An annotated reference library for supporting DNA metabarcoding analysis of aquatic macroinvertebrates in French freshwater environments

Abstract

Freshwater ecosystems are increasingly threatened by human activities, leading to biodiversity loss and ecosystem degradation. Effective biodiversity monitoring, particularly through the use of aquatic macroinvertebrates as bioindicators, is crucial for assessing ecological health. While traditional morphological methods face limitations, DNA metabarcoding offers higher accuracy and efficiency in species identification using environmental DNA. However, the success of metabarcoding is contingent on the quality of reference libraries, which are often incomplete or biased. This study aims to construct a comprehensive COI-based DNA barcode library for freshwater macroinvertebrates in France, specifically targeting short gene regions amplified with fwhF2/fwhR2N primers, suitable for degraded DNA. Data from the PERLA database, Alpine lake surveys, and Opie-benthos were consolidated and refined using TAXREF to produce a final species checklist for France. The resulting library was analyzed for taxonomic completeness and genetic diversity. The checklist consists of 2,841 species across 10 phyla, with Arthropoda being the most diverse. Despite this, 56% of species lack COI-5P sequences in the Barcode of Life Data System (BOLD), with Mollusca and Nematoda showing particularly low coverage. Alignment challenges were identified for certain taxa, particularly within Coleoptera, Diptera, and Malacostraca. This work highlights the need for expanded sequencing efforts to improve barcode coverage and enhance the accuracy of biodiversity assessments in freshwater ecosystems.