The invasive American bullfrog (Lithobates catesbeianus) imperils freshwater biodiversity worldwide. Effective management hinges on early detection of incipient invasions and subsequent rapid response, as established populations are extremely difficult to eradicate. Although environmental DNA (eDNA) detection methods provide a highly sensitive alternative to conventional surveillance techniques, extensive testing is imperative to generate reliable output. Here, we tested and compared the performance of two primer/probe assays to detect and quantify the abundance of bullfrogs in Western Europe in silico and in situ using digital droplet PCR (ddPCR). Although both assays proved to be equally target-specific and sensitive, one outperformed the other in ddPCR detection resolution (i.e., distinguishing groups of target-positive and target-negative droplets), and hence was selected for further analyses. Mesocosm experiments revealed that tadpole abundance and biomass explained 99% of the variation in eDNA concentration. Because per individual eDNA emission rates did not differ significantly among tadpoles and juveniles, and adults mostly reside out of the water, eDNA concentration can be used as an approximation of local bullfrog abundance in natural populations. Seasonal eDNA patterns in three colonized ponds showed parallel fluctuations in bullfrog eDNA concentration. An increase in eDNA concentration was detected in spring, followed by a strong peak coinciding with the breeding season (August, September or October), and continuously low eDNA concentrations during winter. With this study, we report the validation process required for appropriately implementing eDNA barcoding analyses in lentic systems. We demonstrate that this technique can serve as a solid and reliable tool to detect the early stages of bullfrog invasions and to quantify temporal changes in abundance that will be useful in coordinating large-scale bullfrog eradication programs and evaluating their efficiency.

The use of environmental DNA (eDNA) (allochtonous DNA released by macroorganisms) which can be isolated from environmental samples such as water, sediment or even air (Bedwell & Goldberg, 2019), to detect and quantify the occurrence of organisms in the environment, developed spectacularly in the past decade (Bruce et al., 2021;

Biological invasions contribute now more than ever to the global homogenization of fauna and flora. Large‐scale monitoring programs are, therefore, needed to detect incipient invasions and to evaluate management interventions. As conventional monitoring methods are constrained by large costs, environmental DNA (eDNA)‐based methods are increasingly recognized as valuable monitoring tools. However, accurately estimating species abundance from eDNA concentrations in natural systems remains challenging and consequently hinders their integration in management applications. Here, we used droplet digital PCR (ddPCR) in eDNA surveys to estimate the abundance of invasive American bullfrogs (Lithobates catesbeianus). We first introduced bullfrog tadpoles in natural ponds to assess the relationship between abundances and eDNA concentrations under field conditions. Next, we combined eDNA sampling with fyke netting in naturally colonized ponds to investigate whether bullfrog eDNA concentrations can estimate bullfrog capture success and conventional abundance measures obtained via depletion sampling. Finally, we evaluated eradication measures by comparing bullfrog eDNA concentrations before and after fyke netting. We found a strong linear relationship between the numbers of introduced tadpoles and eDNA concentrations (r2 = 0.988). Bullfrog eDNA concentrations were not only linearly related to the catch‐per‐unit‐effort (r2 = 0.739), but also to conventional abundance estimates (r2 = 0.716), particularly when eDNA concentrations were standardized for pond area (r2 = 0.834) and volume (r2 = 0.888). Bullfrog tadpoles were only captured when eDNA concentrations exceeded 1.5 copies µl−1, indicating that quantitative eDNA analyses enable the localization of breeding ponds. We found a significant reduction in eDNA concentrations after fyke netting proportional to the number of captured bullfrogs. These results demonstrate that eDNA quantification is a reliable tool that accurately estimates bullfrog abundance in natural lentic systems. We show that quantitative eDNA analyses can complement the toolbox of natural resource managers and facilitate the coordination of eradication campaigns targeting alien invasive species.