Optimization of wetland environmental DNA metabarcoding protocols for Great Lakes region herpetofauna

Many species of reptiles and amphibians (herpetofauna) rely on wetlands that are being degraded and lost at a high rate. Characterization of herpetofauna diversity in different wetland types may help guide conservation strategies. However, traditional survey methods often involve sampling within small temporal windows and gear deployed may be taxonomically biased, thus, they may fail to accurately characterize species presence/absence and diversity. In contrast, environmental (e)DNA metabarcoding has been shown to effectively survey entire aquatic communities and can provide a useful complement to traditional surveys. The objective of this study was to design and optimize eDNA sampling and laboratory protocols for wetland herpetofauna. Protocols evaluated included different water sampling approaches (point versus transect sampling), seasonality of sampling, and choice of metabarcoding marker (mitochondrial 12S versus 16S rDNA). Samples collected from 10 sites across southern Michigan detected 17 amphibian and five reptile species, including four species of conservation concern (Ambystoma texanum, Clemmys guttata, Rana palustris, and Sternotherus odoratus). We observed no difference in the number of species detected between point and transect samples (p = 0.70), but point sampling required less time (p = 0.03) and allowed significantly larger volumes of water to be filtered (p = 1.13e-5). No difference in species richness was observed between the 12S and 16S mitochondrial DNA markers (p = 0.96). However, a greater number of taxa were identifiable at the species level when using the 16S locus. There was also a significant difference in the number of species detected between early and late summer sampling periods (more species detected in the earlier period; p = 6.31e-6), and some species were only found in the early or late sampling period. We recommend sampling during multiple periods to fully characterize species composition, the use of point sampling, and the 16S mtDNA marker for herpetofauna eDNA metabarcoding studies.