Environmental <scp>DNA</scp> Template Variation: Its Relevance for Species Detection and Conservation

ABSTRACT Accurate species monitoring is foundational for understanding and assessing species extinction risk. Environmental DNA (eDNA) based species detection methods have been proposed as fast and powerful biodiversity monitoring tools. Yet, these methods are susceptible to errors that might hinder the assessment of species extinction risk. Samples may contain low DNA concentrations of the target taxa and/or exhibit high levels of PCR inhibitors, which can yield false negatives. We investigated how adjusting the input sample volume in the eDNA‐based molecular assay improves detection of an endangered fish, Anaecypris hispanica , in highly eutrophic streams. Water samples were filtered and tested using a real‐time PCR (qPCR) assay varying the input volume of eDNA samples (i.e., 0.5X, 1X and 3.3X). From the positive detections obtained with different eDNA input volumes, we built species occurrence maps and estimated geographic range metrics used in species extinction risk assessment. Although the number of sites with positive detections was similar among the input eDNA sample volumes tested, positive detections were not spatially redundant. When comparing the pooled results from all eDNA‐based trials to a fixed 1X eDNA volume, there was a nearly 75% increase in the number of sites with detections, consequently leading to increases in all geographic range metrics (i.e., extent of occurrence, area of occupancy number of locations). Our results highlight that false negatives in eDNA‐based surveys are not to be overlooked. The success of species detection will likely vary on a case‐by‐case basis, depending on the DNA concentration of the target taxa and the concentration of potential inhibitors in bulk eDNA samples, both of which are generally unknown. Improved species detection may be achieved by running, in parallel, qPCR assays with different input volumes of bulk eDNA samples. As interest grows in integrating eDNA‐based tools into species monitoring practices, it is essential to continuously refine protocols and carefully consider study design decisions to ensure robust results, advancing species management and conservation.