Effects of soil preservation for biodiversity monitoring using environmental DNA

Environmental DNA metabarcoding is becoming a key tool for biodiversity monitoring over large geographical or taxonomic scales and for elusive taxa like soil organisms. Increasing sample sizes and interest in remote or extreme areas often require the preservation of soil samples and thus deviations from optimal standardized protocols. However, we still ignore the impact of different methods of soil sample preservation on the results of metabarcoding studies and there is no guidelines for best practices so far. Here, we assessed the impact of four methods of soil sample preservation commonly used in metabarcoding studies (preservation at room temperature for 6h, preservation at 4°C for three days, desiccation immediately after sampling and preservation for 21 days, and desiccation after 6h at room temperature and preservation for 21 days). For each preservation method, we benchmarked resulting estimates of taxon diversity and community composition of three different taxonomic groups (bacteria, fungi and eukaryotes) in three different habitats (forest, river bank and grassland) against results obtained under optimal conditions (i.e. extraction of eDNA right after sampling). Overall, the different preservation methods only marginally impaired results and only under certain conditions. When rare taxa were considered, we detected small but significant changes in MOTU richness of bacteria, fungi and eukaryotes across treatments, while the exclusion of rare taxa led to robust results across preservation methods. The differences in community structure among habitats were evident for all treatments, and the communities retrieved using the different preservation conditions were extremely similar. We propose guidelines on the selection of the optimal soil sample preservation conditions for metabarcoding studies, depending on the practical constraints, costs and ultimate research goals.
Methods
Data has been processed following the pipeline described in Script 1 and Script 2 for Euka02, as example.

环境DNA元条形码（Environmental DNA metabarcoding）正逐渐成为大地理范围、大分类学尺度下生物多样性监测，以及针对土壤生物这类隐秘类群监测的核心工具。随着样本量不断增大，以及科研人员对偏远或极端区域研究的兴趣日益高涨，土壤样本的保存需求愈发凸显，这往往导致实际操作偏离最优标准化流程。然而，目前研究领域仍未明确不同土壤样本保存方法对元条形码研究结果的影响，且迄今尚未形成通用的最佳实践指南。本研究针对元条形码研究中常用的四种土壤样本保存方法展开评估：室温保存6小时、4℃下保存3天、采样后即刻干燥并保存21天、室温放置6小时后再干燥并保存21天。针对每种保存方法，我们以最优条件（即采样后即刻进行环境DNA（eDNA）提取）下的结果为基准，对比分析了森林、河岸、草地三种生境中细菌、真菌与真核生物三类分类群的分类多样性及群落组成估算结果。总体而言，不同保存方法仅在特定条件下对结果造成轻微负面影响。当纳入稀有类群进行分析时，各组处理下细菌、真菌与真核生物的可操作分类单元（MOTU, Molecular Operational Taxonomic Unit）丰富度均出现小幅但显著的变化；而剔除稀有类群后，不同保存方法下的结果均保持稳健。不同生境间的群落结构差异在所有处理组中均清晰可辨，且不同保存条件下获取的群落组成极为相似。基于本研究结果，结合实际操作限制、成本与最终研究目标，我们提出了元条形码研究中土壤样本最优保存条件的选择指南。
方法
本研究的数据处理流程参照脚本1与脚本2（以Euka02为示例）中所述方案执行。