Properties and Limitations of eDNA Substrates for Terrestrial Animal Monitoring.

Environmental DNA (eDNA) constitutes a valuable tool for monitoring terrestrial animal diversity, but outcomes are affected by multiple factors. Among these factors, the choice of sampling substrate and method is especially important and must be aligned with research objectives. We reviewed 245 published studies that utilise eDNA for terrestrial animal monitoring and compiled an overview of the most frequently used environmental substrates. Based on the reviewed literature, we provide a key description of each substrate, as well as its particular properties and limitations related to the detection of animal species across different spatial and temporal scales. We categorise these substrates into three groups: abiotic substrates (soil, water, air, sediment), biotic substrates (invertebrate samples, plant tissues, spiderwebs) and direct-evidence substrates (scat, footprints, shelters, feeding sites). In addition, we identify several key challenges with the interpretation of eDNA-based biodiversity monitoring, including false negatives and false positives, as well as the dynamics of spatial and temporal deviations. The latter concepts, which we propose and define in this review, describe the temporal and spatial discrepancies between the DNA source and its detection in a given sample. We reflect on how these temporal and spatial deviations are expected to affect eDNA data extracted from the different types of substrates and how knowledge of these dynamics can inform effective and accurate biomonitoring. In summary, this review provides a decision basis for designing terrestrial eDNA monitoring studies by summarising the properties and limitations of different substrates and contextualising the interpretation of results in light of substrate-specific challenges.