Discovery of an unrecorded population of Yamato salamander (<i>Hynobius vandenburghi</i>) by GIS and eDNA analysis

Abstract 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 &amp; 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.

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