Development and validation of qpcr assays for use in edna detection of southern two-lined (eurycea cirrigera) and northern dusky (desmognathus fuscus) salamanders

Environmental DNA (eDNA) uses DNA shed from organisms into the environment to detect their presence and provides an effective, non-invasive method to survey organisms in an efficient manner. Recent works have emphasized the need for careful development and both laboratory and field validation of eDNA assays. We developed species specific qPCR assays for Southern Two-lined (Eurycea cirrigera) and Northern Dusky (Desmognathus fuscus) salamanders, two salamanders found throughout much of the eastern USA. We designed primers and probes based on sequences obtained from locally collected specimens and screened for specificity against 19 salamander species that occur sympatrically with these species in various parts of their range. We collected 38 water samples from streams in a minimally disturbed forest in eastern Kentucky, USA, and we analyzed samples for both E. cirrigera and D. fuser's DNA. There were 16 samples that were positive for E. cirrigera and 21 for D. fuscus DNA. We cloned and sequenced four E. cirrigera and five D. fuscus amplicons from filtered water to verify identity. These data add to the growing pool of knowledge concerning eDNA monitoring of salamander species and provide useful reference data as well as valuable molecular tools for future monitoring and range delineation studies.