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.
Highlights
The use of environmental DNA in conservation biology is becoming an increasingly popular non-destructive method for wild species surveying (Thomsen and Willerslev 2015)
It has been used to detect whole communities (Lopes et al 2017; Sasso et al 2017), and cryptic (Torresdal et al 2017; Bálint et al 2018), threatened (Thomsen et al 2012) and invasive species (Dejean et al 2012), even when a species may be present at low population densities (Sasso et al 2017). environmental DNA (eDNA) is becoming an increasingly popular tool for amphibian surveying and there is growing evidence that detection rates may be as efficient as more traditional visual encounter and acoustic surveying (Lopes et al 2017; Bálint et al 2018; Barata et al 2021)
The use of eDNA in Madagascar has huge potential to further our understanding of the amphibian communities in Ambohitantely and elsewhere
Summary
The use of environmental DNA (eDNA) in conservation biology is becoming an increasingly popular non-destructive method for wild species surveying (Thomsen and Willerslev 2015) It has been used on a range of species including fish, amphibians, coral reefs and mammals, and in a range of environments including the ocean, fresh water streams and rivers, ponds, bromeliads and soil (Huerlimann et al 2020). Further research used eDNA to target 30 amphibian species of conservation concern in Brazil, and successfully detected species that had locally disappeared, and one species which has not been seen since 1968 (Lopes et al 2020) These results support that eDNA could perform better for detecting cryptic species which are not recorded using
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