Large-scale hybridization of Japanese populations of Hinamoroko,Aphyocypris chinensis, withA. kikuchiiintroduced from Taiwan

Aphyocypris chinensisis a small cyprinid that is widely distributed in lowland areas of continental China, the Korean Peninsula, and the northwestern part of Kyushu, Japan. However, this species is severely threatened in Japan due to human impacts; thus, several facilities and citizen groups implemented captive breeding and reintroduction/reinforcement programs using several dozen founder fish collected from Tanushimaru, the location with the only known natural population, in the 1980s and 1990s. To determine the phylogenetic position and genetic authenticity of Japanese populations ofA. chinensis, we conducted mtDNA and genome-wide SNP analyses using a total of 254 specimens from 31 wild and captive samples, including specimens of Chinese and KoreanA. chinensisand the close relativeA. kikuchiifrom Taiwan. The mtDNA divergence and phylogeny indicated that the haplotypes found exclusively in Japan were differentiated from the Chinese/Korean haplotypes (uncorrectedp = 2.6% in the cytochromebgene) to the same extent as they were fromA. kikuchiihaplotypes (p = 2.9%). Results from mtDNA sequences and 47-359 SNPs, obtained using the MIG-seq method with different parameter sets, revealed that the initial captive populations and an extinct wild population in Ukiha, adjacent to Tanushimaru, were genuine Japanese populations, whereas all extant captive and wild populations in Japan are hybrids between JapaneseA. chinensisandA. kikuchii, orA. kikuchiiitself. The details of the captive breeding and exchange programs, as well as evidence for the aquarium trade ofA. kikuchiisince the 1990s, strongly suggest that the mixture ofA. kikuchiiinto Japanese populations occurred within the first several years of the establishment of captive populations in 1994. The present case of the highly probable extinction of genuine Japanese populations ofA. chinensisemphasizes the importance of confirmation and management of the genetic authenticity of conservation-targeted species.