Ichthyofaunal Metabarcoding in the Southern Appalachians: Use of eDNA Metabarcoding in Fish Surveys in Lotic Systems of the Great Smoky Mountains National Park with Comparisons to Historic Electrofishing Data

eDNA appears well positioned to play a significant role in the future of biomonitoring, and the need to assess the efficacy of eDNA-based surveys in a variety of habitats is increasing. We conducted an eDNA metabarcoding-based survey of fish communities in the Great Smoky Mountains National Park (GSMNP), located in eastern Tennessee and western North Carolina. The GSMNP, widely recognized as a biodiversity hotspot, encompasses 211,419 hectares of the Southern Appalachian Mountains with elevations up to 2205 meters and is home to approximately 73 species of fish, including 12 families and three species classified as endangered or threatened. We collected 50 water samples in first to sixth order streams at elevations of 336 to 1462 meters, including all major watersheds found in the park. eDNA was amplified utilizing two primer sets which each target differing regions of the 12S mitochondrial gene and generate amplicons of varying size (97 and 225 bp, respectively), and sequencing was conducted to an expected read depth of 400,000 reads per sample per marker. We detected a total of 40 fish species; of these, 36 were detected with the primer set which produces a 97 bp amplicon, and 12 of these 36 were detected only by this primer set. Species assemblages varied between stream orders, and species richness decreased with increasing elevation and increased with increasing stream order. Significant correlations were observed between biomass data from electrofishing monitoring (1984–2023) and eDNA metabarcoding read counts in five of seven species examined, including all salmonids. eDNA metabarcoding was demonstrated to be effective in assessing fish communities in high-elevation lotic systems in the Southern Appalachians, and our results suggest that primers targeting shorter amplicons may exhibit greater efficacy in these ecosystems.