Monitoring within-species genetic diversity of amphibians with eDNA metabarcoding

Ongoing habitat alterations and climate change can lead to a dramatic decline in local populations, and cost-efficient tools to rapidly estimate population genetic diversity in space and time are urgently needed. Here we used the common frog Rana temporaria (Linnaeus, 1758) from 10 wetland sites in the Italian Alps to: i) develop and test a standardized eDNA metabarcoding protocol for the monitoring of within-species genetic diversity (targeting a short fragment of the COI barcode region); and ii) critically compare the results of eDNA metabarcoding with those obtained from traditional genetic sampling. Our results showed that a single temporal sampling replicate performed after spawning (and when egg clutches and/or larval stages are still present in the water) is sufficient to successfully characterize haplotype richness, but two temporal replicates are needed to obtain more accurate information on haplotype frequencies. We then demonstrated that standard genetic variability estimates (haplotype and nucleotide diversity) derived from eDNA metabarcoding are strongly correlated with those derived from traditional genetic data. Similarly, we also found a moderate but yet significant correlation between the population structures inferred from the two considered methods. Thus, our protocol proved to be a fast and effective tool that could be used for the establishment of a surveillance network, with the ultimate goal of monitoring temporal trends in the genetic diversity of pond-breeding amphibians.