Developing and testing environmental DNA metabarcoding protocols for surveying leaf litter amphibians in Brazilian Atlantic Forest

Abstract

Brazil has the highest amphibian diversity in the world, with over 1000 described species. Amphibians are highly sensitive to environmental degradation, facing serious problems of population declines, local extinctions, or even species extinctions. In the Brazilian Atlantic forest, some amphibians associated to the leaf litter are no longer registered, and some were only sampled for their descriptions (i.e. more than 40 years ago). Most of these species are not classified in any level of threat in the IUCN red list, mainly due to the lack of information about their biology and geographical distributions. Their conservation status require more investigations. Monitoring rare amphibians associated with leaf litter is challenging. Search for DNA traces of these species that can remain in the environment (environmental DNA or eDNA) would allow us to monitor a wide geographical range in a short period of time, without the need for direct observation of individuals, and would ultimately increase our ability to survey these species. However, the analysis of DNA traces in leaf litter is an innovative approach. It has not been applied yet to amphibian surveys, and thus requires the development of specific protocols, especially for sampling, and DNA extraction. The aim of this study is therefore to test and validate protocols for leaf litter eDNA analysis in an area in which the anuran fauna is well known. This prior knowledge will gives us support to test the feasibility of an eDNA metabarcoding approach for the detection of amphibians, and to assess the performance of the method to detect both rare and abundant species. The methods developed in this study will be applied hereafter in this project to search for amphibians no longer registered in the Brazilian Atlantic forest. (AU)