Soil organisms are organized in highly diverse communities that provide numerous ecosystem services and contribute decisively to the productivity and resilience of agricultural systems. Over the last decades, however, agricultural intensification has led to a loss of biodiversity, compromising the beneficial functions performed by soil communities. To counteract this decline, a number of agrienvironmental schemes have been implemented to maintain and promote biodiversity. For example, spatial diversification (e.g., flower strips) can effectively promote aboveground biodiversity, whereas little is known on the impact of such measures on belowground communities. Earthworms are an integral component of soil communities as they perform key ecological functions. Earthworms are negatively affected by intensive agricultural management, especially intensive soil management. Consequently, the implementation of perennial structures into agricultural systems (e.g., perennial flower strips and tree rows through agroforestry) is expected to benefit earthworm communities. However, field-based studies validating this assumption, remain scarce. Here, we conducted two studies (Chapters 2 and 3) to evaluate the impact of flower strips and alley-cropping agroforestry on earthworm communities. For that purpose, we sampled earthworms using chemical extraction with allyl isothiocyanate (AITC) under different flower strip mixtures (two annual and two perennial mixtures) and a grassy field margin vegetation at three different sites in Germany (Chapter 2). We found that perennial flower strip mixtures harbored greater earthworm population density and biomass than field margin vegetation, whereas population density and biomass were lower in annual flower strip mixtures as compared to the field margins and perennial flower strip mixtures. The absence of tillage in the field margins and the perennial flower strips as well as high plant diversity of the perennial flower strips are expected to cause the promotion of earthworms. Similar effects of soil management were observed in an alley-cropping agroforestry system in Germany (Chapter 3). Here, we used AITC extraction to sample earthworms in the tree rows, at different distances from the trees into the crop row, and in an adjacent cropland monoculture without trees. We found increased earthworm population density and biomass as well as an altered community composition under the trees as compared to the crop row and the monoculture cropland. The absence of tillage under the trees was most likely the main beneficial factor influencing earthworm communities. In addition, increased above- and belowground litter input in close proximity to the trees might also have promoted earthworms, as some of the recorded positive effects also extended into the crop row. Despite our findings, several knowledge gaps regarding the impact of spatial X diversification measures on earthworms remain (e.g., influence of different flower strip mixtures, tree row orientation, and age of the perennial structures). To fill these knowledge gaps, more field-based studies are required. However, commonly used methods for earthworm sampling and species determination are demanding and expensive. The standardized sampling method for earthworms requires hand sorting of the excavated topsoil and subsequent chemical extraction (e.g., with AITC) of the subsoil. Although this method offers high recovery rates of earthworms, hand sorting is labour-intensive, time-consuming, and destructive towards the sampling site. In Chapter 4 we, therefore, compared this standardized method to a method using only AITC extraction without hand sorting at eleven different sites in Germany. We found AITC extraction without hand sorting to be a viable alternative for investigations regarding anecic earthworms and overall species richness as well as for on-site comparisons of the whole community. Following earthworm sampling, determination of the collected individuals on species level is a necessary step in order to draw conclusions regarding earthworm functions. Species determination is mostly carried out through morphological identification, which is time-consuming, requires taxonomical expertise, and is usually not suitable for the identification of juveniles and cryptic species. Molecular approaches such as DNA barcoding, however, are expensive and hence not commonly used. In Chapter 5, we investigated the potential of high-resolution melting (HRM) curve analysis as a cost-saving alternative to DNA barcoding. In our study, HRM curve analysis enabled the distinction between eight earthworm species commonly found in European agricultural soils. We were also able to distinguish different haplotypes of the earthworm species Allolobophora chlorotica using HRM curve analysis, which indicates the potential of the method to differentiate between cryptic species. Additionally, HRM curve analysis is suitable for the identification of juveniles and damaged individuals and could thus serve as a complementary tool to morphological identification. Overall, the results presented in this thesis show that spatial diversification through perennial flower strips and agroforestry systems generally benefits earthworm communities. Furthermore, it can be concluded that for certain research questions, AITC extraction and HRM curve analysis are viable options to facilitate field-based earthworm research. By this, we hope that remaining knowledge gaps regarding the response of earthworm communities to agricultural management practices can be filled and thereby further practises that preserve the integrity of earthworm communities in agricultural soils can be identified and implemented into agri-environmental schemes.
