# Soil protistology rebooted: 30 fundamental questions to start with

Stefan Geisen, Edward A.D. Mitchell, David S. Wilkinson, Sina Adl, Michael Bonkowski, Matthew Brown, Anna Maria Fiore-Donno, Thierry Heger, Vincent Jassey, Valentyna Krashevska, et al.

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## ► To cite this version:

Stefan Geisen, Edward A.D. Mitchell, David S. Wilkinson, Sina Adl, Michael Bonkowski, et al. Soil protistology rebooted: 30 fundamental questions to start with. Soil Biology and Biochemistry, 2017, 111, pp.94-103. 10.1016/j.soilbio.2017.04.001. hal-02466172

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1

2

Soil protistology rebooted: 30
fundamental questions to start with

3

Stefan Geisen<sup>1, 33*</sup>, Edward A. D. Mitchell<sup>2, 3</sup>, David M. Wilkinson<sup>4,43</sup>, Sina Adl<sup>5</sup>, Michael Bonkowski<sup>6</sup>,

4

Matthew W. Brown<sup>7</sup>, Anna Maria Fiore-Donno<sup>6</sup>, Thierry J. Heger<sup>2</sup>, Vincent E.J. Jassey<sup>8, 9</sup>, Valentyna

5

Krashevska<sup>10</sup>, Daniel J. G. Lahr<sup>11</sup>, Katarzyna Marcisz<sup>12,44</sup>, Matthieu Mulot<sup>2</sup>, Richard Payne<sup>13</sup>, David

6

Singer<sup>2</sup>, O. Roger Anderson<sup>14</sup>, Dan J. Charman<sup>15</sup>, Flemming Ekelund<sup>16</sup>, Bryan S. Griffiths<sup>17</sup>, Regin

7

Rønn<sup>16</sup>, Alexey Smirnov<sup>18</sup>, David Bass<sup>19, 20</sup>, Lassaâd Belbahri<sup>2</sup>, Cédric Berney<sup>21,22</sup>, Quentin Blandenier<sup>2</sup>,

8

Antonis Chatzinotas<sup>23,24</sup>, Marianne Clarholm<sup>25</sup>, Micah Dunthorn<sup>26</sup>, Alan Feest<sup>27</sup>, Leonardo D.

9

Fernández<sup>28</sup>, Wilhelm Foissner<sup>29</sup>, Bertrand Fournier<sup>30</sup>, Eleni Gentekaki<sup>31</sup>, Michal Hajek<sup>32</sup>, Johannes

10

Helder<sup>33</sup>, Alexandre Jousset<sup>34</sup>, Robert Koller<sup>35</sup>, Santosh Kumar<sup>36-37</sup>, Antonietta La Terza<sup>37</sup>, Mariusz

11

Lamentowicz<sup>12</sup>, Yuri Mazei<sup>38,39</sup>, Susana S. Santos<sup>40</sup>, Christophe V.W. Seppey<sup>2</sup>, Frederick W. Spiegel<sup>41</sup>,

12

Julia Walochnik<sup>42</sup>, Anne Winding<sup>40</sup>, Enrique Lara<sup>2</sup>

13

<sup>1</sup> Department of Terrestrial Ecology, Netherlands Institute of Ecology, 6708 PB Wageningen, The

14

Netherlands

15

<sup>2</sup> Laboratory of Soil Biodiversity, University of Neuchatel, Rue Emile-Argand 11, Neuchatel 2000,

16

Switzerland

17

<sup>3</sup> Jardin Botanique de Neuchâtel, Chemin du Perthuis-du-Sault 58, Neuchâtel 2000, Switzerland

18

<sup>4</sup> Natural Science and Psychology, Liverpool John Moores University, Byrom Street, Liverpool L3 3AF,

19

UK

20

<sup>5</sup> Department of Soil Sciences, College of Agriculture and Bioresources, University of Saskatchewan,

21

51 Campus Drive, Saskatoon, Canada
22 ⁶Department of Terrestrial Ecology, University of Cologne, Zuelpicher Str. 47b, 50674 Cologne,

23 Germany

24 ⁷Department of Biological Sciences, Mississippi State University, Mississippi State, MS, 39762

25 ⁸School of Architecture, Civil and Environmental Engineering (ENAC), Ecole Polytechnique Fédérale

26 de Lausanne EPFL, Ecological Systems Laboratory (ECOS), Station 2, 1015 Lausanne, Switzerland

27 ⁹Swiss Federal Institute for Forest, Snow and Landscape Research (WSL), Site Lausanne, Station 2,

28 1015 Lausanne, Switzerland

29 ¹⁰Georg August University Göttingen, J.F. Blumenbach Institute of Zoology and Anthropology,

30 Berliner Str. 28, 37073 Göttingen, Germany

31 ¹¹Department of Zoology, Institute of Biosciences, University of São Paulo, Brazil, 05508-090

32 ¹²Laboratory of Wetland Ecology and Monitoring & Department of Biogeography and Paleoecology,

33 Adam Mickiewicz University, Dziegielowa 27, 61-680 Poznań, Poland

34 ¹³Environment, University of York, York YO105DD, UK

35 ¹⁴Biology and Paleoenvironment, Lamont-Doherty Earth Observatory of Columbia University,

36 Palisades, NY, 10964

37 ¹⁵Department of Geography, College of Life and Environmental Sciences, University of Exeter, Exeter

38 EX4 4RJ, United Kingdom

39 ¹⁶Department of Biology, University of Copenhagen, Universitetsparken 15, 2100 Copenhagen,

