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 siteand 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.

中文翻译：

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使用环境 DNA 和属特异性引物对 Hida 蝾螈 Hynobius kimurae 的季节性监测

在过去的 30 年中，全球两栖动物的多样性和丰度急剧下降，对其栖息地的监测和保护至关重要。然而，诱饵诱捕和标记重新捕获等传统方法成本高昂，形态识别通常需要高水平的分类学专业知识。在这里，Hida 蝾螈 Hynobius kimurae 的季节性监测是通过环境 DNA (eDNA) 分析与 Hynobius 特异性引物和物种特异性 TaqMan 探针进行的。在日本京都府的一条溪流中季节性地进行水采样和目视调查。然后通过实时 PCR 计算 eDNA 的检测率，并通过多尺度占用模型估计 eDNA 位点占用概率。飞騨的eDNA检测率为76.7%，而基于视觉调查的检测率为 23.3%，并且几乎在所有目视确认存在目标物种的地点都检测到了目标 eDNA。此外，与目标 eDNA 的位点和样本水平发生概率相关的因素因目标物种的发育阶段而异。我们的研究结果支持先前的研究，表明 eDNA 分析能够在不损害生物或其栖息地的情况下有效评估两栖动物的分布，并且我们首次比较了两栖动物物种整个生命周期中两栖动物 eDNA 的场地占用概率。