1. Population genetics is a powerful tool for studying evolutionary processes and informing conservation biology. Traditional approaches typically rely on tissue sampling, which poses challenges in aquatic environments where specimen collection is often difficult. Recent efforts to conduct non-invasive studies using environmental DNA (eDNA) have shown promise but face limitations. For instance, the use of a limited number of markers or the unintended capture of DNA fragments from off-target species can hinder their effectiveness for population genetic studies.

2. As a proof of concept of species-specific genome-wide nuclear loci detection from eDNA, we targeted the common frog, Rana temporaria, using a comparative experimental design. Field eDNA replicates were collected from four Alpine ponds, alongside tissue samples from 20 live tadpoles per pond. These tadpoles were also placed in separate tanks to collect eDNA under controlled conditions. Frog DNA from a subset of individuals was analysed through ddRAD genotyping and subsequently used to design custom probes for capturing frog nuclear loci from eDNA samples and individually collected tissues, using a modified hyRAD protocol.

3. Although we observed substantial heterogeneity in the genetic data retrieved across replicates, the number of nuclear loci recovered in eDNA samples using hyRAD was in line with those obtained through classical ddRAD, proving sufficient to investigate genetic variation. We identified a total of 17,617 nuclear single nuclear polymorphisms shared across individual, pond eDNA and tank eDNA samples, enabling us to detect genetic structuring across sampling locations, consistent with individual-based estimates.

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