Environmental DNA (eDNA) metabarcoding of pond water as a tool to survey conservation and management priority mammals

Abstract Environmental DNA (eDNA) metabarcoding is largely used to survey aquatic communities, but can also provide data on terrestrial taxa utilising aquatic habitats. However, the entry, dispersal, and detection of terrestrial species’ DNA within waterbodies is understudied. We evaluated eDNA metabarcoding of pond water for monitoring semi-aquatic, ground-dwelling, and arboreal mammals, and examined spatiotemporal variation in mammal eDNA signals using experiments in captive and wild conditions. We selected nine focal species of conservation and management concern: European water vole, European otter, Eurasian beaver, European hedgehog, European badger, red deer, Eurasian lynx, red squirrel, and European pine marten. We hypothesised that eDNA signals (i.e. proportional read counts) would be stronger for semi-aquatic than terrestrial species, and at sites where mammals exhibited behaviours (e.g. swimming, urination). We tested this by sampling waterbodies in enclosures of captive focal species at specific sites where behaviours had been observed (‘directed’ sampling) and at equidistant intervals along the shoreline (‘stratified’ sampling). We then surveyed natural ponds ( N = 6) where focal species were present using stratified water sampling, camera traps, and field signs. eDNA samples were metabarcoded using vertebrate-specific primers. All focal species were detected in captivity. eDNA signal strength did not differ between directed and stratified samples across or within species, between species lifestyles (i.e. semi-aquatic, ground-dwelling, arboreal), or according to behaviours. Therefore, eDNA was evenly distributed within artificial waterbodies. Conversely, eDNA was unevenly distributed in natural ponds. eDNA metabarcoding, camera trapping, and field signs detected beaver, red deer, and roe deer. Badger and red fox were recorded with cameras and field signs, but not eDNA metabarcoding. However, eDNA metabarcoding detected small mammals missed by cameras and field signs, e.g. water vole. Terrestrial mammal eDNA signals were weaker and detected in fewer samples than semi-aquatic mammal eDNA signals. eDNA metabarcoding has potential for inclusion in mammal monitoring schemes by enabling large-scale, multi-species distribution assessment for priority and difficult to survey species, and could provide early indication of range expansions or contractions. However, eDNA surveys need high spatiotemporal resolution and metabarcoding biases require further investigation before this tool is routinely implemented.