Assessment of the composition of the belowground community
Nematode communities. Nematodes were extracted from 200 g of composite soil sample using the elutriator - cotton wool filter method (Oostenbrink 1960). Nematode suspensions were concentrated and DNA was extracted by a lysis buffer including mammalian DNA as an external standard to monitor losses due to sample handling and DNA purification (Vervoort et al. 2012). DNA extracts were purified using a glass-fiber column-based procedure (Ivanova et al. 2006). Purified DNA extracts were stored at -20 ºC. To assess overall nematode biodiversity across all sites, 5 µl aliquots of all purified extracts were combined. The resulting mixture was analysed by qPCR using 72 nematode taxon-specific primer sets (Table S5). Based on the outcome of the overall biodiversity assessment, 30 nematode taxa were selected for qPCR-based quantification in each of the 98 samples. Two additional qPCR primer sets were used: one primer set was used to assess total nematode densities per sample, and a second primer set was used to quantify DNA levels of the external standard. Quantitative PCR reactions were executed and Ct-values were converted to nematode counts per 200 g soil. For details see Vervoort et al. (2012); Quist et al.(2017).
Microbial communities. Genomic DNA of bacteria, fungi, and protists was extracted from 1.5 gram of each composite soil sample using the Qiagen DNeasy PowerSoil 96-well extraction method. DNA was amplified in triplicate using primers specific to targeted regions within either the 16S or 18S rRNA gene (for prokaryotic and eukaryotic analyses, respectively). A portion of the 16S rRNA gene was amplified using the archaeal- and bacterial-specific primer set 515f/806r (Bates et al. 2011). This 16S rRNA gene primer set is designed to amplify the V4–V5 region of both Archaea and Bacteria, has few biases against specific taxa and accurately represents phylogenetic and taxonomic assignment of sequences (Liu et al. 2007). The 18S rRNA gene was amplified using the eukaryotic-specific primer set F1391 (50-GTACACCGCCCGTC-30) and REukBr (50-TGATCCTTCTGCAGGTTCACCTAC-30). The 18S rRNA gene primer set is designed to amplify the hypervariable V9-region of eukaryotes, with a focus on microbial eukaryotic lineages (Amaral-Zettler et al. 2009), including both protists and fungi. Amplicons were sequenced on two lanes of a 2 x 151 bp sequencing run on the Illumina HiSeq 2500 operating in Rapid Run Mode, following (Caporasoet al. 2012).
Microarthropod communities. To determine the community composition of microarthropods (mites: Acari, and springtails: Collembola), the large intact soil core was extracted using the Tullgren extractors at Lancaster University. Per plot, the batch of extracted microarthropods was collected in 96% ethanol and further processed to enable DNA-based identification. DNA extraction was performed using the DNeasy Blood & Tissue kit. DNA was amplified using the MiteMinBarF7 and MiteMinBarR4 primers, which target a ~200 bp fragment located within the cytochrome oxidase subunit 1 (COI) region, and were specifically designed to cover a wide diversity of microarthropods in NW-European grasslands (de Groot et al. 2016). At Aarhus University (Roskilde, Denmark), amplicons were prepared for in-house paired-end sequencing on an Illumina MiSeq platform, using the Nextera XT indexing kit (Illumina, San Diego, CA, USA). The resulting amplicon libraries were purified using HighPrep™ PCR (Magbio Genomics Inc., Gaithersburg, Maryland, US) beads, quantified and equimolarly pooled, upon sequencing using the 250 bp paired end MiSeq version 2 reagent kit (Illumina, San Diego, CA, USA).