Phylo-MIP: Phylogeny-based Molecular Identification Pipeline for DNA metabarcoding, and assessment of insect communities in subalpine river ecosystems

Environmental DNA metabarcoding is a powerful tool for biodiversity assessment; however, species identification often relies on sequence similarity searches (BLAST), which are limited by incomplete reference databases and result in frequent misidentifications or unclassified taxa. To overcome these challenges, we designed a novel analytical pipeline, Phylo-MIP, which integrates phylogenetic analysis and species delimitation. Phylo-MIP combines sequence alignment, haplotype thinning, phylogenetic tree construction, and multiple species delimitation methods (bPTP, bPTP-ML, mPTP) to objectively identify Molecular Operational Taxonomic Units (MOTUs) without relying on reference databases. As a validation, we applied Phylo-MIP to previously published eDNA data from river in Kanagawa Prefecture, detecting approximately 28% more species than only BLAST-based methods. We also conducted a eDNA survey in the subalpine zone of Kamikochi, Nagano Prefecture, Japan, where Phylo-MIP revealed distinct community structures associated with specific aquatic environments. Phylo-MIP enables non- invasive and efficient biodiversity monitoring, even in protected areas, and offers a reproducible framework for ecological research. This approach has the potential to become a new standard method in river ecology, taxonomy, conservation biology, and ecosystem management.