Assessment of Freshwater Unionidae Using Environmental DNA Metabarcoding in Lentic Ecosystems: Implications for Spatial Sampling Strategies

Environmental DNA (eDNA) metabarcoding has become a powerful, non-invasive method for detecting aquatic organisms. However, optimal sampling strategies for benthic taxa in lentic ecosystems remain unclear. This study evaluated the effectiveness of eDNA metabarcoding in detecting freshwater Unionidae mussels in lake water columns and examined their spatial and seasonal distribution patterns. We validated a mini-barcode primer targeting the mitochondrial 16S rDNA of unionid mussels through controlled laboratory experiments and field tests, confirming reliable amplification and accurate taxonomic assignment of freshwater bivalve DNA. Field surveys were conducted in four lakes within the Nakdong River basin, where eDNA samples were collected from littoral zones and from surface, mid-, and bottom layers of central lake areas during autumn and winter. Metabarcoding analysis identified 79 amplicon sequence variants (ASVs) representing four unionid taxa, with <i>Cristaria plicata</i> and <i>Sinanodonta lauta</i> comprising the majority of reads and ASVs. Overall, the number of Unionidae eDNA reads showed no significant seasonal differences, but there was notable spatial variation among sampling locations. Read numbers were significantly lower in littoral zones compared to central lake areas, with no significant differences detected among depth layers within the central zones. Species-specific analyses revealed contrasting spatial patterns: <i>C. plicata</i> had higher read abundance in mid- and bottom layers, while <i>S. lauta</i> was more frequently detected in surface and littoral samples. These findings suggest that the distribution of freshwater mussel eDNA in lakes is primarily influenced by spatial factors related to habitat preference and hydrodynamic mixing, rather than by seasonal variation during stable periods. This study offers practical insights for designing effective eDNA sampling strategies for benthic invertebrates in lentic ecosystems.