Data from: Not all are free-living: high-throughput DNA metabarcoding reveals a diverse community of protists parasitizing soil metazoa

Protists, the most diverse eukaryotes, are largely considered to be free-living bacterivores, but vast numbers of taxa are known to parasitize plants or animals. High-throughput sequencing (HTS) approaches now commonly replace cultivation-based approaches in studying soil protists, but insights into common biases associated to this method are limited to aquatic taxa and samples. We created a mock community of common free-living soil protists (amoebae, flagellates, ciliates), extracted DNA and amplified it in the presence of metazoan DNA using 454 HTS. We aimed at evaluating whether HTS quantitatively reveals true relative abundances of soil protists and to investigate whether the expected protist community structure is altered by the co-amplification of metazoan-associated protist taxa. Indeed, HTS revealed fundamentally different protist communities from those expected. Ciliate sequences were highly overrepresented, while those of most amoebae and flagellates were underrepresented or totally absent. These results underpin the biases introduced by HTS that prevent reliable quantitative estimations of free-living protist communities. Furthermore, we detected a wide range of non-added protist taxa likely introduced along with metazoan DNA, which altered the protist community structure. Among those, 20 taxa most closely resembled parasitic, often pathogenic taxa. Therewith, we provide the first HTS data in support of classical observational studies that showed that potential protist parasites are hosted by soil metazoa. Taken together, profound differences in amplification success between protist taxa and an inevitable co-extraction of protist taxa parasitizing soil metazoa obscure the true diversity of free-living soil protist communities.

作为多样性最高的真核生物类群，原生生物（Protists）大多被认为是营自由生活的食菌者，但目前已知有大量类群可寄生于植物或动物体内。如今，在土壤原生生物研究中，高通量测序（High-throughput Sequencing, HTS）技术已普遍取代基于培养的研究方法，但目前针对该方法常见偏差的认知仅局限于水生类群与样本。本研究构建了由常见营自由生活的土壤原生生物（变形虫amoebae、鞭毛虫flagellates、纤毛虫ciliates）组成的模拟群落，提取其DNA后，在含有后生动物（metazoan）DNA的体系中，采用454高通量测序进行扩增。本研究旨在评估高通量测序是否能定量反映土壤原生生物的真实相对丰度，并探究与后生动物共生的原生生物类群的共扩增是否会改变预期的原生生物群落结构。实验结果显示，高通量测序得到的原生生物群落与预期结果存在本质差异：纤毛虫序列占比显著偏高，而多数变形虫与鞭毛虫的序列占比偏低甚至完全未被检出。上述结果证实了高通量测序引入的偏差会导致无法对营自由生活的原生生物群落进行可靠的定量评估。此外，本研究还检测到大量未加入模拟群落的原生生物类群，这些类群大概率随后生动物DNA一同引入，进而改变了原生生物群落结构。其中20个类群与寄生性（通常为致病性）的原生生物类群亲缘关系最近。据此，本研究提供了首项高通量测序数据，支持经典观测研究的结论——即土壤后生动物可携带具有潜在致病性的原生生物寄生虫。综上，原生生物类群间扩增效率的显著差异，以及对寄生于土壤后生动物的原生生物类群不可避免的共提取，共同掩盖了营自由生活的土壤原生生物群落的真实多样性。