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

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.