Environmental DNA enables detection of terrestrial mammals from forest pond water

Abstract

Terrestrial animals must have frequent contact with water to maintain their lives, implying that environmental DNA (eDNA) originating from terrestrial animals should be detectable from places containing water in terrestrial ecosystems. Aiming to detect the presence of terrestrial mammals using forest water samples, we applied a set of universal PCR primers (MiMammal, a modified version of fish universal primers) for metabarcoding mammalian eDNA. After verifying the primers usefulness in silico and using water samples from zoo cages of animals with known species compositions, we collected five 500-ml water samples from ponds in two cool-temperate forests in Hokkaido, northern Japan. Using eDNA extracted from the water samples, we constructed amplicon libraries using MiMammal primers for Illumina MiSeq sequencing. MiMammal metabarcoding yielded a total of 75,214 reads, which we then subjected to data pre-processing and taxonomic assignment. We thereby detected species of mammals common to the sampling areas, including deer (Cervus nippon), mouse (Mus musculus), vole (Myodes rufocanus), raccoon (Procyon lotor), rat (Rattus norvegicus) and shrew (Sorex unguiculatus). Previous applications of the eDNA metabarcoding approach have mostly been limited to aquatic/semiaquatic systems, but the results presented here show that the approach is also promising even in forest mammal biodiversity surveys.