Environmental DNA and RNA in aquatic community ecology: Toward methodological standardization

Abstract

Molecular technologies have facilitated the expansion of biodiversity assessments across a broad range of organisms and aquatic systems. Environmental DNA (eDNA) and environmental RNA (eRNA), collectively referred to as environmental nucleic acids (eNAs), have revolutionized biodiversity monitoring due to their noninvasive nature and high-resolution capabilities when compared to traditional survey methods. While eNA applications have grown exponentially over the past decade, methodological inconsistencies hinder reproducibility and comparability. To assess the current state of eNA methodologies in aquatic ecology, we conducted a systematic review of 300 peer-reviewed eNA studies that assess ecological communities across diverse aquatic systems. Of these papers, 291 examine eDNA, four examine eRNA, and five consider both eDNA and eRNA. The small number of eRNA studies compared to eDNA highlights the eRNA field is in its infancy. Of the eNA studies looked at, we found a clear geographic bias, with approximately 50% of all eNA studies occurring in six high-income countries, while less than 10% of eNA studies occur across South America and Africa. Further, we report a lack of methodological standardization across eNA studies, showing high variability in water filtration volume, filter material, filter pore size, eNA extraction method, marker choice, and bioinformatic pipelines. We further highlight that incomplete reference sequence databases for both eRNA and eDNA limit taxonomic assignment and biodiversity inferences. Finally, we identify a systematic issue across eNA studies in community ecology: missing methodological details, which compromise reproducibility, especially in newly emerging eRNA applications. To facilitate the standardization of eNA monitoring across aquatic systems and permit the further integration of eNA applications in biodiversity monitoring, we recommend the improvement of reference databases alongside guidelines that encourage methodological transparency.