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The Quaternary climate affected the present species richness and geographic distribution patterns of amphibians by limiting their activities during the glacial period. The present study examined the phylogenetic relationships of Japanese toads (

The genus Rhabdias StilesHassall, 1905 comprises lung parasites of amphibians and reptiles worldwide. In Japan, 9 species have been recorded, including Rhabdias incerta Wilkie, 1930 which has been reported only in Bufo species. In this study, to assess the diversity of R. incerta, we performed molecular analyses of Rhabdias species sampled from three species/subspecies of Japanese toads namely Bufo japonicus, B. japonicus formosus, and B. torrenticola, collected in various regions of Honshu, Shikoku, and Kyushu, Japan. DNA sequence divergence was compared using mtDNA (COI) and nuclear DNA (28S) to identify possible cryptic species. Morphological analysis was performed through light microscopy and scanning electron microscopy (SEM). The results revealed that Bufo spp. serve as hosts for at least three Rhabdias species. Morphologically, most samples were identified as R. incerta but with a longer body and larger buccal cavity than originally described. Rhabdias incerta appears to be specific to the genus Bufo and is further subdivided into two or three phylogroups based on subspecies divisions and biogeography of their host. Some Rhabdias specimens collected in this study resemble R. tokyoensis Wilkie, 1930, parasitic in hosts from the order Caudata, which suggests host switching. Both molecular and morphological analyses suggested the presence of undescribed and cryptic Rhabdias species within toads collected in Japan. This study was the first to molecularly characterize Rhabdias species in Japan, including novel sequences of R. incerta and two undescribed species.

The Quaternary climate affected the present species richness and geographic distribution patterns of amphibians by limiting their activities during the glacial period. The present study examined the phylogenetic relationships of Japanese toads (Bufo japonicus and B. torrenticola) and the demography of each lineage from the past to the present based on mitochondrial sequences and ecological niche models. Japanese toads are a monophyletic group with two main clades (clades A and B). Clade A represents B. j. formosus, including three clades (clades A1, A2, and A3). Clade B contains three clades, two of which corresponded to B. j. japonicus (clades B1 and B2) and the other to B. torrenticola. Clade B2 and B. torrenticola made a sister group, and, thus, B. j. japonicus is paraphyletic. Clades A and B diverged in the late Miocene 5.7 million years ago (Mya) during the period when the Japanese archipelago was constructed. The earliest divergence between the three clades of clade A was estimated at 1.8 Mya. Clades A1 and A2 may have diverged at 0.8 Mya, resulting from the isolation in the multiple different refugia; however, the effects of the glacial climate on the divergence events of clade A3 are unclear. Divergences within clade B occurred from the late Pliocene to the early Pleistocene (3.2–2.2 Mya). Niche similarity between the parapatric clade in clade B (clades B1 and B2) indicated their allopatric divergence. It was suggested that niche segregation between B. japonicus and B. torrenticola contributed to a rapid adaptation of B. torrenticola for lotic breeding. All clade of Japanese toads retreated to each refugium at a low elevation in the glacial period, and effective population sizes increased to construct the current populations after the Last Glacial Maximum. Furthermore, we highlight the areas of climate stability from the last glacial maximum to the present that have served as the refugia of Japanese toads and, thus, affected their present distribution patterns.