanakia individuals and surveyed mussel density at this well-preserved site. We also developed six nuclear single-nucleotide polymorphism (SNP) markers on different chromosomes to distinguish T. lanceolata and T. limbata. These SNP markers effectively determined individuals as being either one of these species or a hybrid. Based on genotyping, eight individuals were determined to be hybrids, whereas 90 and 173 individuals were determined as purebreds of T. lanceolata and T. limbata, respectively. The proportion of hybrid individuals was 3.0% and was relatively low compared to other regions. In addition, five species of unionids were densely distributed in this stream. Prezygotic isolation between sympatric T. lanceolata and T. limbata was established at this well-preserved site, where unionid mussels are densely populated and provide enough breeding substrate for both species.