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.

环境 DNA (eDNA) 正成为越来越多地用于监测陆地和水生系统中隐匿物种的工具。我们提出了第一种从树洞中提取水用于树栖蛙的 eDNA 研究的方法，以及首次使用 eDNA 在马达加斯加进行两栖动物监测。这项试点研究扩展了先前开发的方法，旨在为从非常小的水样中提取 DNA 提供一个简单的现场协议，与其他收集方法相比，使用相对便宜的试剂盒。我们从马达加斯加 Ambohitantely 特别保护区的树洞中收集了 20 毫升的水，目的是调查极度濒危的树蛙Anodonthyla vallani，我们为该物种开发了物种特异性细胞色素 c 氧化酶 1 引物。虽然我们的两个样品没有检测到A. valllani，但我们成功地从样品中提取了高达 16.6 ng/µl 的 eDNA，并使用 16S rRNA 引物对树蛙Plethodontohyla mihanika 进行了条形码标记在其中一个样本中。尽管只收集了两个样本，但鉴于我们仅从 20 毫升水中提取了青蛙 eDNA，我们强调了来自树洞的 eDNA 在研究神秘栖息地专家两栖动物方面的未来潜力。该方法提供了一种快速、简单且具有成本效益的方法，可以帮助在具有挑战性和耗时的现场条件下监测隐匿物种，应进一步开发用于马达加斯加及其他地区的青蛙调查。新开发的引物可用于进一步的工作，使用这种 eDNA 方法来调查受威胁的Anodonthyla青蛙物种。