Development and optimization of a novel environmental DNA‐based method for moor frog (Rana arvalis) monitoring and comparison with a commercial detection kit

Abstract Moor frogs (Rana arvalis) are protected by the European Union's Habitats Directive, often making them a target for environmental impact assessments. However, moor frog detection with the traditional method using their mating calls is difficult, time‐consuming, and limited to the short mating season. Environmental DNA (eDNA)‐based methods could be beneficial in moor frog detection since moor frogs are shy and co‐occurring common frogs (R. temporaria) are morphologically similar. Additionally, eDNA‐based methods could even extend the detection period of moor frogs. We tested two different DNA isolation methods and two different eDNA detection methods, Sanger sequencing and quantitative PCR (qPCR), and compared the results to the traditional mating call survey method. In addition, we tested whether linear polyacrylamide treatment would improve eDNA detection. We sampled 27 sites in Finland, of which moor frogs were detected in 17 sites by traditional acoustic survey method, in 20 sites with Sanger sequencing‐based eDNA method, and in 21 sites with qPCR‐based eDNA method. We sampled water bodies during and 3–4 weeks after the end of the mating season. Our results show that both tested eDNA methods (Sanger sequencing and qPCR) can be used to detect moor frogs and extend the moor frog detection period by at least 3–4 weeks, but qPCR slightly outperforms Sanger sequencing. Linear polyacrylamide treatment did not improve the results. Detection probabilities were similar or higher with the eDNA‐based methods than with the traditional survey method, as moor frogs were also detected in water bodies in which mating calls were not heard. Based on our results, eDNA‐based detection methods can be highly beneficial in monitoring the presence of moor frogs and can be used to complement traditional surveys.