Development and Validation of a Protocol to Detect the Presence and Abundance of the Streamside Salamander (Ambystoma barbouri) Using Environmental DNA

Nicole A Witzel, Tennessee State University


Low-order intermittent streams represent a habitat type that is largely underappreciated and understudied, despite their unique biodiversity. Species that occupy intermittent streams are often overlooked during evaluations, as these species may only be active for a few months out of the year when these streams are filled with water. These species may give valuable insight into the overall health of the ecosystem. This is specifically true for the Streamside Salamander (Ambystoma barbouri), which inhabits these intermittent stream ecosystems and is state endangered in Tennessee. Surveys that target environmental DNA (eDNA) may provide an effective method to detect species such as A. barbouri. Our objectives were 1) to develop a qPCR assay sensitive and specific to A. barbouri, 2) to successfully use this assay to detect A. barbouri in field samples, and 3) to determine if there was a relationship between salamander biomass and eDNA copies and identify a sampling time frame. Using our qPCR assay, we detected DNA of A. barbouri to 0.0004 ng/µL, while the five other congeners were rarely detected below 40 ng/µL. We collected 1-L water samples from 45 streams from December 2016–May 2017 and April 2018. Of the 45 streams sampled, our assay detected A. barbouri in 24 streams. The detection probability was 0.85 ± 0.05; C.I.: 0.75, 0.95 within a single sampling event that incorporated 5 water samples from each of 17 repeat-visit sites. The sensitivity of this assay was 163/238 (68%), and specificity was 100% (22/22). Using a linear mixed-effects model, we determined that eDNA copy number was logarithmically related to salamander biomass and found a strong link between biomass and eDNA copy number after ~100 copies of DNA in a sample. January 11th through March 22nd represented the critical period of sampling for A. barbouri eDNA with a peak of February 14th. This study provides an efficient, accurate, low-impact method which can be utilized by wildlife agencies to identify the presence and relative abundance of an intermittent stream specialist and could assist in monitoring population status of a state endangered species, as well as evaluate the health of intermittent streams.

Subject Area

Environmental science|Wildlife Management|Molecular biology

Recommended Citation

Nicole A Witzel, "Development and Validation of a Protocol to Detect the Presence and Abundance of the Streamside Salamander (Ambystoma barbouri) Using Environmental DNA" (2020). ETD Collection for Tennessee State University. Paper AAI28154336.