40 Denmark

41 ¹⁷SRUC, Crop and Soil Systems Research Group, West Mains Road, Edinburgh EH9 3JG, United

42 Kingdom

43 ¹⁸Department of Invertebrate Zoology, Faculty of Biology, Saint Petersburg State University,

44 Universitetskaya nab. 7/9, St. Petersburg, 199034, Russia
45 ¹⁹ Department of Life Sciences, The Natural History Museum, London SW7 5BD, United Kingdom

46 ²⁰ Cefas, Barrack Road, Weymouth, Dorset DT4 8UB, United Kingdom

47 ²¹ EPEP—Evolution des Protistes et des Ecosystèmes Pélagiques—team, Sorbonne Universités, UPMC

48 Univ Paris 06, UMR 7144, Station Biologique de Roscoff, Roscoff, France

49 ²² CNRS, UMR 7144, Station Biologique de Roscoff, Roscoff, France

50 ²³ Helmholtz Centre for Environmental Research – UFZ, Department of Environmental Microbiology,

51 Permoserstr. 15, 04318 Leipzig, Germany

52 ²⁴ German Centre for Integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig, Deutscher Platz 5e,

53 04103 Leipzig, Germany

54 ²⁵ Department of Forest Mycology and Plant Pathology, SLU, Uppsala, Sweden

55 ²⁶ Department of Ecology, University of Kaiserslautern, 67663 Kaiserslautern, Germany

56 ²⁷ Faculty of Engineering, University of Bristol, Bristol BS8 1TR, United Kingdom

57 ²⁸ Centro de Investigación en Recursos Naturales y Sustentabilidad (CIRENYS), Universidad Bernardo

58 O'Higgins, Fábrica 1990, 2º piso, Santiago, Chile

59 ²⁹ University of Salzburg, Department of Ecology and Evolution, Hellbrunnerstrasse 34, A-5020

60 Salzburg, Austria

61 ³⁰ Université Bourgogne Franche-Comté, Laboratoire Chrono-Environnement UMR 6249 CNRS, 16

62 route de Gray, 25030 Besançon Cedex, France

63 ³¹ School of Science, Mae Fah Luang University, Chiang Rai, 57100, Thailand

64 ³² Department of Botany and Zoology, Masaryk University, Kotlářská 2, 61137 Brno, Czech Republic

65 ³³ Laboratory of Nematology, Wageningen University, Droevendaalsesteeg 1, 6708 PB, Wageningen,

66 The Netherlands

67 ³⁴ Department of Ecology and Biodiversity, Utrecht University, 3584 CH Utrecht, The Netherlands
68 ³⁵ Forschungszentrum Jülich, IBG-2: Plant Sciences, 52425 Jülich, Germany

69 ³⁶ Department of Biological Sciences, College of Natural Sciences, University of Ulsan, Ulsan 44610,

70 South Korea

71 ³⁷ School of Bioscience and Veterinary Medicine, University of Camerino, Via Gentile III da Varano,

72 62032 Camerino (MC), Italy

73 ³⁸ Department of Hydrobiology, Lomonosov Moscow State University, Leninskiye gory, 1, Moscow

74 119899, Russia

75 ³⁸ Department of Zoology and Ecology, Penza State University, Krasnaya str. 40, 440026 Penza, Russia

76 ⁴⁰ Department of Environmental Science, Aarhus University, Frederiksborgvej 399, 4000 Roskilde,

77 Denmark

78 ⁴¹ Department of Biological Sciences, University of Arkansas, Fayetteville, AR, 72701

79 ⁴² Molecular Parasitology, Institute of Tropical Medicine, Medical University, 1090 Vienna, Austria

80 ⁴³ School of Life Science, University of Lincoln, UK

81 ⁴⁴ Institute of Plant Sciences and Oeschger Centre for Climate Change Research, University of Bern,

82 Altenbergrain 21, CH-3013 Bern, Switzerland

83

84

85 *Corresponding author: Stefan Geisen, Department of Terrestrial Ecology, Netherlands Institute of

86 Ecology, 6708 PB Wageningen, the Netherlands.

87 Tel: +31 (0)317 47 34 00; e-mail: s.geisen@nioo.knaw.nl

88 Running title: Soil protistology rebooted

89 Keywords: Soil protists; Protozoa; Microbial interactions; Food web; Biodiversity; Functional diversity
90 Abstract

91 Protists are the most diverse eukaryotes. These microbes are keystone organisms of soil ecosystems
92 and regulate essential processes of soil fertility such as nutrient cycling and plant growth. Despite
93 this, protists have received little scientific attention, especially compared to bacteria, fungi and
94 nematodes in soil studies. Recent methodological advances, particularly in molecular biology
95 techniques, have made the study of soil protists more accessible, and have created a resurgence of
96 interest in soil protistology. This ongoing revolution now enables comprehensive investigations of the
97 structure and functioning of soil protist communities, paving the way to a new era in soil biology.

98 Instead of providing an exhaustive review, we provide a synthesis of research gaps that should be
99 prioritized in future studies of soil protistology to guide this rapidly developing research area. Based
100 on a synthesis of expert opinion we propose 30 key questions covering a broad range of topics
101 including evolution, phylogenetics, functional ecology, macroecology, paleoecology, and
102 methodologies. These questions highlight a diversity of topics that will establish soil protistology as a
103 hub discipline connecting different fundamental and applied fields such as ecology, biogeography,
104 evolution, plant-microbe interactions, agronomy, and conservation biology. We are convinced that
105 soil protistology has the potential to be one of the most exciting frontiers in biology.

