ferrinetal_c&nmediatewarmingimpactonsoilhexapods

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 soil warming under climate change. 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 (henceforth referred as “+3 °C” and “+6 °C”). <br> 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 (5 replicates in 4 seasons for 3 temperature levels = 60 samples). 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 belonging to DNA remains (i.e. no alive fauna) as previously described (Taberlet et al., 2012; Zinger et al., 2016). <br> 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 hexapod 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 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 219 MOTUs. These retained MOTUs included taxa from classes Insecta and Entognatha, which both belong to the subphylum Hexapoda. 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 (i.e. taxa with distributions outside the palearctic and neartic ecozones). This conservative approach retained a total of 33 identified species. We then used checklists of Icelandic hexapod 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 hexapod communities in the soil.

本研究于2017年8月至2018年6月间，在冰岛ForHot研究站点（Sigurdsson等，2016）开展，地理坐标为64°0′N，21°11′W。供试土壤类型为暗色火山灰土（Brown Andosol，Arnalds，2015），站点年平均气温为5.1℃。2016年邻近村落Eyrarbakki的极端最低、最高气温分别为-12.3℃和21.6℃；同年年均降水量为1153 mm（冰岛气象局，2016）。区域植被为未受人工管理的草原，优势物种为<em>Agrostis capillaris</em> L.、<em>Galium boreale</em> L.及<em>Anthoxantum odoratum</em> L.。维管植物覆盖该区域46%的面积，而苔藓垫覆盖了高达88%的地面。该草原自2008年5月29日起受到地热加温：当年一场地震将地下热水中的地热能量转移至此前未被加热的土壤中（Sigurdsson等，2016）。目前10 cm深度的地下温度呈现永久升温梯度，最高升温达+10℃，同时地表温度小幅上升+0.2℃。此次加温仅造成轻度干扰，季节节律保持不变。土壤湿度仅受轻微影响，体积含水量从40%降至38%；土壤pH值从未加温土壤的5.6上升至加温后最高可达6.3。地热地下水始终留存于基岩中，未抵达根系层，因此未产生直接的生态毒理效应（Sigurdsson等，2016）。这种稳定的实验条件且无实验伪影，为气候变化下的土壤加温研究提供了贴近自然的地下原位实验。该区域自然氮沉降量为1.3 ± 0.1 kg N ha⁻¹ y⁻¹（Leblans等，2014）。

共设置5条样带，每条样带包含3个2 m × 2 m的样地，分别对应3个温度梯度：未加温的对照组、约+3℃的低加温组，以及较对照环境温度高出约+6℃的高加温组（下文分别简称“+3℃组”与“+6℃组”）。

使用土钻采集深度约10 cm的土芯，排除O层（腐殖质层）。分别于2017年8月（生长季末期）、2017年11月（冬季伊始与土壤初始冻结期）、2018年4月（未加温土壤首次解冻期）及2018年6月（生长季早期）进行4次季节性采样。每个加温处理共采集20个土芯样品（3个温度水平×4个季节×5个重复=60个样品）。所有样品采集后立即过筛，去除直径大于2 mm的根系与石块。每份样品取15 g，于野外使用液氮装入塑料袋中冷冻，以立即终止所有生物过程。所有冷冻样品均在实验室进行冷冻干燥。

从15 g土壤样品中提取环境DNA（eDNA，即无活体动物的DNA残留），操作流程参照已发表方法（Taberlet等，2012；Zinger等，2016）。

基于获取的eDNA，采用元条形码（metabarcoding）技术，通过分子操作分类单元（Molecular Operational Taxonomic Units, MOTUs）对土壤六足动物群落进行遗传鉴定。使用Ins16S_l引物对扩增线粒体16S rDNA区域（Ins16S_1-F：5′-TRRGACGAGAAGACCCTATA-3′；Ins16_1-R：5′-TCTTAATCCAACATCGAGGTC-3′；Clarke等，2014）。该引物对专为六足动物元条形码设计，分类学偏差极低，可在六足亚门（Hexapoda）各类群中实现良好的物种水平鉴定（如Kocher等，2017；Talaga等，2017）。

PCR扩增设置3次技术重复，反应体系总体积20 μL，包含10 μL AmpliTaq Gold Master Mix（Life Technologies，美国卡尔斯巴德）、5.84 μL 无核酸酶Ambion水（赛默飞世尔科技，美国沃尔瑟姆）、终浓度0.25 μM的上下游引物、3.2 μg 牛血清白蛋白（罗氏诊断，瑞士巴塞尔），以及2 μL 稀释10倍的DNA模板以降低腐殖质带来的PCR抑制效应。PCR扩增热循环程序为：95℃变性30 s、49℃退火30 s、72℃延伸60 s，共40个循环，最后于72℃进行7 min的终延伸。引物标签之间至少存在5个碱基差异，以最大程度减少测序歧义（Coissac等，2012）。测序多重文库包含提取空白对照、PCR空白对照、未使用的标签组合以及阳性对照（Kocher等，2017）。

测序使用MiSeq平台（Illumina公司，美国圣地亚哥），预设每个样品的测序深度为400 000条读长。序列数据使用OBITOOLS软件（Boyer等，2016）进行处理，过滤掉低质量序列（包含模糊碱基N、比对得分<50、序列长度<140 bp或>320 bp以及单条序列）。剩余序列使用SUMACLUST（Mercier等，2013）以97%的序列相似性阈值聚类为MOTUs。通过BLAST（碱基局部比对搜索工具）对六足动物MOTUs进行分类学注释，将与本地数据库或EMBL参考数据库相似性<80%的MOTUs剔除，最终保留219个MOTUs。这些保留的MOTUs涵盖昆虫纲（Insecta）和内口纲（Entognatha）类群，二者均隶属于六足亚门。

随后采用后处理流程（Zinger等，2021）以最小化PCR和测序错误、污染及假阳性序列，并通过详细整理生态学上不一致的分类注释（即分布区域超出古北界和新北界的类群）。经过该保守流程，最终共鉴定得到33个物种。随后参照冰岛六足动物物种名录及本研究站点既往研究的相关资料（Fjellberg，2007；Holmstrup等，2018），评估本研究采用的eDNA元条形码方案对土壤六足动物群落的描述性能。