Population genetic structure and hybrid zone analyses for species delimitation in the Japanese toad (Bufo japonicus)

Hybridization following secondary contact may produce different outcomes depending on the extent to which genetic diversity and reproductive barriers have accumulated during isolation. The Japanese toad, Bufo japonicus, is distributed on the main islands of Japan. In the present study, we applied multiplexed inter-simple sequence repeat genotyping by sequencing to achieve the fine-scale resolution of the genetic cluster in B. j. japonicus and B. j. formosus. We also elucidated hybridization patterns and gene flow degrees across contact zones between the clusters identified. Using SNP data, we found four genetic clusters in B. j. japonicus and B. j. formosus and three contact zones of the cluster pairs among these four clusters. The two oldest diverged lineages, B. j. japonicus and B. j. formosus, formed a narrow contact zone consistent with species distinctiveness. Therefore, we recommend that these two subspecies be elevated to the species level. In contrast, the less diverged pairs of two clusters in B. j. japonicus and B. j. formosus, respectively, admixed over a hundred kilometers, suggesting that they have not yet developed strong reproductive isolation and need to be treated as conspecifics. These results will contribute to resolving taxonomic confusion in Japanese toads.