Environmental DNA for endangered freshwater mussel monitoring: a review on global synthesis of eDNA methods, challenges, and innovative strategies
Freshwater mussels (family Unionidae) are among the most endangered aquatic species worldwide, making effective monitoring vital for their conservation. Traditional surveys are laborious, time-consuming, and often miss rare or low-density populations. Environmental DNA (eDNA) is a highly sensitive, non-invasive approach for freshwater mussel detection and monitoring; however, uncertainties in ecological interpretation continue to limit its broader application in freshwater biodiversity monitoring. We conducted a systematic review of 49 peer-reviewed studies, identified through an extensive literature search, to assess temporal research trends, methodological advancements, ecological contexts, and regulatory readiness of freshwater mussel eDNA monitoring. Findings show that freshwater mussel eDNA research has surged since 2020, particularly in the United States, which accounts for the largest share of published studies (38.8%). Quantitative polymerase chain reaction (qPCR) was the most frequently applied detection method (46%), followed by metabarcoding (32%), indicating a shift from species-specific detection toward community-level biodiversity assessment. Water was the principal sampling matrix, while freezing and silica-based extraction kits were the most commonly reported preservation and DNA-extraction approaches. The review demonstrates that eDNA greatly enhances the detection of freshwater mussels, particularly rare and endangered species, and supports large-scale biodiversity assessments and conservation planning. However, significant variation exists in sampling strategies, laboratory workflows, molecular markers, bioinformatics pipelines, and quality control practices. Ecological interpretation remains complicated by factors such as hydrological transport, seasonal biological variation, eDNA degradation, PCR inhibition, incomplete reference databases, and unclear relationships between eDNA levels and population size. Overall, this review highlights that eDNA has complemented traditional surveys rather than replacing them. To facilitate regulatory adoption and conservation, ongoing efforts should focus on standardizing protocols, expanding reference databases, improving quantitative analyses, across laboratories, and integrating ecological and hydrological data.