Improving ecological surveys for the detection of cryptic, fossorial snakes using eDNA on and under artificial cover objects

Abstract

Performing ecological surveys for secretive, fossorial snakes is challenging. Traditional survey methods involve visual observation under artificial cover objects (ACOs); this is labor-intensive and requires multiple consistent surveys of suitable habitats. Detection of snake DNA deposited under ACOs represents an innovative method for species detection. However, for terrestrial species, common issues with soil-based methods include the challenges of adequately removing enzyme inhibitors that reduce environmental DNA (eDNA) detection and potential photodegradation of DNA taken from surface samples. These issues may be circumvented by obtaining swabs and soil samples directly from the underside of ACOs for eDNA analysis. We demonstrate the application of this method in surveys of sharp-tailed snake (Contia tenuis), an endangered species under the Canadian Species at Risk Act. We describe the design and validation of a new quantitative real-time polymerase chain reaction (qPCR)-based eDNA eCOTE3 assay with high specificity and sensitivity for sharp-tailed snake. We developed a practical and robust protocol for obtaining eDNA samples by swabbing the underside of ACOs and collecting soil samples under ACOs. Traditional surveys were conducted over two successive years (2018–19) on 220 paired ACOs at 110 sites monitored between 12 and 30 times each. Of the 6,060 ACO visits, only 24 resulted in sharp-tailed snake observations (0.4% success rate) illustrating the considerable difficulty in detecting these snakes. During this same time, 109 swabs were taken directly from the undersides of ACOs and 78 soil samples were collected from a subset of these ACOs. Of the 24 occurrences where sharp-tailed snakes were visually observed, 13 of 23 ACO swabs (57%) and nine of 20 soil samples (45%) tested positive for DNA. eDNA deposition is likely low because of the small size and behavior of this cryptic species, yet DNA was detected from soil exposed to captured snakes for only 10 min. Nevertheless, sharp-tailed snake eDNA was detected at eight sites (9%) from ACO swabs (n = 86) and seven sites (13%) from soil samples (n = 56) where snakes were not observed. This is an overall detection rate of 25% (14/56) for swab and soil samples testing positive in sites where both were tested, representing a substantial reduction in the effort required for detection of this species. Given the time-consuming nature of traditional surveys, eDNA holds great promise as a complementary survey tool for this terrestrial species. While further work is needed to delineate DNA deposition rates, this work represents a significant advance in monitoring a challenging species.