Data from: Genetic barcoding of dark-spored myxomycetes (Amoebozoa)—Identification, evaluation and application of a sequence similarity threshold for species differentiation in NGS studies

Unicellular, eukaryotic organisms (protists) play a key role in soil food webs as major predators of microorganisms. However, due to the polyphyletic nature of protists, no single universal barcode can be established for this group, and the structure of many protistean communities remains unresolved. Plasmodial slime moulds (Myxogastria or Myxomycetes) stand out among protists by their formation of fruit bodies, which allow for a morphological species concept. By Sanger sequencing of a large collection of morphospecies, this study presents the largest database to date of dark-spored myxomycetes and evaluate a partial 18S SSU gene marker for species annotation. We identify and discuss the use of an intraspecific sequence similarity threshold of 99.1% for species differentiation (OTU picking) in environmental PCR studies (ePCR) and estimate a hidden diversity of putative species, exceeding those of described morphospecies by 99%. When applying the identified threshold to an ePCR data set (including sequences from both NGS and cloning), we find 64 OTUs of which 21.9% had a direct match (>99.1% similarity) to the database and the remaining had on average 90.2 ± 0.8% similarity to their best match, thus thought to represent undiscovered diversity of dark-spored myxomycetes.单细胞真核生物——原生生物（protists），作为微生物的主要捕食者，在土壤食物网中发挥着核心作用。然而，由于原生生物具有多系演化的特性，该类群无法建立统一的通用条形码，且多数原生生物群落的结构仍未明确。变形体黏菌（Myxogastria，又称Myxomycetes）在原生生物中独具特色，它们能够形成子实体，这一特征使其可依托形态学物种概念进行分类。本研究通过对大量形态种进行桑格（Sanger）测序，构建了目前规模最大的暗色孢子黏菌（dark-spored myxomycetes）数据库，并评估了用于物种注释的部分18S小亚基（18S SSU）基因标记。本研究确定并探讨了在环境PCR（ePCR）研究中，采用99.1%的种内序列相似性阈值开展物种区分（即操作分类单元（OTU）聚类）的可行性，并估算出暗色孢子黏菌的隐性推定物种多样性较已描述形态种高出99%。当将本研究确定的阈值应用于包含下一代测序（NGS）与克隆测序序列的环境PCR数据集时，共得到64个OTU，其中21.9%的OTU与数据库存在直接匹配（相似性>99.1%），剩余OTU与最佳匹配序列的平均相似性仅为90.2±0.8%，推测这些序列代表了尚未被发现的暗色孢子黏菌多样性。