COI Metabarcoding with a Curated Reference Database and Optimized Protocol Provides a Reliable Species-Level...

DNA metabarcoding is revolutionizing biodiversity research by providing rapid and efficient ways of collecting species occurrence data. However, it has not yet been effectively applied to many taxonomic groups, mainly due to a significant lack of reference sequences and dedicated protocols. One such group is the tardigrades-a charismatic phylum of microinvertebrates known for their extremophilic and cryptobiotic capabilities. In this study, we provide the first curated database of 3194 tardigrade COI sequences sourced from public databases and supplemented with newly produced barcodes. We demonstrate tardigrade metabarcoding in action with optimized PCR primers and a sample processing protocol using 78 samples collected in Poland and Italy. The metabarcoding revealed the presence of more than a hundred operational taxonomic units classified as Tardigrada, representing 23 genera. We compared the metabarcoding results with a morphological survey, which revealed the presence of the same genera, but a lower number of species-level taxa identified morphologically. We observed congruent patterns of tardigrade species richness and taxonomic composition between metabarcoding and morphological surveys in both within-sample and regional fauna composition levels. The metabarcoding had a higher discriminatory power, revealing cryptic diversity, and distinguishing species belonging to taxonomically challenging species complexes. By combining metabarcoding with morphological study, we were able to find rare taxa, including novel biogeographic records and putative species new to science, showing also that this approach can be extremely powerful and effective in meiofauna research.