Over the last decade, the use of eDNA-based detection to monitor aquatic biodiversity in both marine and freshwater systems has rapidly increased¹. The reproducibility of eDNA surveys relies on the assumption that the DNA detected provides an accurate measure of presence of the local community or targeted species at the respective point in time and space^2,3. As many conservation and management strategies have now adopted eDNA-based surveys^4,5, it is urgent to understand the various processes that influence eDNA persistence in aquatic systems so that accurate inferences of a species presence can be made from the detection of its eDNA. Indeed, previous studies highlighted that eDNA stability can vary in systems depending on many parameters, including species-specific eDNA shedding rates, seasonality, and environmental conditions^6–8. When organisms shed DNA into the water column, this gives rise to extra-organismal eDNA (i.e. DNA no longer associated with its organism of origin) and can take the form of at least four states^9,10. These four states include: dissolved DNA, DNA bound to the surfaces of suspended particles^3,6,10, and DNA still encapsulated in either a cell or an organelle¹¹. What we currently lack is a robust understanding of how water chemistry and other environmental parameters affect which eDNA state(s) predominate in specific aquatic environments and how they persist.

The state-of-the-art is to extract eDNA from water and target a single species or whole communities of species using a set of primers and Polymerase Chain Reaction (PCR)¹². However, the presence of eDNA in different states has implications for data interpretation, as detection of species might be influenced by the ‘detectability’ of a specific state that is the result of both the environmental parameters determining the state and the analytical workflow (i.e., preservation, capture, extraction and detection methods) used to isolate the DNA from the water column. Consequently, the relative distribution of eDNA among the different states could affect the probability of detection for a targeted species’ DNA. Therefore, the currently unknown stabilities of eDNA in different states combined with the lack of information on which eDNA states are being detected creates large uncertainty for the spatial and temporal inferences that can be made from extra-organismal eDNA detection^3,13. To reduce this uncertainty, we require a better understanding of the states that eDNA assumes, the processes converting eDNA between different states, and the variations in state-dependent eDNA decay rates.

In this perspective, we describe four principal states of eDNA that are likely in aquatic environments. Based on the presumed chemical behaviour of each state, we discuss how environmental parameters, such as temperature, pH and suspended particles, may influence the conversion of eDNA between states (e.g.,¹⁴). We briefly review what is known about DNA decay, covered in detail elsewhere^3,12,13, and summarize what has been observed from experimental studies on eDNA decay in relation to the environmental parameters of temperature and pH. We then present the results of a literature search to ascertain what states of eDNA are likely being detected using single-species eDNA assays. Lastly, we outline a number of analytic controls, which, if used, will help to assess the loss of specific states from aquatic samples and allow for post hoc observations about the state(s) contributing to species detection. We close with suggestions for future research that would help to fill knowledge gaps regarding the space and time inference that can be made from extra-organismal eDNA species detections.