Quantitative evaluation of intraspecific genetic diversity in a natural fish population using environmental DNA analysis.

Recent advances in environmental DNA (eDNA) analysis using high-throughput sequencing (HTS) enable evaluation of intraspecific genetic diversity in a population. As the intraspecific genetic diversity provides invaluable information for wildlife conservation and management, there is an increasing demand to apply eDNA analysis to population genetics and the phylogeography by quantitative evaluation of intraspecific diversity. However, quantitative evaluations of intraspecific genetic diversity using eDNA is not straightforward because the number of eDNA sequence reads obtained by HTS may not be an index of the quantity of eDNA. In this study, to quantitatively evaluate genetic diversity using eDNA analysis, we applied a quantitative eDNA metabarcoding method using the internal standard DNAs. We targeted Ayu (Plecoglossus altivelis altivelis) and added internal standard DNAs with known copy numbers to each eDNA sample obtained from three rivers during the library preparation process. The sequence reads of each Ayu haplotype were successfully converted to DNA copy numbers based on the relationship between the copy numbers and sequence reads of the internal standard DNAs. In all rivers, the calculated copy number of each haplotype showed a significant positive correlation with the haplotype frequency estimated by a capture-based survey. Furthermore, estimates of genetic indicators such as nucleotide diversity based on the eDNA copy numbers were comparable with those estimated based on a capture-based study. Our results demonstrate that eDNA analysis with internal standard DNAs enables reasonable quantification of intraspecific genetic diversity, and this method could thus be a promising tool in the field of population genetics and phylogeography.