Skip to main content Accessibility help
  • Print publication year: 2016
  • Online publication date: June 2016

9 - Protist systematics, ecology and next generation sequencing

from Part II - Next Generation Biodiversity Science


Protist taxonomy in context

The increasing availability over the past two decades of gene sequence data for protists has fired an energetic and rapidly moving field of taxonomic development and debate, in response to many exciting and often very surprising findings. The classic subdivisions of microbial eukaryotes into four main groups – amoeboid organisms, flagellates, ciliates and sporozoa (a group of parasites) – formulated in the 19th century and current throughout a large part of the 20th appeals because of its simplicity, but almost could not be more wrong. The history of protist taxonomy is not the subject of this chapter, but a diversity of perspectives can be found in (among others) Adl et al. (2005; 2007; 2012), Cavalier-Smith (1998), Corliss (1984), Levine et al. (1980), Walker et al. (2011) and references therein. There are several characteristics of protists that have contributed to this taxonomic turbulence. Their size makes detailed observation non-trivial; individual approaches, skills and tools applied to morphological taxonomic studies have varied significantly over time, and continue to do so. Their single-celled and/or non-differentiated forms do not offer many easily observable characters for either the taxonomist or natural selection to work on. A consequence of the latter is extremely high levels of convergent evolution at different evolutionary scales. Some very striking examples of convergence have been revealed by molecular phylogenetic analyses, which demonstrate the extent to which the pre-molecular subdivision of protists was incorrect (e.g. Nikolaev et al. 2004; Richards and Talbot 2007; Richards et al. 2011; S. D. Brown et al. 2012).

Some other important factors contributing to taxonomic difficulties are (1) the unknown sexual status of most protists and therefore the inapplicability and/or uncertainty of applying the biological species concept (sex is known for some but many are presumed asexual at least in the mid to long term), (2) highly incomplete and patchy knowledge of the diversity of many protist groups and regions of the eukaryote Tree of Life in which knowledge of lineage diversity and biology is generally poor, (3) the absence of a generally agreed or applicable species concept for most protist groups, and (4) difficulty of culturing many lineages.

