title: Not all are free-living: high-throughput DNA metabarcoding reveals a diverse community of protists parasitizing soil metazoa.
abstract: 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 with 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 at investigating 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 over-represented, while those of most amoebae and flagellates were under-represented 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 nonadded protist taxa probably 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.
authors.affiliation: Department of Terrestrial Ecology, Netherlands Institute of Ecology (NIOO-KNAW), PO Box 50, 6700 AB, Wageningen, the Netherlands.|Department of Terrestrial Ecology, Institute of Zoology, University of Cologne, Zülpicher Str 47b, 50674, Cologne, Germany.
authors.forename: S
authors.identifier: http://orcid.org/0000-0003-0734-727X
authors.initials: S
authors.lastname: Geisen
authors.affiliation: ALTERRA - Wageningen UR, P.O. Box 47, 6700 AA, Wageningen, The Netherlands.
authors.forename: I
authors.initials: I
authors.lastname: Laros
authors.affiliation: AllGenetics, Ed. de Servicios Centrales de Investigación, Campus de Elviña s/n, E-15071, A Coruña, Spain.
authors.forename: A
authors.initials: A
authors.lastname: Vizcaíno
authors.affiliation: Department of Terrestrial Ecology, Institute of Zoology, University of Cologne, Zülpicher Str 47b, 50674, Cologne, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Bonkowski
authors.affiliation: ALTERRA - Wageningen UR, P.O. Box 47, 6700 AA, Wageningen, The Netherlands.
authors.forename: G A
authors.initials: GA
authors.lastname: de Groot
mesh_terms: D000656:Amoeba / Q000235:genetics*; D044822:Biodiversity; D056907:Cercozoa / Q000235:genetics*; D016798:Ciliophora / Q000235:genetics*; D058893:DNA Barcoding, Taxonomic; D017753:Ecosystem; D059014:High-Throughput Nucleotide Sequencing; D016830:Kinetoplastida / Q000235:genetics*; D008969:Molecular Sequence Data; D010802:Phylogeny; D012987:Soil / Q000469:parasitology*
chemical_list: D012987:Soil
keywords: 454 Metabarcoding; apicomplexa; high-throughput sequencing; potential parasites; soil metazoan parasites; soil protists