Most soil and litter arthropods are unidentifiable based on current DNA barcode reference libraries.

We are far from knowing all species living on the planet. Understanding biodiversity is demanding and requires time and expertise. Most groups are understudied given problems of identifying and delimiting species. DNA barcoding emerged to overcome some of the difficulties in identifying species. Its limitations derive from incomplete taxonomic knowledge and the lack of comprehensive DNA barcode libraries for so many taxonomic groups. Here, we evaluate how useful barcoding is for identifying arthropods from highly diverse leaf litter communities in the southern Appalachian Mountains (USA). We used 3 reference databases and several automated classification methods on a data set including several arthropod groups. Acari, Araneae, Collembola, Coleoptera, Diptera, and Hymenoptera were well represented, showing different performances across methods and databases. Spiders performed the best, with correct identification rates to species and genus levels of ~50% across databases. Springtails performed poorly, no barcodes were identified to species or genus. Other groups showed poor to mediocre performance, from around 3% (mites) to 20% (beetles) correctly identified barcodes to species, but also with some false identifications. In general, BOLD-based identification offered the best identification results but, in all cases except spiders, performance is poor, with less than a fifth of specimens correctly identified to genus or species. Our results indicate that the soil arthropod fauna is still insufficiently documented, with many species unrepresented in DNA barcode libraries. More effort toward integrative taxonomic characterization is needed to complete our reference libraries before we can rely on DNA barcoding as a universally applicable identification method.