106 1. Introduction

107 Protists are everywhere, in aquatic and terrestrial ecosystems, free-living, and as symbionts
108 (including parasites) of many organisms including humans. These usually single-celled or colonial
109 microorganisms are by far the most diverse eukaryotes (Adl et al., 2012) and their species-numbers
110 might easily exceed 10 million (Global Soil Biodiversity Atlas; www.globalsoilbiodiversity.org). Since
111 the term 'protista' was introduced (Haeckel, 1866), profound taxonomic re-orderings have taken
112 place. The vast majority of eukaryotic lineages has been shown to be protists, with the exception of
113 the derived monophyletic multicellular lineages: animals, plants, and some fungi (Burki, 2014).
Electron microscopy and molecular phylogenies have revealed that both algal and protozoan lineages are intermingled throughout the eukaryote phylogenies (Delwiche, 1999; Burki, 2014), and hence it is less confusing to use Haeckel's broader category of 'protist'. Similarly, the classical protozoan morphological categories: flagellates, testate and naked amoebae – but not ciliates - are not monophyletic but distributed across the eukaryotic tree of life (Adl et al., 2012). A snapshot of the immense morphological and phylogenetic diversity of soil protists is visualized in Fig 1. We therefore recommend to use 'protist' as a term for all single celled phototrophic, mixotrophic and heterotrophic eukaryotes, with the exception of fungi.

The huge diversity of protist species has only recently become evident as many morphospecies recognizable under the microscope were shown to hide many cryptic species (Boenigk et al., 2012a). This 'dark matter of biodiversity' suggests that protist taxon richness has been considerably underestimated. A recent study of environmental eukaryotic diversity based on state-of-the-art high-throughput sequencing (HTS) showed that protists are considerably more diverse than plants and animals in the sunlit zone of oceans (de Vargas et al., 2015). HTS studies of soil protists have shown a wide diversity of non-phagotrophic protists and the diversity of protists in soils is at least as diverse as that in aquatic systems (e.g. Bates et al., 2013; Geisen et al., 2015c). Nevertheless, soil protists are much less well studied than their aquatic counterparts and this gap is increasing (Fig. 2a).

Soil protists have received relatively little attention mainly due to methodological challenges, especially their isolation from the opaque soil matrix. These, however, do not entirely explain why soil protists are relatively less studied than other soil organisms, especially bacteria, fungi and nematodes (Fig. 2b). The volume of work on microbial bacteria and fungi far outweighs protist studies, possibly because of their direct role as primary decomposers, and they represent monophyletic groups that can more easily be studied with various targeted methodological approaches (Foissner, 1987; Mitchell, 2015). Even soil viruses have been subject to more studies than soil protists, despite being extremely challenging to study (Fierer et al., 2007) and their uncertain functional importance in soils. The under-studied nature of soil protists is exemplified by a
140

comparison between research on protists and on soil archaea, a domain erected in 1990 and

141

reported to be functionally important in soil only decade ago (Leininger et al., 2006; Bates et al.,

142

2011). Historically studies mentioning soil protists in the title were eight times more abundant than

143

those including archaea (Fig. 2b, Supplementary Table 2). However, in the last 15 years, this pattern

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entirely changed; studies on soil protists decreased by 15% while those on other common soil

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organisms increased by at least 30%, especially soil archaea which increased by 88% (Fig. 2b,

146

Supplementary Table 2).

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The relative decline of papers on soil protists strongly contrasts with what we now know about their

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ubiquity, diversity, and perhaps more importantly, their functional significance. Soil protists can both

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make an important contribution to primary production (Jassey et al., 2015; Schmidt et al., 2016) and

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play a key role in the decomposition pathways as consumers of bacteria (Clarholm, 1981; de Ruiter et

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al., 1995), fungi, other protists, and small invertebrates; they can also act as parasites of plants and

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animals (Adl and Gupta, 2006; Jassey et al., 2012; Geisen, 2016b). As predators, protists transfer

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nutrients to higher trophic levels in the soil foodweb (de Ruiter et al., 1995; Crotty et al., 2012).

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Protist predation also stimulates microbial activity and nutrient cycling via the microbial loop, thus

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stimulating plant growth (Bonkowski and Clarholm, 2012) and representing an important link

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between aboveground and belowground components.

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The functional significance, abundance, environmental sensitivity, rapid response times and

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increasing ease of analysis of soil protists also makes them invaluable bioindicators of a variety of

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aspects of environmental change (Foissner, 1987; Gupta and Yeates, 1997; Payne, 2013). A particular

160

example of this is in paleoecology, where the hard shells of testate amoebae, diatoms and

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foraminifera are widely used in the reconstruction of past environments and past climate change

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over a range of timescales (Mitchell et al., 2008; Adl et al., 2011; Charman, 2015).

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Most of these applications are, however, based on a few often small-scale studies. Thus significant

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taxonomic and functional aspects remain largely untouched. Our aim in this report is to pool expert
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knowledge and opinion across the diverse field of soil protistology and soil microbial ecology to

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identify major knowledge gaps that need to be addressed and their significance for soil processes

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and ecosystem services.

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## 2. Materials and Methods

169

### 2.1 Approach to identify the 30 most relevant questions

170

Our aim was to review research gaps both in the field of soil protistology and in general soil biology

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with a special focus on protists. In line with recent studies (Sutherland et al., 2013; Seddon et al.,

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2014), we aimed to pool community expertise to identify the most important questions in different

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broad categories. We modified previously-used methods (Sutherland et al., 2013; Seddon et al.,

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2014) to obtain a list of most interesting questions through a democratic, transparent, multi-step

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curation process.

