Innovative approaches in aquatic monitoring: a comparative study between eDNA analysis and morphological techniques in a river ecosystem

Freshwater biomonitoring requires rapid and reliable diagnostic tools to minimize ecological impacts. This study compares morphological identification with environmental DNA (eDNA) metabarcoding data to evaluate which method more efficiently assesses macroinvertebrate communities across contrasting land uses. Benthic macroinvertebrates were collected in the Paraná River basin for morphological analysis, while water and biofilm samples were obtained for eDNA metabarcoding. A total of 125 amplicon sequence variants (ASVs) – high-resolution DNA sequence clusters used as taxonomic units - were detected, most of which were identified to the species or morphospecies level. The eDNA biofilm-derived recovered a higher number of ASVs than water samples, where Annelida, Arthropoda, and Mollusca are the most diverse groups. Diversity metric (richness and community composition) and ecological status classifications were generally comparable between morphological and molecular approaches. The primers employed demonstrated good taxonomic resolution, supporting their usefulness in assessing river quality and enabling the application of region-specific biotic indices. Although metabarcoding performed similarly to morphological methods in assessing diversity patterns and ecological status, 70% of the taxa identified morphologically were not detected molecularly. These limitations, particularly the under-detection of key benthic phyla, indicate that in tropical rivers, an integrative framework combining both methods yields the most accurate evaluation of biodiversity and environmental health.