Detecting fossorial salamanders using eDNA: Development and validation of quantitative and end-point PCR assays for the detection of five species of Ambystoma

In the past decade, quantification of environmental DNA (eDNA) has become firmly established as an effective method for detecting the presence of organisms of research and conservation interest. Salamanders of the family Ambystomatidae are large, fossorial species; adults are rarely encountered above ground outside of their brief reproductive season. Larvae develop rapidly, in ephemeral pools or streams, often with multiple species coexisting in a single habitat. We developed species-specific PCR assays for Ambystoma barbouri , Ambystoma jeffersonianum , Ambystoma opacum , Ambystoma maculatum , and Ambystoma tigrinum based on locally sequenced specimens and tested each in silico and in vitro as well as conducted field surveys, using both end-point and quantitative PCR to test all in situ except A. tigrinum . In silico and in vitro tests confirm specificity of each primer. In situ larvae surveys were conducted and water samples collected from known Ambystoma breeding sites in central and eastern Kentucky, USA. Larvae of one or more species were observed at 12/13 sites. A total of 14 separate species sightings were observed, and in each case, eDNA from the observed species was detected via qPCR. Additionally, nine qPCR detections were observed at sites where species were not field observed. End-point PCR results were less effective in detecting field observed species, and primers appeared to bind to DNA from non-salamander targets. These primers, when used with a probe in a qPCR assay, provide an effective means of determining species presence rapidly and definitively and therefore offer to increase the ease of range delineation and spawning habitat studies.