A window into dark taxa: morphology-compatible DNA extraction for tiny soil arthropods

• Rapid, non-destructive extraction protocol without harmful or hazardous reagents. • Specimen integrity preserved. • Sufficient DNA from minute arthropods for downstream PCR analyses provided. • Integrative taxonomy of fragile, soft-bodied organisms possible. • Hitherto unrecognized diversity in Pauropoda and brachychthoniid mites. Soil is one of the most biodiverse habitats on Earth, hosting organisms that are essential for ecosystem functioning and indicators of soil health. However, most of these taxa are severely understudied, especially ‘dark taxa’ such as mites and pauropods—tiny, fragile organisms for which adequate taxonomic expertise and genetic reference data are lacking. Effective biodiversity monitoring requires integrative approaches that preserve both DNA and morphology, yet most DNA extraction methods are destructive. We present a rapid, non-destructive protocol using the Chelex®-100 ion-exchange resin that enables DNA extraction while maintaining specimen integrity for morphological analysis. Applied to 132 minute arthropods (Pauropoda and Brachychthoniidae), the method achieved 100 % DNA extraction success and > 88 % sequencing success for mitochondrial (COI) and nuclear (D3-28SrRNA) markers. Combining molecular and morphological data from the same individual allowed us to provide first reference DNA barcode data for many of our study taxa and revealed large unknown species diversity. Compared to existing DNA extraction techniques, our protocol is simpler, faster, and better suited for fragile, weakly sclerotized arthropods. It facilitates the development of robust DNA reference libraries from morphologically identified specimens, advancing integrative taxonomy and enabling reliable, DNA-based monitoring of minute and ecologically important, but hitherto understudied soil organisms.