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.