Needle in a haystack? A comparison of eDNA metabarcoding and targeted qPCR for detection of the great crested newt (Triturus cristatus)

Abstract

Environmental DNA (eDNA) analysis is a rapid, cost-effective, non-invasive biodiversity monitoring tool which utilises DNA left behind in the environment by organisms for species detection. The method is used as a species-specific survey tool for rare or invasive species across a broad range of ecosystems. Recently, eDNA and "metabarcoding" have been combined to describe whole communities rather than focusing on single target species. However, whether metabarcoding is as sensitive as targeted approaches for rare species detection remains to be evaluated. The great crested newt Triturus cristatus is a flagship pond species of international conservation concern and the first UK species to be routinely monitored using eDNA. We evaluate whether eDNA metabarcoding has comparable sensitivity to targeted real-time quantitative PCR (qPCR) for T. cristatus detection. Extracted eDNA samples (N = 532) were screened for T. cristatus by qPCR and analysed for all vertebrate species using high-throughput sequencing technology. With qPCR and a detection threshold of 1 of 12 positive qPCR replicates, newts were detected in 50% of ponds. Detection decreased to 32% when the threshold was increased to 4 of 12 positive qPCR replicates. With metabarcoding, newts were detected in 34% of ponds without a detection threshold, and in 28% of ponds when a threshold (0.028%) was applied. Therefore, qPCR provided greater detection than metabarcoding but metabarcoding detection with no threshold was equivalent to qPCR with a stringent detection threshold. The proportion of T. cristatus sequences in each sample was positively associated with the number of positive qPCR replicates (qPCR score) suggesting eDNA metabarcoding may be indicative of eDNA concentration. eDNA metabarcoding holds enormous potential for holistic biodiversity assessment and routine freshwater monitoring. We advocate this community approach to freshwater monitoring to guide management and conservation, whereby entire communities can be initially surveyed to best inform use of funding and time for species-specific surveys.

Keywords: Triturus cristatus; environmental DNA; great crested newt; high‐throughput sequencing; metabarcoding; ponds; real‐time quantitative PCR.

Figure 1

Adult male great crested newt Triturus cristatus. Photograph by Brett Lewis (Lewis Ecology, Brett Lewis Photography)

Figure 2

Comparison of survey methodology for T. cristatus detection in freshwater ponds across the UK. Bars represent proportion of positive and negative T. cristatus ponds by each method with frequency displayed on bars

Figure 3

Venn diagram which summarises the number of positive T. cristatus detections (N = 277) by each method (egg search, qPCR NT, qPCR TA, metabarcoding NT, and metabarcoding TA), and overlap in T. cristatus detection between methods for 506 ponds where all methods were applied. Negative T. cristatus detections (N = 229) are highlighted in red

Figure 4

Relationship between fixed effects (qPCR score, post‐PCR eDNA concentration) and response variable (proportion of T. cristatus reads) in eDNA samples, as predicted by the negative binomial GLMM. The 95% CIs, as calculated using the predicted proportions and standard error for these predictions, are given for each relationship. The observed data (points) are also displayed against the predicted relationships (boxes, line). The proportion of T. cristatus reads within eDNA samples increased as qPCR score increased (a). Conversely, the proportion of T. cristatus reads decreased as post‐PCR eDNA concentration increased (b)

Figure 5

Cost and investigator effort required for targeted qPCR of T. cristatus and eDNA metabarcoding of vertebrate communities from pond water samples