Related content

Powered by UNSILO
Adl, S. M., Leander, B. S., Simpson, A. G., et al. (2007). Diversity, nomenclature, and taxonomy of protists. Systematic Biology, 56, 684–9.
Adl, S. M., Simpson, A. G., Farmer, M. A., et al. (2005). The new higher level classification of eukaryotes with emphasis on the taxonomy of protists. Journal of Eukaryotic Microbiology, 52, 399–451.
Adl, S. M., Simpson, A. G., Lane, C. E., et al. (2012). The revised classification of eukaryotes. Journal of Eukaryotic Microbiology, 59, 429–514.
Álvarez, I. and Wendel, J. F. (2003). Ribosomal ITS sequences and plant phylogenetic inference. Molecular Phylogenetics and Evolution, 29, 417–34.
Amaral-Zettler, L. A., McCliment, E. A., Ducklow, H. W. and Huse, S. M. (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.
Amato, A., Kooistra, W. H., Levialdi, C. F., et al. (2007). Reproductive isolation among sympatric cryptic species in marine diatoms. Protist, 158, 193–207.
Bachy, C., Dolan, J. R., López-García, P., Deschamps, P. and Moreira, D. (2012). Accuracy of protist diversity assessments: morphology compared with cloning and direct pyrosequencing of 18S rRNA genes and ITS regions using the conspicuous tintinnid ciliates as a case study. The ISME Journal, 7, 244–55.
Barraclough, T. G., Birky, C. W. Jr and Burt, A. (2003). Diversification in sexual and asexual organisms. Evolution, 57, 2166–72.
Barraclough, T. G., Hughes, M., Ashford-Hodges, N. and Fujisawa, T. (2009). Inferring evolutionarily significant units of bacterial diversity from broad environmental surveys of single-locus data. Biology Letters, 5, 425–8.
Bass, D. and Boenigk, J. (2011). Everything is everywhere, a 21st century de-reconstruction. In Biogeography of Microscopic Organisms, Is Everything Everywhere? ed. Fontaneto, D.. Systematics Association Special Volume 79. Cambridge, Cambridge University Press, pp. 88–110.
Bass, D., Howe, A. T., Mylnikov, A. P., et al. (2009). Phylogeny and classification of Cercomonadida, Cercomonas, Eocercomonas, Paracercomonas, and Cavernomonas gen. n. Protist 160, 483–521.
Bass, D., Richards, T. A., Matthai, L., Marsh, V. and Cavalier-Smith, T. (2007). DNA evidence for global dispersal and probable endemicity of protozoa. BMC Evolutionary Biology, 7, 162.
Bass, D., Stentiford, G. D., Littlewood, T. D. and Hartikainen, H. (2015). Diverse applications of environmental DNA methods in parasitology. Trends in Parasitology, 31, 499–513.
Bass, D., Yabuki, A., Santini, S., Romac, S. and Berney, C. (2012). Reticulamoeba is a long branched granofilosean (Cercozoa) that is missing from sequence databases. PLoS One, 7, e49090.
Behnke, A., Engel, M., Christen, R., Nebel, M., Klein, R. R. and Stoeck, T. (2011). Depicting more accurate pictures of protistan community complexity using pyrosequencing of hypervariable SSU rRNA gene regions. Environmental Microbiology, 13, 340–9.
Bellemain, E., Carlsen, T., Brochmann, C., Coissac, E., Taberlet, P. and Kauserud, H. (2010). ITS as an environmental DNA barcode for fungi, an in silico approach reveals potential PCR biases. BMC Microbiology, 10, 189.
Berney, C., Romac, S., Mahé, F., Santini, S., Siano, R. and Bass, D. (2013). Vampires in the oceans: predatory cercozoan amoebae in marine habitats. The ISME Journal, 7, 2387–99.
Bik, H. M., Porazinska, D. L., Creer, S., Caporaso, J. G., Knight, R. and Thomas, W. K. (2012). Sequencing our way towards understanding global eukaryotic biodiversity. Trends in Ecology and Evolution, 27, 233–43.
Bittner, L., Gobet, A., Audic, S., et al. (2012). Diversity patterns of uncultured Haptophytes unravelled by pyrosequencing in Naples Bay. Molecular Ecology, 22, 87–101.
Blaxter, M., Mann, J., Chapman, T., et al. (2005). Defining operational taxonomic units using DNA barcode data. Philosophical Transactions of the Royal Society of London B-Biological Sciences, 360, 1935–43.
Boenigk, J., Ereshefsky, M., Hoef-Emden, K., Mallet, J. and Bass, D. (2012). Concepts in protistology: species definitions and boundaries. European Journal of Protistology, 48, 96–102.
Brabender, M., Kiss, A. K., Domonell, A., Nitsche, F. and Arndt, H. (2012). Phylogenetic and morphological diversity of novel soil cercomonad species with a description of two new genera (Nucleocercomonas and Metabolomonas). Protist, 163, 495–528.
