Environmental DNA metabarcoding of Danish soil samples reveals new insight into the hidden diversity of eutardigrades in Denmark

Tardigrades are rarely included in large biodiversity surveys, mainly because of the impracticalities that follow larger sampling and identification of these microscopic animals. Consequently, there is a lack of data on their biogeographical distribution. Here, we analyse environmental DNA sequences of eutardigrades obtained with a metabarcoding protocol on Danish soil samples collected during a national biodiversity project (Biowide). Specifically, we aimed to investigate the applicability of the V4 region (~400 bp) of the 18S rRNA marker gene to assign taxonomy to 96 eutardigrade molecular operational taxonomic units (MOTUs), using three different methods (alignment-, tree- and phylogeny-based methods). Tardigrade reference libraries are currently suffering from insufficient taxon coverage, in some cases challenging the interpretation of data based on similarity searches. This can, to some extent, be accounted for by supplementing identification with algorithms that incorporate a backbone phylogeny and infer models of evolution. Together, the present findings suggest that the V4 region of the 18S rRNA gene offers a promising tool to identify unknown MOTUs of eutardigrades to genus or family level and can, in some cases, be used to assign to species level.