1. To manage biological invasions effectively, the impacts of alien species on the demography and traits of native species must be known, but determining those impacts can be challenging. We used a comparative approach to gain insight into the impacts that an alien toad (Bufo japonicus formosus) might have on native Japanese predatory amphibians. We compared the susceptibility of native predator species to alien toad toxins in the alien-invaded range and the susceptibility of closely related native predator species to the toxins in the alien toad's native range to investigate the impacts of an alien on a native species. Bufo japonicus formosus is native to Honshu, but was recently introduced toHokkaido and Sado. In laboratory experiments, we compared individual mortality of predators exposed to a toad hatchling between novel predators on the toad-invaded islands and ecologically similar congeneric or conspecific species on Honshu, where the toad is native. We also compared (1) the percentage of individuals that consumed a toad hatchling and (2) toxin resistance (i.e. survival and growth of individuals after toad consumption) between these two groups of predators, as mechanistic components behind the susceptibility of the predators to the toxic prey.3. The mortality of Rana pirica from all populations after consumption of a toad hatchling was almost 100%, and that of Hynobius retardatus ranged from 14 to 90%, depending on the population. In contrast, the mortality of Rana ornativentris and Hynobius nigrescens was near 0% regardless of population. These differences between congeneric predators were mostly due to differences in their toxin resistance.4. These results suggest that the alien toad is a potential threat to the novel amphibian predators on Hokkaido, although they also imply that the novel predators on Hokkaido have the potential to develop toxin resistance through adaptive evolution. However, this counteradaptation may have a higher chance of evolving in H. retardatus than in R. pirica because of differences in their genetic backgrounds. K E Y W O R D Sbiological invasion, ecological impact, toad venom, toxin resistance, translocated species | 241 OYAKE Et Al.

The sex chromosomes of most anuran amphibians are characterized by homomorphy in both sexes, and evolution to heteromorphy rarely occurs at the species or geographic population level. Here, we report sex chromosome heteromorphy in geographic populations of the Japanese Tago’s brown frog complex (2n = 26), comprising Rana sakuraii and R. tagoi. The sex chromosomes of R. sakuraii from the populations in western Japan were homomorphic in both sexes, whereas chromosome 7 from the populations in eastern Japan were heteromorphic in males. Chromosome 7 of R. tagoi, which is distributed close to R. sakuraii in eastern Japan, was highly similar in morphology to the Y chromosome of R. sakuraii. Based on this and on mitochondrial gene sequence analysis, we hypothesize that in the R. sakuraii populations from eastern Japan the XY heteromorphic sex chromosome system was established by the introduction of chromosome 7 from R. tagoi via interspecies hybridization. In contrast, chromosome 13 of R. tagoi from the 2 large islands in western Japan, Shikoku and Kyushu, showed a heteromorphic pattern of constitutive heterochromatin distribution in males, while this pattern was homomorphic in females. Our study reveals that sex chromosome heteromorphy evolved independently at the geographic lineage level in this species complex.

Background Biodiversity loss is a serious environmental problem, and human activities might be primarily responsible for the marked decline in animal populations globally. Amphibians, in particular, have significantly decreased in number in recent decades. One example is the endangered Yamato salamander (Hynobius vandenburghi), which is distributed in Central Japan and has a very restricted distribution in Gifu Prefecture. Aims We aimed to discover new populations of H. vandenburghi using a combination of GIS and environmental DNA (eDNA) analysis. Materials & Methods Firstly, we designed two primer sets for amplifying Hynobius species targeting mitochondrial 12S rRNA and cytochrome b genes. Next, we performed aquarium experiments to detect H. vandenburghi DNA in aquarium water. We also conducted sequential eDNA detection surveys in five known habitats in Gifu City, Japan, 17 times from January to August 2016. Following these basic eDNA studies, we used GIS to characterize the vegetation and topography of known habitats of H. vandenburghi. We collected water samples in the potential habitats identified by GIS and analyzed eDNA for the presence of H. vandenburghi using the designed primers. Finally, we conducted physical collection surveys in these potential habitats. Results We successfully developed two Hynobius‐specific primer sets and detected H. vandenburghi eDNA in aquarium water. The eDNA of the target species was detected in almost all (94%–100%) samples from four habitats, whereas only 29% of samples were positive for one habitat. We identified five potential habitats by GIS analysis. The DNA of H. vandenburghi was detected in three of five potential habitats. Finally, we discovered a new population in one of the potential sites. Discussion Our approach combining GIS and eDNA enabled the detection of novel population of an endangered amphibian species. This study was conducted by high school students under the supervision of teachers with the help of university researchers, suggesting the applicability of eDNA studies as a tool of citizen science. Conclusion The combination of GIS and eDNA will allow to detect cryptic populations on which conservation efforts may be focused and to alert people to the need for conservation action.

Toads of the Family Bufonidae possess neurotoxins (bufadienolides) which are generally considered to be synthesized de novo. Thus, invasive toad species pose a threat to native predators via toxic effects. However, the influence of diet on toad toxicity is poorly understood. We evaluated the effect of diet on toxicity of embryos of the invasive toad Bufo japonicus formosus in Japan, using native tadpoles (Rana pirica) as a bioassay. Specifically, we compared the toxicity of embryos spawned by captive toads reared entirely on non-toxic prey versus embryos spawned by wild toads. Embryos of captive-reared toads and wild toads were comparable in terms of toxic effects: all tadpoles that consumed a toad embryo died, irrespective of origin of embryos. Embryo toxicity appears to be via parental (likely maternal) provisioning. Our study indicates that adult B. j. formosus produce toxins (likely bufadienolides) in amounts sufficient for embryos to be fatal to native predator tadpoles irrespective of whether the adult diet contains toxins. Thus, the toxic effects of invasive B. j. formosus on R. pirica, and possibly on other sensitive native predator species, are likely to be widespread and occur regardless of variation in prey biota in invaded areas in Japan.

The present study reports spontaneous tyrosinase gene mutations identified in oculocutaneous albinos of three Japanese wild frog species, Pelophylax nigromaculatus, Glandirana rugosa and Fejervarya kawamurai. This represents the first molecular analyses of albinic phenotypes in frogs. Albinos of P. nigromaculatus collected from two different populations were found to suffer from frameshift mutations. These mutations were caused by the insertion of a thymine residue within each of exons 1 and 4, while albinos in a third population lacked three nucleotides encoding lysine in exon 1. Albinos from the former two P. nigromaculatus populations were also associated with splicing variants of mRNA that lacked either exons 2-4 or exon 4. In the other two frog species examined, missense mutations that resulted in amino acid substitutions from glycine to arginine and glycine to aspartic acid were identified in exons 1 and 3, respectively. The two glycines in F. kawamurai and G. rugosa, and the lysine deleted in one P. nigromaculatus albino, were highly conserved in vertebrates, which suggested that they were situated in regions of critical importance to tyrosinase function. In fact, the glycine of G. rugosa is located within a predicted copperbinding domain. The five mutations identified in the present study are candidates for causing the albinic phenotypes, and, if directly confirmed, they are all unique among vertebrates, which suggests that molecular analysis of albino frogs could contribute to research on albinos in humans and vertebrates by providing new information about tyrosinase structure and transcript processing.