Abstract
The high numbers and diversity of protists in soil systems have long been presumed, but their true diversity and community composition have remained largely concealed. Traditional cultivation-based methods miss a majority of taxa, whereas molecular barcoding approaches employing PCR introduce significant biases in reported community composition of soil protists. Here, we applied a metatranscriptomic approach to assess the protist community in 12 mineral and organic soil samples from different vegetation types and climatic zones using small subunit ribosomal RNA transcripts as marker. We detected a broad diversity of soil protists spanning across all known eukaryotic supergroups and revealed a strikingly different community composition than shown before. Protist communities differed strongly between sites, with Rhizaria and Amoebozoa dominating in forest and grassland soils, while Alveolata were most abundant in peat soils. The Amoebozoa were comprised of Tubulinea, followed with decreasing abundance by Discosea, Variosea and Mycetozoa. Transcripts of Oomycetes, Apicomplexa and Ichthyosporea suggest soil as reservoir of parasitic protist taxa. Further, Foraminifera and Choanoflagellida were ubiquitously detected, showing that these typically marine and freshwater protists are autochthonous members of the soil microbiota. To the best of our knowledge, this metatranscriptomic study provides the most comprehensive picture of active protist communities in soils to date, which is essential to target the ecological roles of protists in the complex soil system.
Similar content being viewed by others
Log in or create a free account to read this content
Gain free access to this article, as well as selected content from this journal and more on nature.com
or
Accession codes
References
Adl SM, Simpson AGB, Lane CE, Lukeš J, Bass D, Bowser SS et al. (2012). The revised classification of eukaryotes. J Eukaryot Microbiol 59: 429–514.
Adl SM, Habura A, Eglit Y . (2014). Amplification primers of SSU rDNA for soil protists. Soil Biol Biochem 69: 328–342.
Altizer S, Harvell D, Friedle E . (2003). Rapid evolutionary dynamics and disease threats to biodiversity. Trends Ecol Evol 18: 589–596.
Amaral-Zettler LA, McCliment EA, Ducklow HW, Huse SM . (2009). A method for studying protistan diversity using massively parallel sequencing of V9 hypervariable regions of small-subunit ribosomal RNA genes. PloS One 4: e6372.
Arndt H, Dietrich D, Auer B, Cleven E-J, Gräfenhan T, Weitere M et al. (2000). Functional diversity of heterotrophic flagellates in aquatic ecosystems. In: Leadbeater BSC, Green JC et al. The Flagellates: Unity, Diversity and Evolution. Taylor & Francis: London, pp 240–268.
Bachy C, Dolan JR, Lopez-Garcia P, Deschamps P, Moreira D . (2013). Accuracy of protist diversity assessments: morphology compared with cloning and direct pyrosequencing of 18 S rRNA genes and ITS regions using the conspicuous tintinnid ciliates as a case study. ISME J 7: 244–255.
Baldwin DS, Colloff MJ, Rees GN, Chariton AA, Watson GO, Court LN et al. (2013). Impacts of inundation and drought on eukaryote biodiversity in semi-arid floodplain soils. Mol Ecol 22: 1746–1758.
Bamforth SS . (2007). Protozoa from aboveground and ground soils of a tropical rain forest in Puerto Rico. Pedobiologia 50: 515–525.
Bass P, Bischoff PJ . (2001). Seasonal variability in abundance and diversity of soil gymnamoebae along a short transect in southeastern USA. J Eukaryot Microbiol 48: 475–479.
Bass D, Cavalier-Smith T . (2004). Phylum-specific environmental DNA analysis reveals remarkably high global biodiversity of Cercozoa (Protozoa). Int J Syst Evol Micr 54: 2393–2404.
Bates ST, Clemente JC, Flores GE, Walters WA, Parfrey LW, Knight R et al. (2013). Global biogeography of highly diverse protistan communities in soil. ISME J 7: 652–659.
Benhamou N, Rey P, Picard K, Tirilly Y . (1999). Ultrastructural and cytochemical aspects of the interaction between the mycoparasite Pythium oligandrum and soilborne plant pathogens. Phytopathol 89: 506–517.
