Exploration of Environmental DNA (eDNA) to Detect Kirtland’s Snake (Clonophis kirtlandii)

Simple Summary Small and difficult to find species of conservation concern such as the Kirtland’s Snake require significant survey effort using traditional methods. Surveying for DNA shed into the environment, or environmental DNA, we set out to improve detection probability and efficiency to aid in future conservation efforts for this species. Field surveys revealed temporal and spatial variation in Kirtland’s Snake activity. More snakes were found in the spring, during the first field season, and in areas with abundant grass, herbaceous vegetation, and shrubs. We collected environmental samples and developed a molecular assay to detect eDNA across this spatial and temporal gradient of snake activity. We also tested the persistence of DNA in the microenvironment snakes are expected to use by introducing feces into artificial burrows. We were able to detect snake eDNA in only a single environmental sample and found that eDNA in artificial burrows appears to decline within a week. We explored the potential methodological and biological causes of this low detection success to aid future research employing eDNA detection as a survey method in snakes. Abstract Environmental DNA (eDNA) surveys utilize DNA shed by organisms into their environment in order to detect their presence. This technique has proven effective in many systems for detecting rare or cryptic species that require high survey effort. One potential candidate for eDNA surveying is Kirtland’s Snake (Clonophis kirtlandii), a small natricine endemic to the midwestern USA and threatened throughout its range. Due to its cryptic and fossorial lifestyle, it is also a notoriously difficult snake to survey, which has limited efforts to understand its ecology. Our goal was to utilize eDNA surveys for this species to increase detection probability and improve survey efficiency to assist future conservation efforts. We conducted coverboard surveys and habitat analyses to determine the spatial and temporal activity of snakes, and used this information to collect environmental samples in areas of high and low snake activity. In addition, we spiked artificial crayfish burrows with Kirtland’s Snake feces to assess the persistence of eDNA under semi-natural conditions. A quantitative PCR (qPCR) assay using a hydrolysis probe was developed to screen the environmental samples for Kirtland’s Snake eDNA that excluded closely related and co-occurring species. Our field surveys showed that snakes were found in the spring during the first of two seasons, and in areas with abundant grass, herbaceous vegetation, and shrubs. We found that eDNA declines within a week under field conditions in artificial crayfish burrows. In environmental samples of crayfish burrow water and sediment, soil, and open water, a single detection was found out of 380 samples. While there may be physicochemical and biological explanations for the low detection observed, characteristics of assay performance and sampling methodology may have also increased the potential for false negatives. We explored these outcomes in an effort to refine and advance the successful application of eDNA surveying in snakes and groundwater microhabitats.