Three sampling strategies reveal spatial and methodological patterns in macroinvertebrate diversity in freshwater habitats

Abstract

Summary Assessing macroinvertebrate diversity is crucial for freshwater ecosystem management. Environmental DNA (eDNA) metabarcoding has emerged as a powerful, non-invasive tool that complements conventional morphological methods. In this study, we compared morphological sampling, ethanol-derived eDNA from preserved specimens, and water-based eDNA to evaluate macroinvertebrate diversity in the Burhatong and Hailan Rivers of the Tumen River Basin, China. Water-derived eDNA revealed the highest species richness and α-diversity, underscoring method-specific detection patterns. Despite these biases, strong correlations between metabarcoding read abundance and morphological data supported the reliability of all three approaches for within-habitat (α) diversity assessment. Consistent β-diversity patterns across methods further validated their comparability. Among environmental factors, total suspended solids (TSS) emerged as the dominant driver, with α-diversity peaking at moderate TSS levels, consistent with the intermediate disturbance hypothesis. By integrating morphological identification, ethanol-eDNA, and water-eDNA metabarcoding, this study demonstrated that despite method-specific biases, all the approaches captured consistent ecological gradients in riverine macroinvertebrate communities along an East Asian international border.