Seasonal monitoring of Hida salamander Hynobius kimurae using environmental DNA with a genus-specific primer set

Abstract

The diversity and the abundance of amphibians have dramatically declined globally over the past 30 years, and the monitoring and conservation of their habitats is essential. However, traditional methods such as bait trapping and mark-recapture are costly, and morphological identification usually requires a high level of taxonomic expertise. Here, seasonal surveillances of Hida salamander Hynobius kimurae were performed by means of environmental DNA (eDNA) analysis with Hynobius-specific primers and a species-specific TaqMan probe. Water sampling and visual surveys were conducted seasonally in a stream in Kyoto Prefecture, Japan. Detection rates of eDNA were then calculated by real-time PCR, and eDNA site occupancy probability was estimated by multi-scale occupancy modeling. The eDNA-based detection rate of Hida salamander was 76.7%, whereas the visual survey-based detection rate was 23.3%, and target eDNA was detected at almost all sites where the presence of target species was visually confirmed. Moreover, factors relating to the site- and sample-level occurrence probabilities of the target eDNA differed depending on the developmental stage of the target species. Our findings support previous studies showing that eDNA analysis enables an effective assessment of amphibian distributions without damaging the organisms or their habitat, and we compare for the first time the site occupancy probability of amphibian eDNA throughout the life cycle of an amphibian species. The present study contributes to the development of eDNA analysis as a tool for understanding the distribution and seasonal activity of amphibian species and will thus aid in the planning of conservation measures and habitat restoration for these species.