176

The participants in this process are involved in a wide range of research areas, with self-determined

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primary research area expressed as being ecology (62%), palaeoecology (12%), evolution (9%),

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biogeography (6%), phylogeny (6%), taxonomy (3%), parasitology (3%). Each participant formulated

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up to 10 questions that they believed were most relevant for their future research. The resulting 368

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questions were then compiled via an integrative group effort into consensus questions and placed

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into six major categories following a discussion by 16 of the participants at the German Society for

182

Protozoology meeting in February 2016. We included very broad, general questions as well as highly

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specialised topics into similar scaled consensus questions.

184

The resulting consensus questions were then re-evaluated and groupings adjusted in a vote. These

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questions (Supplementary Table 1) were sent out to all 47 participants, who individually indicated up

186

to 12 priority questions with at least one being allocated in each of the six following categories: (i)

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Morphology, Phylogeny, Taxonomy, Evolution and Physiology, (ii) Diversity, Community Composition
188 and Biogeography, (iii) Interactions among Protists and other Organisms, (iv) Functions of Protists, (v)

189 Global Change, Bioindicators and Applications, and (vi) Methodology.

190 All 47 participants were asked to provide their key scientific expertise and literature references for
191 studies that (partly) addressed individual questions. Finally, minor comments raised by individual
192 participants during the vote were integrated to clarify the questions and give consistent formatting
193 without changing the meaning of the questions that had been voted upon.

194 All individual votes were combined and five questions per category chosen to result in the final list of
195 30 key questions. When more than one question received the same number of votes (as present in
196 categories 1, 2, and 4), these questions were sent out to all 47 participants for another vote on the
197 selected questions only.

198 2.2 Potential limitations

199 Biases in broad-scale studies are impossible to avoid (Sutherland et al., 2013). On the other hand, the
200 more interdisciplinary the panel of authors is in terms of cultural and societal background and
201 specific scientific expertise, the more biases are reduced. Researchers working on soil protists are
202 often ecologists, whereas taxonomists, phylogeneticists, and physiologists more often focus on
203 aquatic taxa that are easier to isolate and cultivate. Indeed, participants who indicated ecology as
204 their first expertise dominated our list of participants (62%). Ecology, however, is a broad field and
205 our division into finer categories such as biogeography, palaeoecology, community structure, and
206 interactions resulted in a broad diversification into different subcategories. Additionally, 21% of the
207 participants indicated topics such as taxonomy, phylogeny, evolution, and physiology as their main
208 expertise corroborating the wide diversity of research fields among the co-authors.

209 The majority of participants are PhDs (Professor: 38%; Graduated scientists: 29%; Post-doc: 24%)
210 with an average number of publications on protists of 43 (minimum = 1; maximum = 230). A high
211 proportion of the participants work on multiple ecosystems (41%). Many focus on testate amoebae
212 (41%) although 26% of them work on multiple morphogroups (ciliates, heterotrophic flagellates,
213 amoebae, etc.). A majority (74%) of participants have a European background, but Asia, North and

214 South America are also well represented, thus reducing potential impacts of geographic origin.

215 Furthermore, most participants have international collaborations that partly compensate for gaps in
216 the geographic distribution of individuals.

217 Despite these potential limitations, we found few biases in the way participants replied to questions

218 (Supplementary Results 1). Most participants (70%) selected questions evenly distributed across the
219 six categories, except a small group of people mostly constituted of researchers from the same
220 institute and/or with the same kind of expertise (phylogeny, taxonomy and evolutionary; see

221 Supplementary Results 1). This small group allocated 45% of their votes to the category (ii). Except

222 this small bias, most participants selected questions regardless of their experience, age, geographic
223 background, and most importantly, their expertise and group of interest.

224 Questions were differently formulated, hence we had to make decisions and remove some nuances

225 as we merged similar questions. This resulted in some discussions about how questions should be

226 best stated and consequently combined and grouped into non-predetermined categories. However,
227 we preferred to receive non-restricted questions to stimulate lateral thinking as previously suggested
228 (Sutherland et al., 2013); due to intensive exchange and to a democratic group effort at all steps of
229 the procedure, we are convinced that we have reached a consensus format.
230 **3. Results and Discussion**

231 **3.1 The 30 most relevant consensus questions**

232 From the 107 questions in the final vote, 94% received at least one, 79% two, 67% three and 50% five votes showing that the pre-selected questions had a wide general appeal to the scientific experts
233 involved (Supplementary Table 1). Therefore, all questions seem to be relevant for future studies
234 that focus on soil protists. However, as we aimed at providing a highly specific list of the major
235 research gaps and open challenges in soil protistology, we only provide the top-ranked 30 questions
236 classified in six major categories that most researches voted upon.
237

238 **3.2 Categories**

239 I Morphology, Phylogeny, Taxonomy, Evolution and Physiology

1 How long can protists survive in an encysted form? What are the tolerances of (encysted) protists to stress and what is the importance of cysts for ecosystem resilience?

2 How much morphological and genetic variability exists within soil protists?

3 How do species that occur in both aquatic and soil systems adapt to differing demands?

4 What are the phylogenetic relations of true soil to aquatic protist taxa and how often have soils been colonized by aquatic protists and vice versa?

