Enhancing groundwater metazoan metabarcoding through primer performance assessment and reference library updates

Groundwater is among the planet's most essential natural resources and a crucial part of the global freshwater cycle. It also harbours a specialized aquatic fauna, the stygofauna, which plays a pivotal role in subterranean ecosystem functioning. DNA metabarcoding provides a powerful tool to better uncover the previously largely hidden aquifer communities. It does so by circumventing taxonomic impediments and uncovering cryptic species, simultaneously enabling the assessment of the impacts of pollution and climate change. However, the application of (e)DNA metabarcoding for groundwater metazoans is hindered by two major obstacles: the scarcity of validated primer pairs and the limited taxonomic coverage of reference databases. In this study, we sought to strengthen the molecular toolbox for groundwater biodiversity research by tackling these two challenges. We demonstrate that the primer pair fwhF2 / fwhR2n, targeting the mitochondrial COI region, provides the most effective amplification of central European groundwater invertebrates, particularly crustaceans. In addition, we contributed targeted updates to public databases (for this study assembled in the Groundwater invertebrate Database; GwinD), particularly for 30 Copepoda, five Bathynellacea, and five Ostracoda species. The toolbox was applied in a case study, demonstrating its utility for (e)DNA metabarcoding of central European groundwater communities while highlighting persisting limitations for several non-crustacean groups.