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

Abstract

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.

A New Species of Crossodactylodes from the Espinhaço Mountain Range, Southeastern Brazil (Anura: Leptodactylidae: Paratelmatobiinae)

“…For these reasons, the use of mouth-operated aspirators substantially increases the chance of collecting specimens. The use of environmental DNA (eDNA) can also improve the detection of phytotelm species (Barata et al 2021; Mullin et al 2022), such as bromeliad-dwelling frogs. Barata et al (2021) demonstrated that C. itambe can have a high detection rate (0.97–0.98) by extracting and amplifying eDNA from water samples of as little as 120 mL.…”

First population estimates of two Critically Endangered frogs from an isolated forest plateau in Madagascar

“…Additionally, by using acoustic surveys, estimates will only account for, and will therefore be limited to, the number of active males in the population, which can potentially introduce bias into inferences to the whole population, especially when the sex ratio is unknown (a 50:50 sex ratio is a naïve assumption). Alternative methods are available that could be used to improve parameter estimates, such as using automated recording devices (Measey et al, 2017) or environmental DNA (Lopes et al, 2017), which has since been trialled for A. vallani at Ambohitantely (Mullin et al, 2021b). Both of these tools eliminate the requirement for the observer to be at the site at the same time as the species, enabling data collection on much larger spatial and temporal scales and improving the robustness of any inferences made, although both tools require significant technical expertise.…”

Conceptual analysis: What signals might plant canopies send via stemflow?

“…The environmental DNA (eDNA) left by canopy fauna on the surfaces of leaves and bark or within canopy aquatic microhabitats may also enter stemflow. Continuing with the tree frog example, the dendrotelmata or phytotelmata in which many tree frogs rely for food, shelter and/or breeding have recently been found to house detectable levels of their eDNA (Barata et al, 2021;Mullin et al, 2022). During large storms, these canopy aquatic microhabitats overflow and drain down tree stems (as discussed in greater detail in the following sections), potentially delivering the eDNA to the surface at the base of the tree stem.…”