5 How widespread is sex in soil protists?

240

241 Linking the individual topics of this category is one of the major tasks confronting soil protistology.

242 The coupling of morphology and phylogeny is crucial to obtain a stable taxonomic framework for protists. This is, for instance, crucial to answer evolutionary questions on the origin of eukaryotes
243 (López-García and Moreira, 2015). Soil protists may have an important role to play in such research
244 as most taxa likely remain unknown and novel higher-level taxonomic groups are continuously being discovered (Berney et al., 2015; Bass et al., 2016; Singer et al., 2016; Tice et al., 2016). Soil protists
245
246
247 might fill remaining phylogenetic gaps from better-studied aquatic taxa to improve phylogenetic resolution within and between protist clades, as strict soil protist clades seem to be common (Bass et al., 2016). Sequencing whole genomes will reveal ancient traits of eukaryotes and potential changes in their function during the evolution of eukaryotes. In this respect, soil protists must certainly play a key role for understanding the evolution of the eukaryotic cell and, therefore, of life as a whole.

252 While the morphological and phylogenetic framework for ciliates is reasonably well established (Lynn, 2008; Foissner, 2016), it remains rudimentary for other morphogroups as well as phylogenetic clades of protists (Kosakyan et al., 2016). The taxonomy of the groups has profoundly benefited from (mainly) 18S rRNA gene-based characterisations that have often led to drastic changes in phylogenetic placements of individual species, genera, families or even orders (Boenigk et al., 2012a; Berney et al., 2015; Bass et al., 2016). This is exemplified by the morphologically and functionally diverse Cercozoa, which was the first protist clade inferred solely based on molecular phylogenetic information, and has become home to ever more morphologically different organisms (Bass et al., 2016). Therefore, the true extent of morphological and genetic variability in different groups of soil protists remains largely unknown and is a key missing gap for future studies (Q2).

262 A key feature of soil protist species is their capacity for cyst formation as this allows them to resist constantly changing conditions, especially with respect to moisture and temperature. Furthermore, given that protists can excyst after decades, even millennia (Shmakova et al., 2016), cyst formation may protect species from becoming extinct at local or even at the global scales, influence population dynamics and maintain biodiversity (Corliss and Esser, 1974; Jones and Lennon, 2010). The importance of the cyst bank in ecosystem functioning and resilience remains largely unknown and have consequently been identified as a key element for future studies (Q1). More generally, this applies to all specific (physiological) adaptations of soil protists in comparison to their aquatic relatives (Q3, Q4) and to reproduction (Q5)..
6 What is the real diversity and community structure of soil protists in different systems (e.g. soils, rhizosphere, (plant) endosphere)?

7 How similar are the diversity patterns of soil protists and other soil biota along ecological gradients, and to what extent do different environmental factors shape their respective diversity?

8 What abiotic environmental factors influence the distribution and community composition of protists, and how?

9 How cosmopolitan are protists and how many endemic soil protist species are there?

10 What are dominant groups of soil protists in terms of turnover, abundance and biomass?

272 We are progressively shedding light into the soil 'black box'; however, knowledge on protists lags behind that of other groups (Fig. 2) (Wilkinson, 2008). Traditional studies have focused exclusively on a few of the 'classic' morphogroups, especially ciliates and testate amoebae, at least partly due to their ease of isolation and feature-rich morphologies (Foissner, 1999). Despite dominating many soil protist communities in terms of numbers and diversity, flagellates and naked amoebae have remained understudied, due to their often smaller sizes, lack of diagnostic features when studied by light microscopy, and the need to establish specialised enrichment cultivation for their isolation from soils (Berthold and Palzenberger, 1995; Foissner, 1999; Smirnov and Brown, 2004; Tikhonenkov et al., 2010). The development of molecular tools such as DNA barcoding and metabarcoding has considerably improved the situation in the last decade (Pawlowski et al., 2012) and allowed a phylogenetically based (morphogroup-independent) and consequently much more detailed analysis of the entirety of soil protist communities. These studies have revealed an enormous diversity of protists inhabiting soils, a phylogenetic diversity that might be similar to that of bacteria (e.g., (Bates et al., 2013; Geisen et al., 2015c; Mahé et al., 2017). Also, groups of protists previously almost unknown from soils have been shown to be common e.g. choanoflagellates, foraminifera, dinoflagellates, parasitic apicomplexans and pathogenic oomycetes (Bates et al., 2013; Geisen et al.,
289

2015c; Grossmann et al., 2016; Mahé et al., 2017). Therefore, we are only beginning to understand
the diversity of soil protists (Q10), which part is active, how this diversity differs in different soil
environments (Q6), how protist communities are structured by, e.g., abiotic factors (Q7, Q8) (Geisen
et al., 2014a; Lentendu et al., 2014; Geisen et al., 2015c; Dupont et al., 2016).

290

291

292

293

In addition, the biogeography of (soil) protists has been addressed in some studies, but it is still
unclear which and how many groups display a restricted biogeography and what the factors are that
shape these distributions (Q8, Q9). Although distribution of bacteria may support the hypothesis that
"everything is everywhere, but, the environment selects" (Baas-Becking, 1934), its extrapolation to
protists has been countered, particularly, by work on ciliates and testate amoebae (Foissner, 2006,
2008; Fernandez et al., 2016). The diversity and biogeographical distribution of protists, is, therefore,
one of degree (rather than all cosmopolitan or all limited) and the possibility exists that the
investigation of cryptic diversity within morphospecies will allow a finer-scale resolution of these
questions.

