Evaluating amphibian community composition in the southeastern U.S. using eDNA metabarcoding

Metabarcoding utilizes environmental DNA (eDNA) shed by living organisms to allow for the detection of many related species at once from an environmental substrate such as soil or water. We explored the tools required for amphibian metabarcoding within the southeast US and implemented a comparative study that examined the utility of metabarcoding in lieu of traditional visual encounter surveys and the ability of the metabarcoding strategy to detect temporal variation in the composition of pond-breeding amphibian assemblages. We tested a previously developed mitochondrial ribosomal RNA gene assay (12S rRNA) for the ability to detect and discriminate among southeastern amphibian species. We successfully detected 11 amphibian taxa either to the genus or species level at our study sites with the 12S assay, indicating the potential to adequately describe amphibian assemblages across the southeastern U.S. However, a lack of a comprehensive 12S gene sequence reference database for all southeastern amphibians and the inability of the 12S assay to distinguish between certain taxa at the species level indicates caution should be used in implementing this strategy for important conservation and management purposes. In our study, the combination of both visual encounter surveys and metabarcoding yielded the highest levels of detection. Our results suggest that the development of a reliable amphibian metabarcoding strategy will improve our ability to efficiently and rapidly assess amphibian assemblages at ephemeral pools compared to traditional methods.