Efficient eDNA-based assessment of mitochondrial lineage diversity in wild brown trout populations

Among the numerous applications of environmental DNA (eDNA), the assessment of genetic diversity in wild populations of target taxa remains moderately investigated. Focusing on 10 selected sites inhabited by brown trout (Salmo trutta complex) in central Italian watercourses, we: (1) developed a non-invasive genetic monitoring protocol combining environmental DNA sampling, amplicon sequencing with custom-designed primers targeting a 192 bp fragment of the mitochondrial Control Region (CR) - whose evolutionary lineages are informative for brown trout phylogeography and genetic conservation status-and a curated reference database; (2) validate it comparing CR lineages assemblages revealed by eDNA with those obtained through traditional CR genotyping of trouts collected by electrofishing in recent surveys, while accounting for uncertainty associated with sample size of genotyped individuals. The two methods returned strongly correlated per-site raw frequencies of CR lineages (Pearson r = 0.93, p < 0.0001), and remarkably similar lineage assemblages (Mantel r = 0.832, p = 0.0001). Concordances between (semi-quantitative) lineage assemblages retrieved by the two methods increased at increasing trout densities and decreased at warmer water temperatures. Discordances mostly involved low-frequency lineages that were not detected by traditional CR genotyping. This study supports the effectiveness of eDNA for reliably characterising mitochondrial intra-population diversity in brown trout, a widely distributed and actively managed taxon of important conservation and economic value, offering a valuable tool to assist the genetic monitoring of wild populations, including the identification and quantification of exotic lineages.