Berney C, Fahrni J, Pawlowski J . (2004). How many novel eukaryotic 'kingdoms'? Pitfalls and limitations of environmental DNA surveys. BMC Biol 2: 13.
Berney C, Geisen S, Van Wichelen J, Nitsche F, Vanormelingen P, Bonkowski M et al. Expansion of the 'Reticulosphere': diversity of novel branching and network-forming amoebae helps to define Variosea (Amoebozoa). Protist.
Boenigk J, Pfandl K, Stadler P, Chatzinotas A . (2005). High diversity of the ‘Spumella-like’ flagellates: an investigation based on the SSU rRNA gene sequences of isolates from habitats located in six different geographic regions. Environ Microbiol 7: 685–697.
Bonkowski M . (2004). Protozoa and plant growth: the microbial loop in soil revisited. New Phytol 162: 617–631.
Cavalier-Smith T, Chao EEY, Oates B . (2004). Molecular phylogeny of Amoebozoa and the evolutionary significance of the unikont Phalansterium. Eur J Protistol 40: 21–48.
Chatzinotas A, Schellenberger S, Glaser K, Kolb S . (2013). Assimilation of cellulose-derived carbon by microeukaryotes in oxic and anoxic slurries of an aerated soil. Appl Environ Microbiol 79: 5777–5781.
Chen M, Chen F, Yu Y, Ji J, Kong F . (2008). Genetic diversity of eukaryotic microorganisms in Lake Taihu, a large shallow subtropical lake in China. Microb Ecol 56: 572–583.
Chou H-H, Holmes MH . (2001). DNA sequence quality trimming and vector removal. Bioinformatics 17: 1093–1104.
Clarholm M . (1985). Interactions of bacteria, protozoa and plants leading to mineralization of soil nitrogen. Soil Biol Biochem 17: 181–187.
Clarholm M, Bonkowski M, Griffiths B . (2007). Protozoa and other protista in soil. In: Van Elsas J, Jansson J, Trevors J (eds), Modern Soil Microbiology 2nd edn CRC, Boca Raton London New York. CRC Press: Boca Raton, FL, USA, pp 148–175.
Coince A, Caël O, Bach C, Lengellé J, Cruaud C, Gavory F et al. (2013). Below-ground fine-scale distribution and soil versus fine root detection of fungal and soil oomycete communities in a French beech forest. Fungal Ecol 6: 223–235.
Darriba D, Taboada GL, Doallo R, Posada D . (2012). jModelTest 2: more models, new heuristics and parallel computing. Nat Methods 9: 772–772.
De Jonckheere JF, Gryseels S, Eddyani M . (2012). Knowledge of morphology is still required when identifying new amoeba isolates by molecular techniques. Eur J Protistol 48: 178–184.
de Ruiter PC, Moore JC, Zwart KB, Bouwman LA, Hassink J, Bloem J et al. (1993). Simulation of nitrogen mineralization in the below-ground food webs of two winter wheat fields. J Appl Ecol 30: 95–106.
de Ruiter PC, Neutel AM, Moore JC . (1995). Energetics, patterns of interaction strengths, and stability in real ecosystems. Science 269: 1257–1260.
Ekelund F, Rønn R . (1994). Notes on protozoa in agricultural soil with emphasis on heterotrophic flagellates and naked amoebae and their ecology. FEMS Microbiol Rev 15: 321–353.
Ekelund F, Patterson DJ . (1997). Some heterotrophic flagellates from a cultivated garden soil in Australia. Arch Protistenkd 148: 461–478.
Ekelund F, Rønn R, Griffiths BS . (2001). Quantitative estimation of flagellate community structure and diversity in soil samples. Protist 152: 301–314.
Epstein S, López-García P . (2008). “Missing” protists: a molecular prospective. Biodivers Conserv 17: 261–276.
Fenchel T . (2010). The life history of Flabellula baltica Smirnov (Gymnamoebae, Rhizopoda): Adaptations to a spatially and temporally heterogeneous environment. Protist 161: 279–287.
