Seasonality, DNA degradation and spatial heterogeneity as drivers of eDNA detection dynamics.

In recent years, eDNA-based assessments have evolved as valuable tools for research and conservation. Most eDNA-based applications rely on comparisons across time or space. However, temporal, and spatial dynamics of eDNA concentrations are shaped by various drivers that can affect the reliability of such comparative approaches. Here, we assessed (i) seasonal variability, (ii) degradation rates and (iii) micro-habitat heterogeneity of eDNA concentrations as key factors likely to inflict uncertainty in across site and time comparisons. In a controlled mesocosm experiment, using the white-clawed crayfish as a model organism, we found detection probabilities of technical replicates to vary substantially and range from as little as 20 to upwards of 80% between seasons. Further, degradation rates of crayfish eDNA were low and target eDNA was still detectable 14-21 days after the removal of crayfish. Finally, we recorded substantial small-scale in-situ heterogeneity and large variability among sampling sites in a single pond of merely 1000m2 in size. Consequently, all three tested drivers of spatial and temporal variation have the potential to severely impact the reliability of eDNA-based site comparisons and need to be accounted for in sampling design and data analysis of field-based applications.

Development and application of eDNA-based tools for the conservation of white-clawed crayfish.

eDNA-based methods represent non-invasive and cost-effective approaches for species monitoring and their application as a conservation tool has rapidly increased within the last decade. Currently, they are primarily used to determine the presence/absence of invasive, endangered or commercially important species, but they also hold potential to contribute to an improved understanding of the ecological interactions that drive species distributions. However, this next step of eDNA-based applications requires a thorough method development. We developed an eDNA assay for the white-clawed crayfish (Austropotamobius pallipes), a flagship species of conservation in the UK and Western Europe. Multiple subsequent in-situ and ex-situ validation tests aimed at improving method performance allowed us to apply eDNA-based surveys to evaluate interactions between white-clawed crayfish, crayfish plague and invasive signal crayfish. The assay performed well in terms of specificity (no detection of non-target DNA) and sensitivity, which was higher compared to traditional methods (in this case torching). The eDNA-based quantification of species biomass was, however, less reliable. Comparison of eDNA sampling methods (precipitation vs. various filtration approaches) revealed that optimal sampling method differed across environments and might depend on inhibitor concentrations. Finally, we applied our methodology together with established assays for crayfish plague and the invasive signal crayfish, demonstrating their significant interactions in a UK river system. Our analysis highlights the importance of thorough methodological development of eDNA-based assays. Only a critical evaluation of methodological strengths and weaknesses will allow us to capitalise on the full potential of eDNA-based methods and use them as decision support tools in environmental monitoring and conservation practice.

Combining ddPCR and environmental DNA to improve detection capabilities of a critically endangered freshwater invertebrate.

Isogenus nubecula is a critically endangered Plecoptera species. Considered extinct in the UK, I. nubecula was recently rediscovered (in one location of the River Dee, Wales), after 22 years of absence. In a similar way to many other species of Perlodidae, I. nubecula could be utilised as a bio-indicator, for assessing water quality and health status of a given freshwater system. However, conventional monitoring of invertebrates via kick-sampling, is invasive and expensive (time consuming). Further, such methods require a high level of taxonomic expertise. Here, we compared the traditional kick-sampling method with the use of eDNA detection using qPCR and ddPCR-analyses. In spring 2018, we sampled eDNA from twelve locations on the River Dee. I. nubecula was detected using kick-sampling in five of these locations, three locations using both eDNA detection and kick-sampling and one location using eDNA detection alone - resulting in a total of six known and distinct populations of this critically endangered species. Interestingly, despite the eDNA assay being validated in vitro and in silico, and results indicating high sensitivity, qPCR analysis of the eDNA samples proved to be ineffective. In contrast, ddPCR analyses resulted in a clear detection of I. nubecula at four locations suggesting that inhibition most likely explains the large discrepancy between the obtained qPCR and ddPCR results. It is therefore important to explore inhibition effects on any new eDNA assay. We also highlight that ddPCR may well be the best option for the detection of aquatic organisms which are either rare or likely to shed low levels of eDNA into their environment.