Assessment of Hyporheic Macroinvertebrate Biodiversity Within Gravel Bars Before and After Flooding: Comparison of Morphology, Bulk‐Sample, and eDNA Metabarcoding

Studies validating the efficiency of environmental DNA (eDNA) metabarcoding for hyporheic biodiversity assessment are still limited. Hence, we investigated species diversity in the hyporheic zone beneath gravel bars, focusing on lateral locations along the gravel bar transect and the effects of flooding. We compared the accuracy of metabarcoding with morphological identification, specifically focusing on eDNA metabarcoding and bulk‐sample metabarcoding data to understand the effect of river surface water contamination on eDNA analysis in the hyporheic zone. Despite challenges such as primer bias and surface water contamination, our metabarcoding analysis of both bulk‐sample and eDNA metabarcoding yielded results consistent with morphological identification, providing evidence that the hyporheic zone has a more stable community structure than the river benthic zone in response to flooding. Furthermore, the homogeneity of hyporheic community structures across lateral locations based on eDNA and bulk‐sample metabarcoding suggests that the hyporheic community may be particularly vulnerable to biodiversity loss compared to the river benthic community under extreme flood disturbance. Notably, eDNA metabarcoding demonstrated higher sensitivity for detecting low abundance and elusive taxa compared to both bulk‐sample metabarcoding and morphological identification. Overall, our results highlight the potential of eDNA metabarcoding as a comprehensive, less invasive, and more time‐efficient tool for assessing biodiversity in the hyporheic zone.