Field SG, Michiels NK . (2005). Parasitism and growth in the earthworm Lumbricus terrestris: fitness costs of the gregarine parasite Monocystis sp. Parasitology 130: 397–403.
Finlay BJ, Black HIJ, Brown S, Clarke KJ, Esteban GF, Hindle RM et al. (2000). Estimating the growth potential of the soil protozoan community. Protist 151: 69–80.
Fiore-Donno AM, Kamono A, Chao EE, Fukui M, Cavalier-Smith T . (2010). Invalidation of Hyperamoeba by transferring its species to other genera of Myxogastria. J Eukaryot Microbiol 57: 189–196.
Foissner W . (1987). Soil protozoa: fundamental problems, ecological significance, adaptations in ciliates and testaceans, bioindicators, and guide to the literature. Prog Protistol 2: 69–212.
Foissner W . (1999). Soil protozoa as bioindicators: pros and cons, methods, diversity, representative examples. Agr Ecosyst Environ 74: 95–112.
Geisen S, Bandow C, Römbke J, Bonkowski M . (2014a). Soil water availability strongly alters the community composition of soil protists. Pedobiologia 57: 205–213.
Geisen S, Fiore-Donno A, Walochnik J, Bonkowski M . (2014b). Acanthamoeba everywhere: high diversity of Acanthamoeba in soils. Parasitol Res 113: 3151–3158.
Geisen S, Kudryavtsev A, Bonkowski M, Smirnov A . (2014c). Discrepancy between species borders at morphological and molecular levels in the genus Cochliopodium (Amoebozoa, Himatismenida), with the description of Cochliopodium plurinucleolum n. sp. Protist 165: 364–383.
Geisen S, Weinert J, Kudryavtsev A, Glotova A, Bonkowski M, Smirnov A . (2014d). Two new species of the genus Stenamoeba (Discosea, Longamoebia): cytoplasmic MTOC is present in one more amoebae lineage. J Eukaryot Microbiol 50: 153–165.
Glockling SL, Marshall WL, Gleason FH . (2013). Phylogenetic interpretations and ecological potentials of the Mesomycetozoea (Ichthyosporea). Fungal Ecol 6: 237–247.
Gong J, Dong J, Liu X, Massana R . (2013). Extremely high copy numbers and polymorphisms of the rDNA operon estimated from single cell analysis of oligotrich and peritrich ciliates. Protist 164: 369–379.
Goodey T . (1915). Note on the remarkable retention of vitality by protozoa from old stored soils. Ann Appl Biol 1: 395–399.
Gouy M, Guindon S, Gascuel O . (2010). SeaView version 4: a multiplatform graphical user interface for sequence alignment and phylogenetic tree building. Mol Biol Evol 27: 221–224.
Hong S, Bunge J, Leslin C, Jeon S, Epstein SS . (2009). Polymerase chain reaction primers miss half of rRNA microbial diversity. ISME J 3: 1365–1373.
Howe AT, Bass D, Vickerman K, Chao EE, Cavalier-Smith T . (2009). Phylogeny, taxonomy, and astounding genetic diversity of Glissomonadida ord. nov., the dominant gliding zooflagellates in soil (Protozoa: Cercozoa). Protist 160: 159–189.
Huang X, Madan A . (1999). CAP3: A DNA sequence assembly program. Genome Res 9: 868–877.
Huelsenbeck JP, Ronquist F . (2001). MRBAYES: bayesian inference of phylogenetic trees. Bioinformatics 17: 754–755.
Jeon S, Bunge J, Leslin C, Stoeck T, Hong S, Epstein S . (2008). Environmental rRNA inventories miss over half of protistan diversity. BMC Microbiol Biol 8: 222.
Kaiser C, Koranda M, Kitzler B, Fuchslueger L, Schnecker J, Schweiger P et al. (2010). Belowground carbon allocation by trees drives seasonal patterns of extracellular enzyme activities by altering microbial community composition in a beech forest soil. New Phytol 187: 843–858.
