Targeting terrestrial vertebrates with eDNA: Trends, perspectives, and considerations for sampling

Terrestrial vertebrates are experiencing worldwide population declines and species extinctions. To effectively conserve remaining populations and species, rapid, cost effective and scalable methods are needed to complement longstanding monitoring methods. Increasingly, environmental DNA (eDNA) based approaches are being used for terrestrial vertebrate biomonitoring within a range of environments. However, as we move eDNA biomonitoring onto land, we are presented with a new set of challenges. This necessitates the development of best-practice eDNA sample collection guidelines for terrestrial systems with the purpose of detecting terrestrial vertebrates. To address these needs we conducted a systematic literature review of 143 peer-reviewed papers applying eDNA to terrestrial vertebrate monitoring (excluding Lissamphibia) that were published between 2012 and 2023. We summarize the use of eDNA for terrestrial vertebrate biomonitoring, focusing on study design and field techniques. Over the decade we observe a steady growth in the annual number of publications, with 3 in 2012 and 33 in 2023. The majority of the reviewed studies targeted terrestrial mammals within temperate forest regions. While an equal number of studies focused on a metabarcoding approach to assess community taxon composition and/or species-specific eDNA detection methods, novel uses are increasingly published. These include studies of animal behaviour and population genetics. We record three types of sampling strategies, eight different substrate types and seven different preservation methods, suggesting there is no one size fits all eDNA based sampling methodology when detecting terrestrial vertebrates. With a multitude of study aims, across different environments, and target organisms with different ecologies, the standardization of eDNA sampling approaches in terrestrial systems is extremely challenging. We summarize in a table known factors influencing eDNA detection within terrestrial environments. Furthermore, we identify five key considerations to be addressed when sampling for eDNA studies targeting terrestrial vertebrate species, with the aim of guiding decision making.