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295

296

297

298

299

300

301

302

III Interactions among Protists and other Organisms

11

How do protist taxa affect the composition of the soil microbiome and what other important
interactions take place?

12

What are the biotic interactions of soil protists with other taxonomic groups, and how are
protists linked within the soil food web?

13

What is the relative contribution of nutrient cycling (i.e. the microbial loop) versus modification
of the rhizosphere microbiome in protist-induced stimulation of plant growth?

14

What are the mechanisms by which individual soil protist species affect plant performance, and
do those mechanisms differ between plant species?

15

What is the impact of protists on the community functioning of other soil microbes?

303
304 Soil protists are still predominantly considered as being mainly bacterivorous (Bradford, 2016; Geisen, 2016a). Differential feeding by protists stimulated by bacterial volatiles modifies the community composition of bacteria (Bonkowski, 2004; Glücksman et al., 2010; Schulz-Bohm et al., 2017), which results in functional changes in the bacterial community structure (see next section).
305
306
307
308 Many free-living bacteria can, in turn, defend themselves against certain protist predators and even kill them (Greub and Raoult, 2004; Jousset et al., 2006). Several bacteria, viruses, and even other protists can also parasitize protist hosts (Barker and Brown, 1994; Raoult and Boyer, 2010).
309
310
311 The prey spectrum of protists has, however, repeatedly been shown to be much more diverse than bacteria. Indeed, archaea (Ballen-Segura et al., 2017), fungi (Gupta and Germida, 1988; Ekelund, 1998; Adl and Gupta, 2006; Geisen et al., 2016), other protists (Page, 1977; Jassey et al., 2012), and nematodes (Bjørnlund and Rønn, 2008; Geisen et al., 2015b) constitute prey for diverse protist species. Recently, HTS approaches have revealed the ubiquitous presence and dominant roles of protist parasites and pathogens in soils, and they likely represent a key component controlling other soil organisms including larger soil metazoans (animals) and plants (Geisen et al., 2015a; Dupont et al., 2016; Geisen, 2016b). This draws attention to the enormous complexity and importance of protist interactions with other organisms (Bonkowski, 2004).
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318
319

320 Due to our limited knowledge of protist diversity and because most studies have used only one or
321 few protists as models, we lack understanding about most aspects of how soil protist communities
322 interact with other organisms. Disentangling the diverse interactions of protists with other soil
323 organisms (Q11, Q12, Q15), the exact mechanisms (Q14) and the resulting importance for
324 functioning (Q13, Q14), therefore, are key knowledge gaps necessitating future research.
325

IV Functions of Protists

16 What is the importance of soil protists in biogeochemical cycling?

17 How much functional redundancy is there in the soil protist community?

18 Does increased protist diversity affect ecosystem functioning?
19 What is the comparative importance of eukaryotic microbes vs. prokaryotes in driving key soil processes?

20 Which individual functions are performed by distinct groups, and what is the entire functional diversity of soil protists?

326
327 Many acknowledged functions of soil protists are attributed to interactions with other organisms as outlined above. Especially important is the role of protists in driving the microbial loop, i.e. releasing nutrients (particularly nitrogen) bound in bacterial prey. The microbial loop has been demonstrated both in aquatic (Azam et al., 1983) and soil systems (Clarholm, 1985). This ground-breaking research identified protists as important drivers of the global ecosystem. Subsequent work on the microbial loop demonstrated that differential feeding by protists on bacterial prey is beneficial for plant growth (Bonkowski, 2004; Rosenberg et al., 2009). The main focus in earlier studies was, however, mainly on nitrogen cycling, and the importance of protists for cycling of other elements such as carbon and phosphorus has been relatively neglected, with few exceptions (Cole et al., 1977; Gupta and Germida, 1988; Treonis and Lussenhop, 1997; Frey et al., 2001; Murase et al., 2011; Eisenhauer et al., 2012; Jassey et al., 2015). Protists might even play a role in silica cycling as some use Si as reinforcing elements or in an exoskeleton (Aoki et al., 2007; Creevy et al., 2016). More thorough investigations about the functional roles of additional protist species and communities as a whole will likely reveal insights into the importance of protists in biogeochemical nutrient cycling. This was identified by most participants of this study as *the* most important question for future research (Q16).

343 In contrast to free-living protists, plant pathogenic protists, such as oomycetes or plasmodiophorids, have, not surprisingly, attracted considerable attention due to their agro-economic impact (Anderson et al., 2004; Bell et al., 2006; Neuhauser et al., 2014). These were, however, until very recently often considered as 'fungi' (Schardl and Craven, 2003; Gams et al., 2011). Similarly, soil protists with
immediate relevance for human diseases such as those directly harmful to humans (Schuster, 2002;
Siddiqui and Ahmed Khan, 2012; Geisen et al., 2014b) and those that act as “Trojan horses”
harbouring human-pathogenic bacteria (Brown and Barker, 1999; Molmeret et al., 2005) have
received considerable attention. In turn, the role of protists in plant disease control due to, e.g.,
increasing bacterial biocontrol agents (Jousset, 2012) or by directly feeding on plant pathogens (Old
and Chakraborty, 1986; Geisen et al., 2016) has received comparatively little attention. In line with
their importance in nutrient cycling and as biocontrol agents, the role of individual protist species
and that of protist diversity for the general functioning of soils and ecosystems (Q17, Q18, Q20), also
in comparison to other groups of microbes (Q19), were identified as important questions to be
addressed in future studies.

V Global Change, Bioindicators and Applications

21 How do changing climatic patterns affect the diversity of, community structure of and ecosystem services provided by soil protists?

