Environmental DNA (eDNA) analysis is a rapid, cost-effective, non-invasive biodiversity monitoring tool which detects species using DNA deposited in the environment by organisms. The method is a recognised species-specific survey tool for rare or invasive species in a broad range of ecosystems, and employed for survey of protected great crested newt (Triturus cristatus) in the UK and Europe. More recently, eDNA has been combined with ‘metabarcoding’ to reveal entire communities from environmental samples. However, this method of mass species detection can fail to detect rare or cryptic species. We examined the sensitivity of this community sequencing approach for single species compared to targeted real-time quantitative PCR (qPCR) and evaluated the potential of eDNA metabarcoding for holistic biodiversity assessment in freshwater ponds. Pond eDNA samples (N = 532) previously screened by qPCR for T. cristatus were re-analysed for all vertebrate species using high-throughput sequencing technology.
eDNA metabarcoding (34%) and qPCR (36%) had comparable sensitivity for T. cristatus detection but application of sequence thresholds to minimise false positives reduced metabarcoding detection (28%). Nonetheless, the proportion of sequences identified as T. cristatus in each sample positively correlated with qPCR score (number of positive qPCR replicates) but was negatively associated with post-PCR eDNA concentration of samples. eDNA metabarcoding detected a wealth of vertebrate biodiversity from all groups (excluding reptiles) alongside T. cristatus, allowing identification of significant positive and negative associations between T. cristatus and other species. Broadly, presence of T. cristatus was reduced by increased number of fish species but enhanced by greater number of waterfowl species. Application of environmental metadata to eDNA metabarcoding results revealed predictors of T. cristatus presence and vertebrate species richness in ponds. eDNA metabarcoding holds enormous potential for biodiversity assessment in ponds and fully encapsulates molecular ecology. We advocate this community approach to freshwater monitoring to guide management and conservation, and generate targets for species-specific survey.