Environmental DNA is more effective than soil-pit hand sorting in evaluating earthworm biodiversity responses to more regenerative agricultural management

Restoring and maintaining soil biodiversity is important for the sustainability of our food systems, particularly as the health of agricultural soils continues to degrade at alarming rates. Earthworms are key components of soil biodiversity and provide many benefits to agricultural soil functioning, but effective and standardised approaches to monitor their populations are lacking. In this study, the feasibility of using soil environmental DNA (eDNA) metabarcoding for surveying earthworm populations in agroecosystems was explored. Soil samples were collected from arable fields with temporary grass-clover ley strips sown into them, to investigate the effects of different land use treatments on earthworm communities. Extracellular earthworm eDNA was extracted, amplified and sequenced using next-generation sequencing, and earthworm populations were also sampled using a traditional hand-sorting approach. This allowed the performance of the eDNA method to be assessed and quantitative comparisons to be made between the different methods. The eDNA method detected significantly higher local species richness than the traditional hand-sorting approach, with the site occupancy proportions indicating that eDNA was markedly better at detecting anecic (deep burrowing) earthworms. The high sensitivity of eDNA sampling enabled the detection of differences in the earthworm communities resulting from different land-use treatments in the same field, with significantly higher earthworm diversity detected in the 33-month old ley strips than in the adjacent arable soils. Both relative abundances and site occupancy proportions were used as potential proxies for abundance, and the performance of each of these measures and the implications for further work are discussed. eDNA sampling has potential as a powerful tool for monitoring agricultural earthworm diversity and soil health, and could be utilised on farms to promote better management and soil care. ### Competing Interest Statement The authors have declared no competing interest.