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Molecular approaches are offering a supplement to, or even the possibility of replacing morphological identification of soil fauna, because of advantages for throughput, coverage and objectivity. We determined ecological indices of nematode community data from four sets of duplicate soil cores, based on morphological identification of nematodes after elutriation from 200 g soil and high throughput sequencing (HTS) targeting nematodes both after being elutriated from soils and DNA extracted directly from 10 g soil. HTS (at genus and species level) increased the taxonomic resolution compared to morphology (at family level). DNA extracted from elutriated nematodes identified more nematode taxa than when extracted from soil, due to an enrichment in nematode sequences. Each method also gave a different ecological footprint for the nematode community. Standardisation to previously determined indices based on morphological identification is needed in order to provide more meaningful information about soil quality and for ecological monitoring.

Protists are the most diverse eukaryotes. These microbes are keystone organisms of soil ecosystems and regulate essential processes of soil fertility such as nutrient cycling and plant growth. Despite this, protists have received little scientific attention, especially compared to bacteria, fungi and nematodes in soil studies. Recent methodological advances, particularly in molecular biology techniques, have made the study of soil protists more accessible, and have created a resurgence of interest in soil protistology. This ongoing revolution now enables comprehensive investigations of the structure and functioning of soil protist communities, paving the way to a new era in soil biology. Instead of providing an exhaustive review, we provide a synthesis of research gaps that should be prioritized in future studies of soil protistology to guide this rapidly developing research area. Based on a synthesis of expert opinion we propose 30 key questions covering a broad range of topics including evolution, phylogenetics, functional ecology, macroecology, paleoecology, and methodologies. These questions highlight a diversity of topics that will establish soil protistology as a hub discipline connecting different fundamental and applied fields such as ecology, biogeography, evolution, plant-microbe interactions, agronomy, and conservation biology. We are convinced that soil protistology has the potential to be one of the most exciting frontiers in biology.

Soils provide many ecosystem services that are ultimately dependent on the local diversity and below ground abundance of organisms. Soil biodiversity is affected negatively by many threats and there is a perceived policy requirement for the effective biological monitoring of soils at the European level. The aim of this study was to evaluate and recommend policy relevant, cost-effective soil biological indicators for biodiversity and ecosystem function across Europe. A total of 18 potential indicators were selected using a logical-sieve based approach. This paper considers the use of indicators from the 'top down' (i.e. concerned with the process of indicator selection), rather than from the 'bottom up' detail of how individual indicators perform at specific sites and with specific treatments. The indicators assessed a range of microbial, faunal and functional attributes, newer nucleic acids based techniques, morphological approaches and process based measurements. They were tested at 6 European experimental sites already in operation and chosen according to land-use, climatic zone and differences in land management intensity. These were 4 arable sites, one each in Atlantic, Continental, Mediterranean and Pannonian climate zones, and 2 grassland sites, one each in Atlantic and Continental zones. At each site we sampled three replicated plots of contrasting management intensity and, while the treatments varied from site to site, their disturbance effects were quantified in terms of land use intensity. The field sampling and laboratory analysis were standardised through a combination of ISO protocols, or standard operating procedures if the former were not available. Sites were sampled twice, in autumn 2012 and spring or autumn 2013, with relative costs of the different indicators being determined each time. A breakdown of the cost effectiveness of the indicators showed the expected trade-off between effort required in the field and effort required in the laboratory. A

Soil organisms are considered drivers of soil ecosystem services (primary productivity, nutrient cycling, carbon cycling, water regulation) associated with sustainable agricultural production. Soil biodiversity was highlighted in the soil thematic strategy as a key component of soil quality. The lack of quantitative standardised data at a large scale has resulted in poor understanding of how soil biodiversity could be incorporated into legislation for the protection of soil quality. In 2011, the EcoFINDERS (FP7) project sampled 76 sites across 11 European countries, covering five biogeographical zones (Alpine, Atlantic, Boreal, Continental and Mediterranean) and three land-uses (arable, grass, forestry). Samples collected from across these sites ranged in soil properties; soil organic carbon (SOC), pH and texture. To assess the range in biodiversity and ecosystem function across the sites, fourteen biological methods were applied as proxy indicators for these functions. These methods measured the following: microbial diversity: DNA yields (molecular biomass), archaea, bacteria, total fungi and arbuscular mycorrhizal fungi; micro fauna diversity: nematode trophic groups; meso fauna diversity: enchytraeids and Collembola species; microbial function: nitrification, extracellular enzymes, multiple substrate induced respiration, community level physiological profiling and ammonia oxidiser/nitrification functional genes. Network analysis was used to identify the key connections between organisms under the different land use scenarios. Highest network density was found in forest soils and lowest density occurred in arable soils. Key taxomonic units (TUs) were identified in each land-use type and in relation to SOC and pH categorisations. Top-connected taxonomic units (i.e. displaying the most co-occurrence to other TUs) were identified for each land use type. In arable sites this was dominated by bacteria and fungi, while in grassland sites bacteria and fungi were most con