Lahr DJG, Grant J, Katz LA . (2013). Multigene phylogenetic reconstruction of the Tubulinea (Amoebozoa) corroborates four of the six major lineages, while additionally revealing that shell composition does not predict phylogeny in the Arcellinida. Protist 164: 323–339.
Lanzén A, Jørgensen SL, Bengtsson MM, Jonassen I, Øvreås L, Urich T . (2011). Exploring the composition and diversity of microbial communities at the Jan Mayen hydrothermal vent field using RNA and DNA. FEMS Microbiol Ecol 77: 577–589.
Lanzén A, Jørgensen SL, Huson DH, Gorfer M, Grindhaug SH, Jonassen I et al. (2012). CREST – Classification Resources for Environmental Sequence Tags. PloS one 7: e49334.
Lara E, Berney C, Ekelund F, Harms H, Chatzinotas A . (2007). Molecular comparison of cultivable protozoa from a pristine and a polycyclic aromatic hydrocarbon polluted site. Soil Biol Biochem 39: 139–148.
Lara E, Mitchell EA, Moreira D, López García P . (2011). Highly diverse and seasonally dynamic protist community in a pristine peat bog. Protist 162: 14–32.
Lares-Jiménez LF, Booton GC, Lares-Villa F, Velázquez-Contreras CA, Fuerst PA . (2014). Genetic analysis among environmental strains of Balamuthia mandrillaris recovered from an artificial lagoon and from soil in Sonora, Mexico. Exp Parasitol 145 (Supplement): S57–S61.
Latijnhouwers M, de Wit PJGM, Govers F . (2003). Oomycetes and fungi: similar weaponry to attack plants. Trends Microbiol 11: 462–469.
Lejzerowicz F, Pawlowski J, Fraissinet-Tachet L, Marmeisse R . (2010). Molecular evidence for widespread occurrence of Foraminifera in soils. Environ Microbiol 12: 2518–2526.
Lentendu G, Wubet T, Chatzinotas A, Wilhelm C, Buscot F, Schlegel M . (2014). Effects of long-term differential fertilization on eukaryotic microbial communities in an arable soil: a multiple barcoding approach. Mol Ecol 23: 3341–3355.
Lesaulnier C, Papamichail D, McCorkle S, Ollivier B, Skiena S, Taghavi S et al. (2008). Elevated atmospheric CO2 affects soil microbial diversity associated with trembling aspen. Environ Microbiol 10: 926–941.
Martin FN, Abad ZG, Balci Y, Ivors K . (2012). Identification and detection of Phytophthora: reviewing our progress, identifying our needs. Plant Dis 96: 1080–1103.
Medinger R, Nolte V, Pandey RV, Jost S, Ottenwälder B, Schlötterer C et al. (2010). Diversity in a hidden world: potential and limitation of next generation sequencing for surveys of molecular diversity of eukaryotic microorganisms. Mol Ecol 19: 32–40.
Meisterfeld R, Holzmann M, Pawlowski J . (2001). Morphological and molecular characterization of a new terrestrial allogromiid species: Edaphoallogromia australica gen. et spec. nov. (Foraminifera) from northern Queensland (Australia). Protist 152: 185–192.
Monchy S, Sanciu G, Jobard M, Rasconi S, Gerphagnon M, Chabé M et al. (2011). Exploring and quantifying fungal diversity in freshwater lake ecosystems using rDNA cloning/sequencing and SSU tag pyrosequencing. Environ Microbiol 13: 1433–1453.
Moon van der Staay SY, Tzeneva VA, Van Der Staay GW, De Vos WM, Smidt H, Hackstein JH . (2006). Eukaryotic diversity in historical soil samples. FEMS Microbiol Ecol 57: 420–428.
Moussa M, Tissot O, Guerlotté J, De Jonckheere J, Talarmin A et al. (2015). Soil is the origin for the presence of Naegleria fowleri in the thermal recreational waters. Parasitol Res 114: 311–315.
Page FC . (1988) A new key to freshwater and soil gymnamoebae with instructions for culture. Freshwater Biological Association: Ambleside.