22 Which protist clades can be used as bioindicators to assess soil properties, ecosystem state, and anthropogenic impacts? How could this be implemented?

23 Why are some species more sensitive to environmental change than others, why do some respond faster to environmental factors?

24 How can protists be used for nutrient mobilization and biocontrol in cropping systems?

25 What is the importance of soil protists for biodiversity conservation and ecosystem management and restoration? Should we protect particular species or habitats?

358

Protist communities are often studied as bioindicators of past and present climatic conditions, land
use changes and pollution (Gupta and Yeates, 1997; Mitchell et al., 2008). Abiotic changes affect
protists in species-specific ways, thus forming the basis for their use as bioindicators (Fournier et al.,
2012). They may, for instance, provide information on soil state in agro-ecosystems (Foissner, 1997,
1999; Bharti et al., 2015). Testate amoebae and their subfossil remains have been used to evaluate wetland hydrological conditions, applied, for instance, in studies of peatland restoration (Marcisz et al., 2014) and reconstruction of Holocene environmental change (Turner et al., 2014; Lamentowicz et al., 2015; Payne et al., 2015). However, more generally, there has been little progress on evaluating protists as bioindicators even though reliable indicators to assess soil quality continue to be of high relevance (Griffiths et al., 2016) as also revealed here (Q22). Application of protists for stimulating plant performance in terms of nutrition, growth, productivity and disease suppression holds great promise but has received little attention (Q24)

Effects of ongoing global climate change and human impact on the environment are the focus of increasing scientific attention. Global warming has been shown to alter the abundance and community structure of protists (Tsyganov et al., 2011; Jassey et al., 2013) in the limited number of studies that have been done. Predicted changes in precipitation regime will likely affect water availability, which will impact protist communities directly (Clarholm, 1981; Bates et al., 2013; Geisen et al., 2014a). Elevated atmospheric CO₂ has also been shown to increase abundance and changes community structure of rhizosphere protists, possibly due to increased plant productivity and enhanced release of root organic exudates (e.g., (Treonis and Lussenhop, 1997; Anderson and Griffin, 2001; Rønn et al., 2002)). Increased air pollution by nitrogen, sulphur, tropospheric ozone and metals are also likely to alter protist abundance and diversity (Meyer et al., 2012; Payne et al., 2012; Payne et al., 2013). Most of these studies focused on testate amoebae, but it is important to study how global environmental changes affect entire protist communities (Q21, Q23, Q25) as these changes are likely to have significant impacts on ecosystem functioning/services and, consequently, on human welfare, and may provide more informative markers of environmental change.

VI Methodology

26 What is the most practical taxonomic unit to measure protist diversity?
27 How can we standardize and calibrate cultivation based and molecular methods to reliably quantify soil protist abundance, diversity and activity?

28 How should sampling be performed to adequately evaluate soil protist diversity?

29 At what scales (temporal, spatial/physical, morphological, phylogenetic) should we study protists to fully understand their diversity and function in soil; which one should be prioritized?

30 How can we infer functional traits of soil protists based on morphology or phylogenetic affiliation, and what taxonomic resolution is needed?

387

388 Diverse methods are used to study community structures of soil protists. Even with respect to more

389 classical culturing and morphological observational techniques, the application of methods of non-

390 protistological disciplines, such as mycology, have the potential of broadening our perspectives on

391 the soil protist community (Spiegel et al., 2004). However, especially recent developments in

392 molecular sequencing technologies, have changed and will continue to change our knowledge about

393 protist diversity and community structure in soils (Bates et al., 2013; Geisen et al., 2015c). However,

394 some issues relating to HTS-based efforts remain as they provide relative abundances of taxa without

395 providing information on absolute abundances. For example PCR-based HTS efforts have been shown

396 to artificially alter the observed community structure of soil protists, a problem which needs to be

397 solved to decipher their real community structure (Geisen et al., 2015a). PCR-free 'omics-

398 approaches', i.e. metagenomics and metatranscriptomics, might resolve some of these issues (Geisen

399 et al., 2015c; Jacquiod et al., 2016). Indeed, these sequence-based omics approaches and sequence-

400 independent metaproteomics provide valuable information not only on taxonomic diversity but also

401 on their potential functions (Prosser, 2015). Calibrating, standardizing and adopting community-

402 defined methodologies to study soil protists will, consequently, be key for cross-study comparisons

403 (Q27) and correct sampling and analyses through different scales need to be defined a *priory* (Q28,

404 Q29). Furthermore, it is essential to identify the most meaningful taxonomic levels to use in the study

405 of diversity and functioning of soil protists (Q26), but even the definition of a species remains a
406 challenge (Boenigk et al., 2012b) and integrating morphology to phylogeny to function remains
407 missing (Q30).

408 In addition, medical and novel imaging techniques applied to soil are revolutionising *in situ* work
409 allowing us to study protist species in undisturbed soil and on plant roots. These include applications
410 of NanoSIMS technology to precisely locate isotopic markers and isotopic composition of material in
411 fixed preparations and to study dynamics of nutrient fluxes (Stockdale et al., 2009), which allows
412 tracing nutrient flow from microbial prey to protist predator and further in the food web in high
413 resolution. This will allow detailed investigations how protists selective interact in microsites with their
414 prey, how nutrients become released and where they are translocated. Applications of a variety of X-
415 ray based synchrotron spectroscopy and tomography with undisturbed soil is becoming technically
416 feasible and permits the study of dynamics and fluxes at a very fine resolution without interfering
417 with the matrix (Keyes et al., 2013). The ability to use soils with intact fine roots, and examining
418 undisturbed natural soil communities finally provides access to rhizosphere processes. Techniques to
419 measure and analyse chemically soil community molecular interactions and communications are now
420 only a few steps away.

