Environmental DNA is more effective than hand sorting in evaluating earthworm biodiversity recovery under regenerative agriculture

Regenerating soil biodiversity can help to reverse declines in soil health caused by cultivation and continuous arable cropping, and support sustainable food production and agro-ecosystem services. Earthworms are key functional components of soil biodiversity, with different ecological categories and species delivering specific beneficial soil functions. Conventional monitoring by hand-sorting from soil pits is highly labour intensive, can reliably identify only adults to species, and may under-record anecics (deep-burrowers). Here, we compare soil environmental DNA (eDNA) metabarcoding using two different primer sets and next-generation sequencing, with earthworm hand-sorting from standard soil-pits, in four conventionally managed arable fields into which strips of grass-clover ley had been introduced three years earlier. Earthworm populations had been recorded by hand-sorting in the previous three years and our goal was to assess the effects of the three-year leys compared to arable cropping using both hand-sorting and eDNA. The eDNA method found the same eight earthworm species as hand-sorting, but had greater power for detecting anecic earthworms and quantifying local species richness. Earthworm abundance increased by over 55% into the third year of the leys, surpassing abundances in adjacent permanent grasslands, helping to explain the observed soil health regeneration. Both overall relative read abundances and site occupancy proportions of earthworm eDNA were found to have potential as proxies for abundance, and the performance of each of these measures and the implications for further work are discussed. We show that eDNA can improve earthworm diversity monitoring and recommend its wider use both to better understand soil management effects on earthworm populations, and to guide agricultural policy and practice decisions affecting soil health.