Pawlowski J, Audic S, Adl S, Bass D, Belbahri L, Berney C et al. (2012). CBOL protist working group: barcoding eukaryotic richness beyond the animal, plant, and fungal kingdoms. PLoS Biol 10: e1001419.
Pawlowski J . (2013). The new micro-kingdoms of eukaryotes. BMC Biol 11: 40.
Phillips AJ, Anderson VL, Robertson EJ, Secombes CJ, van West P . (2008). New insights into animal pathogenic oomycetes. Trends Microbiol 16: 13–19.
Radax R, Rattei T, Lanzen A, Bayer C, Rapp HT, Urich T et al. (2012). Metatranscriptomics of the marine sponge Geodia barretti: tackling phylogeny and function of its microbial community. Environ Microbiol 14: 1308–1324.
Robinson BS, Bamforth SS, Dobson PJ . (2002). Density and diversity of protozoa in some arid Australian soils. J Eukaryot Microbiol 49: 449–453.
Ruiz A, Frenkel J, Cerdas L . (1973). Isolation of Toxoplasma from soil. J Parasitol 59: 204–206.
Schaefer M, Schauermann J . (1990). The soil fauna of beech forests: comparison between a mull and a moder soil. Pedobiologia 34: 299–314.
Schröter D, Wolters V, de Ruiter PC . (2003). C and N mineralisation in the decomposer food webs of a European forest transect. Oikos 102: 294–308.
Schuster FL, Visvesvara GS . (2004). Free-living amoebae as opportunistic and non-opportunistic pathogens of humans and animals. Int J Parasitol 34: 1001–1027.
Siddiqui R, Ahmed Khan N . (2012). Biology and pathogenesis of Acanthamoeba. Parasit Vectors 5: 1–13.
Smirnov AV, Brown S . (2004). Guide to the methods of study and identification of soil gymnamoebae. Protistology 3: 148–190.
Smirnov AV, Nassonova E, Berney C, Fahrni J, Bolivar I, Pawlowski J . (2005). Molecular phylogeny and classification of the lobose amoebae. Protist 156: 129.
Smirnov AV, Nassonova ES, Cavalier-Smith T . (2008). Correct identification of species makes the amoebozoan rRNA tree congruent with morphology for the order Leptomyxida Page 1987; with description of Acramoeba dendroida n. g., n. sp., originally misidentified as ‘Gephyramoeba sp. Eur J Protistol 44: 35–44.
Smirnov AV, Chao E, Nassonova ES, Cavalier-Smith T . (2011). A revised classification of naked lobose amoebae (Amoebozoa: Lobosa). Protist 162: 545–570.
Sokal RR . (1961). Distance as a measure of taxonomic similarity. Syst Zool 10: 70–79.
Stamatakis A . (2006). RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22: 2688–2690.
Stoeck T, Breiner H-W, Filker S, Ostermaier V, Kammerlander B, Sonntag B . (2014). A morphogenetic survey on ciliate plankton from a mountain lake pinpoints the necessity of lineage-specific barcode markers in microbial ecology. Environ Microbiol 16: 430–444.
Stoupin D, Kiss AK, Arndt H, Shatilovich AV, Gilichinsky DA, Nitsche F . (2012). Cryptic diversity within the choanoflagellate morphospecies complex Codosiga botrytis - phylogeny and morphology of ancient and modern isolates. Eur J Protistol 48: 263–273.
Tikhonenkov DV, Mylnikov AP, Gong YC, Feng WS, Mazei Y . (2012). Heterotrophic flagellates from freshwater and soil habitats in subtropical China (Wuhan Area, Hubei province). Acta Protozool 51: 65.
Tong S, Vørs N, Patterson D . (1997). Heterotrophic flagellates, centrohelid heliozoa and filose amoebae from marine and freshwater sites in the Antarctic. Polar Biol 18: 91–106.
Turner TR, Ramakrishnan K, Walshaw J, Heavens D, Alston M, Swarbreck D et al. (2013). Comparative metatranscriptomics reveals kingdom level changes in the rhizosphere microbiome of plants. ISME J 7: 2248–2258.
