Aquatic eDNA can advance monitoring of a small‐bodied terrestrial salamander and amphibian pathogen

Abstract Successful conservation efforts for threatened species depend on accurate characterization of their distribution, habitat use, and threats. However, surveillance can be challenging for species with small size, cryptic coloring, or elusive behavior. Environmental DNA (eDNA) monitoring can provide a sensitive and noninvasive alternative to traditional surveillance techniques by detecting trace DNA shed by a target species into their environment. We evaluated the efficacy of eDNA monitoring for the four‐toed salamander (Hemidactylium scutatum), a terrestrial salamander threatened throughout much of its range. Additionally, we integrated eDNA monitoring for ranavirus, a widespread pathogen of ectothermic vertebrates, to efficiently evaluate a potential disease threat to H. scutatum. We designed a novel species‐specific quantitative PCR (qPCR) assay for H. scutatum, multiplexed the assay with a previously developed ranavirus qPCR assay, and validated the multiplexed tests in silico, in vitro, and in situ. We collected aquatic eDNA weekly for 8 weeks from pools with historical reports of H. scutatum (n = 6) and contemporary reports of H. scutatum on associated uplands (n = 4). We identified H. scutatum eDNA at low quantities (< 12 copies/reaction) on multiple sampling days in two pools with historic species presence. Ranavirus eDNA was widespread; nine pools tested positive including the pools where H. scutatum eDNA was detected. Ranavirus eDNA quantities increased, peaked, then decreased (i.e., were not sporadic in time) in several pools but never rose in abundance to levels associated with ranavirosis in other studies. Our results suggest that H. scutatum eDNA is distributed sporadically at low quantities in pools; we recommend that future surveillance efforts prioritize increased frequency and quantity of sample collection per site visit and include repeat surveys of candidate sites. Further work to characterize Ranavirus eDNA movement, temporal trends, relationship to infectious virion, and decay is needed to help advance this tool.