Data_Sheet_2_Unearthing the Potential of Soil eDNA Metabarcoding—Towards Best Practice Advice for Invertebrate Biodiversity Assessment.CSV

Metabarcoding has proven to be a powerful tool to assess ecological patterns and diversity from different habitats. Terrestrial invertebrate diversity is frequently based on bulk samples, which require comparatively high sampling effort. With environmental DNA (eDNA) metabarcoding, field sampling effort can be reduced while increasing the number of recovered organism groups. However, a proof of concept is missing for several invertebrate groups, hampering the development of best-practice advice for these groups. This study aims to provide recommendations on key aspects for the processing of soil samples, from sampling effort to choice of DNA extraction method and marker genes. This study uses eDNA metabarcoding as a tool for assessing invertebrate biodiversity in soil samples, specifically comparing two DNA extraction methods (with and without a lysis step) and two genes, 18S and COI markers. The results show that the choice of marker and DNA extraction method (including a lysis step) significantly affect species detection rates and concomitantly observed invertebrate community composition. Combining methods, by using larger amounts of starting material and including a lysis step resulted in an increase of invertebrate species numbers. Together, these methods improved the detection of species with known lower population densities and allowed the assessment of temporary mesofauna. Furthermore, the choice of marker significantly influenced the diversity levels found. The 18S marker allowed the detection of a higher number of annelid and nematode OTUs, while the COI marker was more suitable for detecting changes in arthropod community structure, especially at the species level. This study makes significant advances to the field of invertebrate biodiversity assessment, particularly using metabarcoding tools by addressing several methodological considerations that are key for accurate ecological appraisals.

元条形码测序（Metabarcoding）已被证实为评估不同生境生态格局与生物多样性的有力工具。陆生无脊椎动物多样性研究多基于混合样本，此类样本的采集往往需要投入较高的人力成本。借助环境DNA（environmental DNA, eDNA）元条形码测序技术，可在提升目标类群检出数量的同时降低野外采样工作量。然而目前仍有多个无脊椎动物类群缺乏概念验证研究，这阻碍了针对这些类群的最佳实践指南制定。本研究旨在为土壤样本处理的关键环节提供建议，覆盖从采样工作量确定到DNA提取方法与标记基因选择的全流程。本研究以环境DNA元条形码测序为工具，评估土壤样本中的无脊椎动物生物多样性，并专门对比了两种DNA提取方法（分别带有裂解步骤与不带裂解步骤）以及两种标记基因——18S与COI。研究结果表明，标记基因与DNA提取方法（含裂解步骤）的选择，会显著影响物种检出率，同时也会改变观测到的无脊椎动物群落组成。通过结合使用更多起始样本量并添加裂解步骤的处理方案，可提升无脊椎动物物种的检出数量。此类联用方法不仅改善了对已知种群密度较低物种的检出效果，还可实现对暂时性中型土壤动物群的评估。此外，标记基因的选择会显著影响所测得的多样性水平：18S标记可检出更多环节动物门与线虫动物门的操作分类单元（operational taxonomic unit, OTU），而COI标记则更适用于检测节肢动物群落结构的变化，尤其是在物种水平上。本研究针对无脊椎动物生物多样性评估领域，尤其是元条形码测序工具的应用取得了重要进展，通过解决若干对精准生态评估至关重要的方法学问题，为该领域的研究提供了关键支撑。