Tveit A, Schwacke R, Svenning MM, Urich T . (2013). Organic carbon transformations in high-Arctic peat soils: key functions and microorganisms. ISME J 7: 299–311.
Urich T, Lanzén A, Qi J, Huson DH, Schleper C, Schuster SC . (2008). Simultaneous assessment of soil microbial community structure and function through analysis of the meta-transcriptome. PloS One 3: e2527.
Urich T, Schleper C . (2011). The “Double-RNA” approach to simultaneously assess the structure and function of a soil microbial community. de Brujin FJ. Handbook of Molecular Microbial Ecology I. John Wiley & Sons, Inc.: Hoboken, NJ, USA, pp 587–596.
Urich T, Lanzén A, Stokke R, Pedersen RB, Bayer C, Thorseth IH et al. (2014). Microbial community structure and functioning in marine sediments associated with diffuse hydrothermal venting assessed by integrated meta-omics. Environ Microbiol 16: 2699–2710.
Visvesvara GS, Schuster FL, Martinez AJ . (1993). Balamuthia mandrillaris, N. G., N. Sp., agent of amebic meningoencephalitis in humans and other animals. J Eukaryot Microbiol 40: 504–514.
Visvesvara GS, Moura H, Schuster FL . (2007). Pathogenic and opportunistic free living amoebae: Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri, and Sappinia diploidea. FEMS Immunol Med Mic 50: 1–26.
Weber AAT, Pawlowski J . (2013). Can abundance of protists be inferred from sequence data: a case study of Foraminifera. PloS one 8: e56739.
Zwart KB, Burgers SLGE, Bloem J, Bouwman LA, Brussaard L, Lebbink G et al. (1994). Population dynamics in the belowground food webs in two different agricultural systems. Agr Ecosyst Environ 51: 187–198.
Acknowledgements
We thank Ave Tooming-Klunderud and colleagues at the Centre for Ecological and Evolutionary Synthesis (CEES, University of Oslo, Norway) for 454-sequencing. We thank Sarah Jennings (NIOO, Netherlands) for help in cDNA generation from Rothamsted soil and thank Christoph Bayer (Vienna) for help in sequence processing. Christa Schleper (Vienna) is thanked for her generous and continuous support. Special thanks to Fionn Clissman for his helpful suggestions on the wording. Part of this work was supported by a research grant from the EU-project ‘EcoFINDERS’ No. 264465 and by a research council of norway grant 191696/V49.
Author Contributions
TU, SG and MB designed research. TU, AT, MS and IC collected the soil samples. Metatranscriptomes were generated by TU and AT. TU processed metatranscriptome data, analysed by SG and TU. SG and TU generated Amoebozoa-CREST. SG, TU and MB wrote the manuscript, aided by all authors.
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
Competing interests
The authors declare no conflict of interest.
Additional information
Supplementary Information accompanies this paper on The ISME Journal website
Supplementary information
Rights and permissions
About this article
Cite this article
Geisen, S., Tveit, A., Clark, I. et al. Metatranscriptomic census of active protists in soils. ISME J 9, 2178–2190 (2015). https://doi.org/10.1038/ismej.2015.30
Received:
Revised:
Accepted:
Published:
Issue date:
DOI: https://doi.org/10.1038/ismej.2015.30
This article is cited by
-
Soil microbial ecology through the lens of metatranscriptomics
Soil Ecology Letters (2024)
-
Metagenomics and metatranscriptomics as potential driving forces for the exploration of diversity and functions of micro-eukaryotes in soil
3 Biotech (2023)
-
Protists: the hidden ecosystem players in a wetland rice field soil
Biology and Fertility of Soils (2023)
-
Microeukaryotic gut parasites in wastewater treatment plants: diversity, activity, and removal
Microbiome (2022)
-
Contrasting protist communities (Cercozoa: Rhizaria) in pristine and earthworm-invaded North American deciduous forests
Biological Invasions (2022)
