# Properties and Limitations of eDNA Substrates for Terrestrial Animal Monitoring

## TL;DR

This paper reviews the use of environmental DNA (eDNA) for monitoring terrestrial animals, highlighting the importance of substrate choice and its impact on detection accuracy.

## Contribution

The paper introduces the concepts of spatial and temporal deviations in eDNA monitoring and categorizes substrates to guide study design.

## Key findings

- eDNA substrates are grouped into abiotic, biotic, and direct-evidence types with distinct properties and limitations.
- Spatial and temporal deviations between DNA sources and detection in samples are key challenges in eDNA monitoring.
- The review provides a decision framework for selecting substrates based on research objectives and expected accuracy.

## Abstract

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.