Thorough high‐throughput sequencing analyses unravels huge diversities of soil parasitic protists

Microbes are by far the most abundant and diverse organisms in soils. Among them, prokaryotic bacteria and archaea as well as eukaryotic fungi constitute the base of the soil food web (Hunt et al., 1987; de Ruiter et al., 1995). Thanks to continuously improving molecular tools, especially high-throughput sequencing (HTS) approaches, deep insights into the previously unknown diversity and community structure of these microbes has now been obtained. For instance, a huge diversity of previously undescribed groups among these microbes were detected in soils; many of those were later shown to provide important ecological functions and ecosystem services. For instance, archaea were shown to represent surprisingly abundant soil prokaryotes (Roesch et al., 2007) and to be key ammonium oxidizers in soils (Leininger et al., 2006). HTS-based methods also helped at disentangling environmental drivers that structure prokaryotic and fungal community compositions; pH was shown to be the major driver of prokaryotic communities (Lauber et al., 2009; Rousk et al., 2010; Griffiths et al., 2011), while fungi seem equally affected by a wider range of variables including pH, plant species and climatic factors (Tedersoo et al., 2014; 2016; Treseder et al., 2014). Comparably little is known about soil protists, which represent the often forgotten remaining microbial group (Caron et al., 2008). The majority of the few studies investigating the community structure of soil protists applied time-consuming microscopic determinations that require profound taxonomic expertise [e.g. (Finlay and Fenchel, 2001; Scherber et al., 2010; Domonell et al., 2013; Geisen et al., 2014)]. Molecular approaches, nowadays more and more based on HTS, start replacing those classical approaches (Baldwin et al., 2013; Bates et al., 2013; Lentendu et al., 2014; Geisen et al., 2015b; Fiore-Donno et al., 2016). Still, HTS targeting protists are extremely underrepresented in comparison to other microbes (Mitchell, 2015). The major reason for the profound gap stems from the immense, paraphyletic diversity of protists that span over the entire eukaryotic tree of life with plants, fungi and metazoans representing monophyletic branches at different places therein (Adl et al., 2012; Burki et al., 2016). Therefore, designing universal protistspecific primers is impossible. This is of little relevance when studying microbes in aquatic systems, where protists can be easily extracted and focused upon through subsequent size-filtering (de Vargas et al., 2015). Close attachment of many protists to soil particles eliminates straight-forward extraction, while directly applying universal eukaryotic primers to extracted DNA/RNA will ultimately result in sequencing mostly fungi due to their dominance in soils. To avoid this issue, more focused group specific primers targeting e.g. Cercozoa, Kinetoplastida, Chrysophyceae, acanthamoebae and Myxomycetes have been applied (Lentendu et al., 2014; Bass et al., 2016; Fiore-Donno et al., 2016). Continuous improvements in sequencing technologies with decreased costs at higher sequencing depth enabled the use of general eukaryotic primers to target soil protists. Bates et al. (2013) were the first using this approach and found biogeographic patterns of protist communities that were mainly explained by soil moisture. The key novelty of this initial survey was the finding of an unimagined diversity of soil protists similar to that of bacteria, with many sequences most closely matching potential parasites such as apicomplexans (Bates et al., 2013). This finding was supported by follow-up DNAbased HTS studies (Ramirez et al., 2014; Grossmann et al., 2016) as well as metatranscriptomic approaches (Urich et al., 2008; Geisen et al., 2015b). In this issue of Environmental Microbiology, Dupont et al. (2016) provide further evidence for the widespread diversity of parasitic protists in soils. One of the key novelties in this study is the use of phylogenetic analyses of manually mined HTS data; the authors elegantly reveal an immense diversity of largely unknown parasitic *For correspondence. E-mail s.geisen@nioo.knaw.n; Tel. 131 (0)317 40 634; Fax 131 317473675.