ferrinetal_c&nmediatewarmingimpactonsoilinsects

This study was conducted at the ForHot research site in Iceland (Sigurdsson et al., 2016) between August 2017 and June 2018 (64°0′N, 21°11′W). Soil type was a Brown Andosol (Arnalds, 2015). Mean annual temperature at the site was 5.1 °C. The coldest and warmest temperatures in the neighboring village of Eyrarbakki in 2016 were -12.3 °C and 21.6 °C, respectively. Average annual precipitation for the same year was 1153 mm (Icelandic Meteorological Office, 2016). The vegetation was an unmanaged grassland dominated by <em>Agrostis capillaris</em> L., <em>Galium boreale </em>L. and<em> Anthoxantum odoratum</em> L.. Vascular plants cover 46% of the area over a moss mat which covers up to 88% of the ground. This grassland has been geothermally warmed since 29 May 2008, when an earthquake transferred geothermal energy from hot groundwater to previously unheated soils (Sigurdsson et al., 2016). Belowground temperatures at 10 cm depth now display a permanent warming gradient reaching +10 °C, with a discreet increase in aboveground temperature of +0.2 °C. The warming has only been mildly disruptive, with seasonality remaining unchanged. Soil humidity was only marginally affected, with volumetric water content changing from 40% to 38%, and water pH increased from 5.6 in unheated soil to up to 6.3 after warming. Geothermal groundwater has remained in the bedrock and has not reached the root zone, thus avoiding direct eco-toxicological effects (Sigurdsson et al., 2016). The resulting stable conditions and lack of artifacts provide a realistic natural belowground experiment on climate change. A N addition experiment was conducted in the same grassland but in areas free from this geothermal warming. Natural N deposition in the area is 1.3 ± 0.1kg N ha-1 y-1 (Leblans et al., 2014). Five transects were established, each one consisting of three 2 x 2 m plots, and each plot at different temperature: an unheated control, a low warming level of ca. +3 °C and a higher warming level of ca. +6 °C above the ambient reference in the control. Soil cores were collected using an auger to a depth of ~10 cm, excluding the O horizon. Soil cores were sampled seasonally four times: August 2017, corresponding to late growing season; November 2017, at start of winter and initial soil freezing; April 2018, with the first soil thaw in un-warmed soils, and June 2018, in the early part of the growing season. We thus collected a total of 20 core samples for each warming treatment (five replicates in four seasons for three temperature levels = 60 samples). The experimental N addition followed a similar approach to the observational study of soil warming, with five replicate transects, with a control plot and an experimental plot where 150 kg N ha-1 had been added annually (in three doses) as NH4-NO3 since 2014. Likewise, a soil core from each replicate was collected for each of the four seasonal samplings, for a total of 20 samples per treatment plot. All samples were immediately sieved to remove roots and stones larger than 2 mm. Fifteen grams of each sample were then frozen in plastic bags in liquid N in the field to immediately stop all biological processes. All frozen samples were freeze-dried in the laboratory. eDNA was extracted from 15 g soil samples as previously described (Taberlet et al., 2012; Zinger et al., 2016). The soil hexapod communities were genetically characterized based on Molecular Operational Taxonomic Units (MOTUs) using the retrieved eDNA and applying a metabarcoding approach. We amplified the 16S mitochondrial rDNA region using the Ins16S_l primer pair (Ins16S_1-F: 5′-TRRGACGAGAAGACCCTATA-3′; Ins16_1-R: 5′-TCTTAATCCAACATCGAGGTC-3′; Clarke et al. 2014). This primer pair, specifically designed for insect metabarcoding, introduces a very limited taxonomic bias and performs very well for identifications at the species level throughout the Hexapoda subphylum (e.g. Kocher et al., 2017; Talaga et al., 2017). PCR amplification was performed in triplicate in 20-μL mixtures consisting of 10 μL of AmpliTaq Gold Master Mix (Life Technologies, Carlsbad, USA), 5.84 μL of nuclease-free Ambion water (Thermo Fisher Scientific, Waltham, USA), 0.25 μM each primer, 3.2 μg of bovine serum albumin (Roche Diagnostic, Basel, Switzerland) and 2 μl of DNA template that was diluted 10-fold to reduce PCR inhibition by humic substances. The thermal profile of the PCR amplification was 40 cycles of denaturation at 95 °C (30 s), annealing at 49 °C (30 s) and elongation at 72 °C (60 s), with a final elongation step at 72 °C for 7 min. Tags had at least five differences between them to minimize ambiguities (Coissac et al., 2012). The sequenced multiplexes comprised extractions/PCR blank controls, unused tag combinations and positive controls (Kocher et al., 2017). The PCR products were then sequenced using the MiSeq platform (Illumina Inc., San Diego, USA), with the expected sequencing depth set at 400 000 reads per sample. The sequences were processed using OBITOOLS software (Boyer et al., 2016). Low-quality sequences (containing Ns, alignment scores &lt;50, lengths &lt;140 bp or &gt;320 bp and singletons) were excluded. The remaining sequences were clustered into MOTUs using SUMACLUST (Mercier et al., 2013) at a threshold of sequence similarity of 97%. The final number of MOTUs after curation was 11785. The hexapod MOTUs were taxonomically assigned using Blast. MOTUs showing &lt;80% similarity with either the local or the EMBL reference databases were removed, leading to 590 MOTUs. These retained MOTUs included taxa from classes Insecta and Entognatha, which both belong to the subphylum Hexapoda. Hereafter we may refer to both classes as insects <em>sensu latto</em> for simplicity. We then applied a post-processing pipeline (Zinger et al., 2021) to minimize PCR and sequencing errors, contaminations and false-positive sequences, and by detailed curation of ecologically incongruent assignments (e.g. taxa with distributions clearly outside the study region). This highly conservative approach retained only 40 species. We then used checklists of Icelandic insect and springtail species and information from previous studies at the same study site (Fjellberg, 2007; Holmstrup et al., 2018) to assess the performance of our eDNA metabarcoding protocol to properly describe the insect communities in the soil.