Hybridization between an endangered freshwater fish and an introduced congeneric species and consequent genetic introgression.

Artificial transplantation of organisms and consequent invasive hybridization can lead to the extinction of native species. In Matsuyama, Japan, a native bitterling fish, Tanakia lanceolata, is known to form hybrids with another bitterling species, T. limbata, which was recently introduced from western Kyushu, Japan. These bitterlings spawn in the gills of two freshwater unionid species, Pronodularia japanensis and Nodularia douglasiae nipponensis, which have rapidly declined on the Matsuyama Plain in the past 30 years. To gauge the effect of invasive hybridization, we determined the genetic introgression between T. lanceolata and T. limbata and analyzed the morphology of these species and their hybrids to infer their niche overlap. We collected adult individuals of Tanakia spp. and genotyped them based on six microsatellite loci and mitochondrial cytochrome b sequences. We analyzed their meristic characters and body shapes by geometric morphometrics. We found that 10.9% of all individuals collected were hybrids. Whereas T. lanceolata were more densely distributed downstream and T. limbata were distributed upstream, their hybrids were widely distributed, covering the entire range of native T. lanceolata. The body height and anal fin length of T. limbata were greater than those of T. lanceolata, but their hybrids were highly morphologically variable, covering both parental morphs, and were widely distributed in the habitats of both parental species. Hybridization has occurred in both directions, but introduced T. limbata females and native T. lanceolata males are more likely to have crossed. This study shows that invasive hybridization with the introduced T. limbata is a potential threat to the native population of T. lanceolata via genetic introgression and replacement of its niche in streams.

Hybridization between two bitterling fish species in their sympatric range and a river where one species is native and the other is introduced.

The distributions of two bitterling fish (subfamily: Acheilognathinae), Tanakia lanceolata and T. limbata, overlap in western Japan. Acheilognathinae fish lay their eggs in the gills of freshwater bivalves, and the early juvenile stage develops in the gills. Populations of freshwater bivalves are declining worldwide, which has limited the number of spawning substrate for bitterlings. T. limbata has been artificially introduced to some rivers in Ehime, Japan, where it coexists with native T. lanceolata, and some hybrids have been observed. We collected both species from several sites in western Japan, and from the Kunichi River system in Ehime, and analyzed genetic population structure based on six microsatellite loci and sequences of the mitochondrial cytochrome b gene. Structure analysis identified three genetically distinct populations: T. lanceolata, T. limbata "West Kyushu", and T. limbata "Setouchi". Two clades of T. limbata were also supported by molecular phylogenetic analyses based on cytochrome b. Hybrids in Ehime originated mostly from interbreeding between male T. lanceolata and female T. limbata "West Kyushu", and made up 10.2% of all collected fish, suggesting that hybrids occurred frequently between females of colonizing species and males of native species. On the other hand, interspecific hybrids were detected at rates of 40.0%, 20.0%, and 17.6% in the Ima River (Fukuoka), Midori River (Kumamoto), and Kase River (Saga), respectively, which are naturally sympatric regions. We found a few T. limbata "Setouchi" in the Midori and Kase Rivers, which were supposed to be introduced from other regions, coexisting with native T. limbata "West Kyushu", and this cryptic invasion may have triggered the interspecific hybridization. These results suggest that artificial introduction of a fish species, a decline in the unionid population, and degradation of habitat have caused broad hybridization of bitterlings in western Japan.