Clarifying genetic relationships and intraspecific variation of lentic-breeding Japanese salamanders (Amphibia, Caudata, Hynobiidae) based on mitochondrial DNA and genome-wide SNP

Clarifying taxonomic status of species can be crucial for understanding biodiversity and informing conservation efforts. This study focused on Hynobius setouchi, H. vandenburghi, and H. owariensis, which are lentic breeding salamander species endemic to Japan and which were split or resurrected recently from populations previously recognized as H. nebulosus. Since the three species are closely related and occupy geographically adjacent ranges, interspecific hybridization may have occurred. However, detailed investigations have yet to be conducted, and the genetic distinctiveness of the three species is unclear near the boundaries of their ranges. Regarding intraspecific variation, H. setouchi consisted of highly diverged lineages based on mitochondrial DNA (mtDNA), but the genetic relationships of intraspecific variations have not been evaluated. This study aimed to clarify the genetic relationships of the three species and intraspecific variations by genetic approach. We performed phylogenetic and population structure analyses based on mtDNA and genome-wide single nucleotide polymorphisms. Genetic analyses resulted in clades in each species and few interspecific genetic introgressions. Regarding intraspecific variability, H. setouchi showed gene flow between the lineages and indicated the absence of strong reproductive isolation between intraspecific populations. Past geographic events and dynamics of large rivers would have promoted geographical isolation and speciation of these salamanders. Hynobius setouchi, in particular, would have formed complex population structures due to repeated transgression and regression of the Seto Inland Sea.