Diversity of Niphargus (Crustacea, Amphipoda) in France, as revealed by DNA barcoding approach

Niphargus is the most speciose amphipod genus inhabiting groundwater of the Western Palearctic. The genus is renowned for its notorious taxonomy, associated with large within- but often negligible between-species morphological variation. Results of molecular taxonomy suggested that morphological variation captured less than half of its species diversity, calling for a molecular revision of past taxonomic and faunistic works. Molecular assessment of species diversity within Niphargus has been conducted since the 2000s, but it remains spatially incomplete given the genus’ extensive geographic range. Here, we provide the first comprehensive assessment of the diversity of Niphargus in France using molecular markers. We sequenced the COI mitochondrial marker from 436 individuals collected from 234 locations. Using Assemble Species by Automated Partitioning, we identified 99 molecular operational taxonomic units (MOTU), among which 74 could represent potential new species. In turn, we selected one individual per MOTU, amplified nuclear markers 28S and Histone 3, aligned them with an additional 142 MOTUs from across the genus range, and reconstructed their phylogenetic relationships using the Maximum Likelihood method. We found that several nominal species occurring in France are polyphyletic. Furthermore, Niphargus from France comprised lineages derived from deep and terminal splits, some of which showed signs of evolutionary radiation. The phylogenetic relationships are consistent with the hypothesis that Niphargus originated in the western part of its range. This study addressed a knowledge gap in the molecular and spatial coverage of Niphargus , provided new barcodes for monitoring the diversity of the genus, and identified future directions for research.