Abstract
AbstractEnvironmental DNA (eDNA), or DNA that is shed into the environment by an organism, can be used to detect the presence of cryptic species. However, eDNA methodology requires validation of an assay in both laboratory and field environments. Here, we describe the development of a quantitative PCR (qPCR) assay and field protocol for detecting a secretive amphibian, the Streamside Salamander (Ambystoma barbouri). This fossorial species is rarely encountered because adults are only active for several months during the winter when they breed and deposit eggs underneath rocks within intermittent streams. We designed and validated a qPCR assay for A. barbouri against five ambystomatid congeners and the Southern Two‐lined Salamander (Eurycea cirrigera), which occur within the range of A. barbouri in the focal study area. We detected DNA of A. barbouri in the laboratory to 0.0004 ng/µl, while all other congeners were rarely detected below 40 ng/µl. We collected 1‐L water samples from 45 streams from December 2016 to May 2017 and during April 2018 to validate our methods in the field. Our assay was effective at detecting A. barbouri in 24 of 45 streams. We confirmed physical presence of A. barbouri (adults, larvae, and/or eggs) at 21 out of 24 of these positive sites. The detection probability was 0.85 ± 0.05; CI: 0.75, 0.95 within a single sampling event that incorporated 5 water samples from each of 17 repeat‐visit sites. The per water sample sensitivity was 68% (163/238) and specificity was 100% (22/22). The per site visit sensitivity was 85% (46/54) and specificity was 100% (6/6). Lastly, the per site sensitivity was 95% (21/22) and specificity was 100% (3/3). Collectively, this protocol outlines an efficient and cost‐effective method to detect A. barbouri and provides a technique to rapidly identify sites where breeding is occurring and to determine the distribution of this species.
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