Power and limitations of environmental DNA metabarcoding for surveying leaf litter eukaryotic communities

Abstract

Leaf litter habitats shelter a great variety of organisms, which play an important rolein ecosystem dynamics. However, monitoring species in leaf litter is challenging, especiallyin highly diverse environments such as tropical forests, because individualsmay easily camouflage themselves or hide in the litter layer. Identifying species basedon environmental DNA (eDNA) would allow us to assess biodiversity in this microhabitat,without the need for direct observation of individuals. We applied eDNAmetabarcoding to analyze large amounts of leaf litter (1 kg per sample) collected inthe Brazilian Atlantic forest. We compared two DNA extraction methods, one totaland one extracellular, and amplified a fragment of the mitochondrial 18S rRNA genecommon to all eukaryotes, to assess the performance of eDNA from leaf litter samplesin identifying different eukaryotic taxonomic groups. We also amplified twofragments of the mitochondrial 12S rRNA gene to specifically test the power of thisapproach for monitoring vertebrate species, with a focus on anurans. Most of theeukaryote sequence reads obtained were classified as Fungi, followed by Metazoa,and Viridiplantae. Most vertebrate sequences were assigned to Homo sapiens; onlytwo sequences assigned to the genus Phyllomedusa and the species Euparkerella brasiliensiscan be considered true detections of anurans in our eDNA samples. The detectionof taxa varied depending on the DNA extraction method applied. Our resultsdemonstrate that the analysis of eDNA from leaf litter samples has low power formonitoring vertebrate species and should be preferentially applied to describe activeand abundant taxa in terrestrial communities, such as Fungi and invertebrates.