Influence of short-term water sample storage on environmental RNA metabarcoding-based biodiversity assessment

Environmental RNA-based metabarcoding has emerged as a powerful tool for biodiversity assessment across diverse disciplines. During field sampling and sample processing, collected water samples often require short-term storage. However, the effects of different storage conditions on biodiversity recovery remain largely unexplored. Here we evaluated the impacts of various storage temperatures (4, 10, 20 °C, and air temperature) and durations (1-72 h) on fish biodiversity recovery from eRNA samples. Our findings revealed that taxon richness declined significantly with increasing storage time and temperature, with storage time having a greater impact than temperature. No taxa were detected after 72 h of storage under any tested temperature condition. Although high-abundance taxa were generally more resilient, they still showed substantial declines in detectability over time, with a 9.5 %-35.7 % reduction in taxon richness observed after just 1 h of storage. Low-abundance taxa experienced a faster and more pronounced decline, with many detected only transiently. Both storage time and temperature, as well as taxon abundance, significantly affected detection rates, with taxon abundance having the strongest effect, followed by storage time and storage temperature. In addition to affecting taxon detection, short-term storage significantly impacted community structure and reduced the reproducibility of replicates. These findings highlight the potential for increased false negatives during short storage periods or even brief sample processing times. Meanwhile, these results provide crucial empirical evidence for developing standardized handling procedures in aquatic eRNA research and contribute to the broader methodological framework for reliable biodiversity monitoring using eRNA.