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).