First extraction of eDNA from tree hole water to detect tree frogs: a simple field method piloted in Madagascar

Environmental DNA (eDNA) is becoming an increasingly used tool for monitoring cryptic species within terrestrial and aquatic systems. We present the first method for extracting water from tree holes for eDNA studies of tree-dwelling frogs, and the first use of eDNA for amphibian monitoring in Madagascar. This pilot study expands on a previously developed method and aims to provide a simple field protocol for DNA extraction from very small water samples, using a relatively inexpensive kit compared to other collection methods. We collected 20 ml of water from tree holes in Ambohitantely Special Reserve in Madagascar, with the aim to survey for the Critically Endangered tree frog Anodonthyla vallani, and we developed species specific cytochrome c oxidase 1 primers for this species. While our two samples did not detect A. vallani, we successfully extracted up to 16.6 ng/µl of eDNA from the samples and using 16S rRNA primers barcoded the tree frog Plethodontohyla mihanika in one of the samples. Despite just two samples being collected, we highlight the future potential of eDNA from tree holes for investigating cryptic habitat specialist amphibians given we extracted frog eDNA from just 20 ml of water. The method provides a rapid, simple, and cost-effective method which can assist cryptic species monitoring in challenging and time-consuming field conditions and should be developed further for frog surveying in Madagascar and beyond. The newly developed primers can be used for further work using this eDNA method to survey threatened Anodonthyla frog species.