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.

Comparison of fish detections, community diversity, and relative abundance using environmental DNA metabarcoding and traditional gears

“…Findings here support previous work demonstrating the sensitivity of eDNA assays (Turner, Miller, et al, 2014); however, our study applied an eDNA metabarcoding approach, which has not been as well vetted in the literature (but see Harper et al, 2018). In lakes where both traditional gear and eDNA assays detected AIS, eDNA methods often had higher detection rates, indicating that eDNA metabarcoding surveys are a sensitive method for early AIS detection.…”

Fish diversity assessment in the headwaters of the Volga River using environmental DNA metabarcoding

“…For species identification, the polymerase chain reaction (PCR) primers used should bind in highly conserved regions of the genes, whereas the amplified sequence should exhibit adequate species‐specific variability, and these three specific markers are known to have these characteristics. Using several markers increases the reliability of species identification, while minimizing taxonomic bias due to varying mismatches (Evans et al, ; Hänfling et al, ; Harper et al, ; Valentini et al, ). All three primer sets, used for the first‐step PCR, were previously described: primers for Cyt b (L14735/H15149c) were designed by Burgener and Hübner () (Table ), and primers for 12S and 16S partial genes were developed by Evans et al () (Table ).…”

“…Using several markers increases the reliability of species identification, while minimizing taxonomic bias due to varying mismatches Hänfling et al, 2016;Harper et al, 2018;Valentini et al, 2016). All three primer sets, used for the first-step PCR, were previously described: primers for Cyt b (L14735/ H15149c) were designed by Burgener and Hübner (1998) PCR products were run on 2% agarose gel with peqGREEN dye (PEQLAB).…”

“…Overall, the spatial distribution and preservation of eDNA of different species is not yet fully understood owing to a number of factors involving the condition of the DNA itself and its complex interactions with numerous abiotic factors (Barnes et al, 2014;Dejean et al, 2011;Pilliod, Goldberg, Arkle, & Waits, 2014;Pont et al, 2018 ;Wilcox et al, 2016). The shedding rates of eDNA also vary across species, sexes, ages, seasons, and environmental characteristics, and a combination of biases, during library preparation and PCR, can differentially affect the amplification of eDNA across species (Harper et al, 2018;Kelly, Port, Yamahara, & Crowder, 2014). Furthermore, rare species can be more difficult to detect using an eDNA metabarcoding approach compared with a qPCR method.…”

Limited dispersion and quick degradation of environmental DNA in fish ponds inferred by metabarcoding

“…This tool removes the need to select target organisms a priori with the use of generic PCR primers that amplify multiple taxa, thus facilitating detection of invasive or threatened species when conducting holistic biodiversity assessment and routine freshwater monitoring (Thomsen & Willerslev, ). Encouragingly, Harper et al () demonstrated that Triturus cristatus (great crested newt) detection via metabarcoding with no threshold is equivalent to qPCR with a stringent detection threshold. eDNA metabarcoding has also been applied to large‐scale investigations of spatial or temporal variation in marine and freshwater communities, with some studies indicating that communities can be distinguished from 100 m to 2 km due to stream discharge or tidal patterns (Civade et al, ; Kelly, Gallego, & Jacobs‐Palmer, ; Li, Evans, et al, ; O’Donnell et al, ; Port et al, ).…”