Cailyn M. Zamora, Nathanael B. J. Harper, Maxwell P. Bui-Marinos, Paul M. Craig, Mark R. Servos, Andrew C. Doxey, John P. Giesy, Barbara A. Katzenback (2022). Validation of environmental DNA barcoding assays for Southern Ontario amphibian species. Proceedings of the GWF Annual Open Science Meeting, May 16-18, 2022.

Effective monitoring of populations of amphibians is necessary for conservation and reversing declines in populations of amphibians. Due to diverse life histories, cryptic nature, and low population density of threatened species, current methods of monitoring, such as visual and auditory surveys, are fraught with challenges. Organisms constantly shed biological material resulting in the accumulation of genetic material in the environment, termed environmental DNA (eDNA), which can be collected, concentrated by filtration, and identified and quantified. Environmental DNA barcoding is an emerging, sensitive, method for detection of the presence of species by identification of unique sequences of nucleic acid, specific to a target species, which obviates the need for direct observations of taxa. While eDNA barcoding assays have been designed for 24 Ontario amphibians and validated under laboratory settings (in vitro), these assays have yet to be validated for use with eDNA collected from the field, which poses a barrier to their uptake and application for monitoring efforts by end-users. The goal of this research is to determine if eDNA barcoding is a reliable and efficient method to detect amphibians in vernal pool habitats in Southern Ontario compared to conventional surveys. Target species will include vernal pool obligate breeders, including wood frog, spotted salamander, and species that inhabit various wetland types, including spring peeper, green frog, and American toad. It is hypothesized that eDNA barcoding will provide equal or greater accuracy for detection of the presence/absence of specific amphibians, compared to conventional survey methods. From April to July 2019, conventional (visual/auditory) and eDNA surveys for amphibians in vernal pools were conducted in collaboration with the rare Charitable Research Reserve, Cambridge, ON. Audio recorders collected call data daily at dawn and dusk. Visual surveys were conducted weekly concurrently with collection of eDNA. Three 1-L samples of surface waters water were collected from sites around vernal pools and kept on ice until filtered within 12 hours of collection. Assays to detect eDNA barcodes, based on species-specific sequences were validated for effectiveness and overall efficiency at low concentrations of eDNA. Presence/absence of eDNA for selected species will be compared to results of conventional survey methods. Both methods will be used to assess status and trends in distributions of amphibians on spatial and temporal scales. Results will provide evidence needed to support the uptake of these methods for ongoing monitoring of populations of amphibians by end users in academia, government, and private industry.

AOSM2022 Next generation solutions to ensure healthy water resources for future generations (PI:JP Giesy) First Author: Cailyn M. Zamora, Dept. of Biology, University of Waterloo, Waterloo, ON, Canada Additional Authors: Nathanael B. J. Harper, Dept. of Biology, University of Waterloo, Waterloo, ON, Canada. Maxwell P. Bui-Marinos, Dept. of Biology, University of Waterloo, Waterloo, ON, Canada. Paul M. Craig, Dept. of Biology, University of Waterloo, Waterloo, ON, Canada. Mark R. Servos, Dept. of Biology, University of Waterloo, Waterloo, ON, Canada. Andrew C. Doxey, Dept. of Biology, University of Waterloo, Waterloo, ON, Canada. John P. Giesy, Dept. of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, SK, Canada. Barbara A. Katzenback, Dept. of Biology, University of Waterloo, Waterloo, ON, Canada.