Evaluation of Preservation and Extraction Methods on <scp>eDNA</scp> Yield and Detection Probability After Long‐Term Storage

Establishing best practices for the overall workflow of environmental DNA (eDNA) sampling is necessary to increase reproducibility and precision in estimates of biodiversity across studies. Rigorous comparisons between eDNA sample preservation strategies for long-term storage durations are lacking, and previous studies have primarily evaluated DNA yield rather than detection success, despite detection being of critical importance when studying rare or elusive species. Here, we assessed the efficacy of common preservation media, storage temperatures and DNA extraction methods on eDNA yield and detection probability after one- and four-years of storage. We found that frozen and ethanol-preserved filters had significantly higher DNA concentrations and detection rates than samples preserved in Longmire's lysis buffer when DNA extraction methods differed among the treatment groups. Substantial inhibition was observed in the Longmire's samples when using a phenol-chloroform-isoamyl alcohol (PCI) extraction method, but did not occur when Longmire's samples were extracted using a Qiagen DNeasy Blood & Tissue Kit. PCI extraction also caused reduced yield and detection rates in ethanol-preserved samples, demonstrating its relative inefficiency for eDNA recovery. eDNA yield and detection rates were highly stable over one- and four-years of storage for all preservation strategies except for ethanol samples stored at room temperature, in which concentrations, but not detection rates, declined significantly after 4 years. Overall, frozen and Longmire's preserved samples had higher yield than ethanol-preserved samples, although detections were high across all media. Our study contributes valuable information towards the optimisation and standardisation of long-term storage protocols of filtered eDNA samples.