421 3.3 (Partial) knowledge gaps and future directions

422 In this paper we provide a guide to 30 highly relevant questions for future studies in soil protistology.
423 Research has already been conducted on many of these questions. Literature searches and personal
424 knowledge of the literature allowed us to identify studies that addressed 91 % of the initial and 97 %
425 of the final questions. However, many of these studies focus on organisms other than soil protists
426 (e.g. aquatic protists or non-protist microbes), and may not be directly applicable to the situation
427 with soil protists. The fact that these 30 questions have been identified by our pool of experts
428 strongly implies that previous research has been insufficient to provide conclusive answers. In
429 Supplementary Table 1 we provide an extensive bibliography of previous research relevant to
addressing these questions. This bibliography will be a valuable literature guide to the current state
of the art on soil protistology.

We are beginning to understand many aspects of soil protist biology, as we are identifying the
hyperdiverse nature of protist communities, determining their (a)biotic drivers, deciphering
interactions with other organisms, and shedding light on their importance in ecosystem dynamics. So
far, however, we are only seeing the tip of the iceberg. Addressing many of the 30 questions
highlighted here will undoubtedly reveal novel insights, not only into soil protists, but also into other
organisms, soils, and fundamental ecological processes. We hope that these questions will be used to
catalyse soil protistology and to build research agendas for the future. More specifically, we
encourage both protistologists and researchers in closely related fields to consider these questions
carefully and to use them to develop new and innovative individual and collaborative projects. With
newly available techniques, an increase in knowledge and a growing awareness of the importance of
soil protists, we are at the start of a bright future for soil protist research!

Acknowledgements

We thank Alex K. Tice for providing pictures of *Acrasis* and *Rosculus* included in Fig.1. SG thanks Wim van der Putten and his ERC-Adv grant 260-55290 (SPECIALS). We further acknowledge funding by the Swiss Contribution to the enlarged European Union (Project CLIMPEAT: PSPB-013/2010) to ML, KM, MM, VEJJ and EADM, the Russian Scientific Fund (no 14-14-00891) to RJP and YuM, the São Paulo Research Foundation (FAPESP) (Young Investigator Award no 2013/04585-3) to DJGL, the Swiss National Science Foundation to VEJJ (no 315260_149807), EL, QB and DS (grants no 143960 and 163254), EADM & TH (no 141188 and 163431), the Chilean Comisión Nacional de Investigación Científica y Tecnológica (CONICYT) (doctoral grants no 21110037 and 78130011), the Universidad de Concepcion (Chile) and the Wildlife Conservation Society (Karukinka-grant no. 2012) to LDFP, and the University of Neuchâtel to LDFP & CVWS. This is publication XXXX of the NIOO-KNAW.
All authors declare no conflict of interest.
455 Author contributions

456 SG, EADM and EL designed the study; all authors sent initial questions. SG, TH, VEJJ, DJGL, DS and DW

457 compiled the information and analysed data for Fig. 2. JW, MB, AF, EL, VK, ALT, SK, FS, AS, FE, SG

458 provided photographs that DS, SG, EL and EADM compiled in Fig. 1. SG, EL, EADM, MB, VK, KM, DJGL,

459 MWB, MM, DMW, BSG, SA, RR, AMFD integrated, modified and established the final questions being

460 sent out to all participants. All authors voted on the final list of questions and provided references for

461 separate questions. SG and EL wrote the first draft of the manuscript and all authors contributed

462 substantially to revisions.
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768
769 **Figures**

770 Fig. 1: Common free-living soil protists as visualized by size (lengths), morphology and phylogenetic
771 affiliation. Note, soil protists belong to a wide range of supergroups (in brackets), whereas animals
772 are only placed in the supergroup Opisthokonta. Furthermore, soil protists span a much wider size
773 range as commonly assumed. With the exception of ciliates, morphogroups are not phylogenetically
774 conserved and are placed in different eukaryotic supergroups. Most soil protists can occur in
775 different life forms including active form (amoeba, flagellate, ciliate), but most form cysts, while
776 some can form special reproduction structures (sorocarps and fruiting bodies).

777 Fig. 2: (a) Overview of studies specifically mentioning protists in the title in 5-year intervals since
778 1980. Soil studies represent only about a fraction of aquatic studies (separated into freshwater,
779 marine and those that more broadly indicate aquatic) showing a strong increase in protist research in
780 aquatic, but not soil environments; (b) Comparison of soil studies specifically mentioning protists in
781 the title with those on other micro-sized organisms including viruses (blue filled circles), archaea
782 (green open circles), bacteria (red diamonds), fungi (orange crosses) and nematodes (green
783 triangles). See Supplementary Methods for details on the search.
<figure><img src="image_3.png" /></figure>

Naked amoeba
*Acrasis kona*

(HETEROLOBOSEA, EXCAVATA)

Cyst of

Acanthamoeba sp.

(AMOEBOZOA)

Flagellate

Neoheteromita globosa

(RHIZARIA; SAR)

Naked amoeba
*Rosculus* sp.

(RHIZARIA; SAR)

Cyst of

*Rosculus* sp.

(RHIZARIA; SAR)

Cyst of *Acrasis kona*

(HETEROLOBOSEA, EXCAVATA)
<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>