Brown, M. W., Kolisko, M., Silberman, J. D. and Roger, A. J. (2012). Aggregative multicellularity evolved independently in the eukaryotic supergroup Rhizaria. Current Biology, 22, 1123–7.
Brown, S. D., Armstrong, K. F. and Cruickshank, R. H. (2012). Molecular phylogenetics of a South Pacific sap beetle species complex (Carpophilus spp., Coleoptera, Nitidulidae). Molecular Phylogenetics and Evolution, 64, 428–40.
Buée, M., Reich, M., Murat, C., et al. (2009). 454 Pyrosequencing analyses of forest soils reveal an unexpectedly high fungal diversity. New Phytologis, 184, 449–56.
Burki, F. (2014). The eukaryotic tree of life from a global phylogenomic perspective. Cold Spring Harbor Perspectives in Biology, 6(5), a016147.
Caisová, L., Marin, B. and Melkonian, M. (2011). A close-up view on ITS2 evolution and speciation: a case study in the Ulvophyceae (Chlorophyta, Viridiplantae). BMC Evolutionary Biology, 11, 262.
Cavalier-Smith, T. (1998). A revised six-kingdom system of life. Biological Reviews of the Cambridge Philosophical Society, 73, 203–66.
Cavalier-Smith, T. and Chao, E. E. (2010). Phylogeny and evolution of Apusomonadida (Protozoa, Apusozoa): new genera and species. Protist, 161, 549–76.
Chambouvet, A., Richards, T. A., Bass, D. and Neuhauser, S. (2015). Revealing micro-parasite diversity using brute force molecular techniques and gently persuasive microscopy in aquatic environments. In Parasite Diversity and Diversification, ed. Morand, S., Brasnov, B. R. and Littlewood, D. T. J.. Cambridge, Cambridge University Press.
Chan, C. X. and Ragan, M. A. (2013). Next-generation phylogenomics. Biology Direct, 8, 3.
China Plant BOL Group, Li, D. Z., Gao, L. M., et al. (2011). Comparative analysis of a large dataset indicates that internal transcribed spacer (ITS) should be incorporated into the core barcode for seed plants. Proceedings of the National Academy of Sciences of the United States of America, 108, 19641–6.
Coleman, A. W. (2007). Pan-eukaryote ITS2 homologies revealed by RNA secondary structure. Nucleic Acids Research, 35, 3322–9.
Coleman, A. W. (2009). Is there a molecular key to the level of “biological species” in eukaryotes? A DNA guide. Molecular Phylogenetics and Evolution, 50, 197–203.
Corliss, J. O. (1984). The kingdom Protista and its 45 phyla. Biosystems, 17, 87–126.
Creer, S., Fonseca, V. G., Porazinska, D. L., et al. (2010). Ultrasequencing of the meiofaunal biosphere: practice, pitfalls and promises. Molecular Ecology, 19 (Suppl 1), 4–20.
del Campo, J., Not, F., Forn, I., Sieracki, M. E. and Massana, R. (2013). Taming the smallest predators of the oceans. The ISME Journal, 7(2), 351–8.
de Vargas, C., Audic, S., Henry, N., et al. (2015). Eukaryotic plankton diversity in the sunlit ocean. Science, 348(6237). doi: 10.1126/science.1261605.
Dunthorn, M., Otto, J., Berger, S. A., et al. (2014). Placing environmental next-generation sequencing amplicons from microbial eukaryotes into a phylogenetic context. Molecular Biology and Evolution, 31, 993–1009.
Dupont, A. Ö. C., Griffiths, R. I., Bell, T. and Bass, D. (2016). Differences in soil micro-eukaryotic communities over soil pH gradients are strongly driven by parasites and saprotrophs. Environmental Microbiology, in press.
Edgcomb, V., Orsi, W, Bunje, J. et al. (2011). Protistan microbial observatory in the Cariaco Basin, Caribbean. I. Pyrosequencing vs Sanger insights into species richness. The ISME Journal, 5, 1344–56.
Evans, S. N. and Matsen, F. A. (2012). The phylogenetic Kantorovich-Rubinstein metric for environmental sequence samples. Journal of the Royal Statistical Society. Series B, Statistical Methodology, 74, 569–92.
Fonseca, V. G., Carvalho, G. R., Sung, W., et al. (2010). Second-generation environmental sequencing unmasks marine metazoan biodiversity. Nature Communications, 1, 98.
Fonseca, V. G., Nichols, B., Lallias, D., et al. (2012). Sample richness and genetic diversity as drivers of chimera formation in nSSU metagenetic analyses. Nucleic Acids Research, 40, e66.
Garzón, C., Geiser, D. M. and Moorman, G. W. (2005). Amplified fragment length polymorphism analysis and internal transcribed spacer and coxII sequences reveal a species boundary within Pythium irregulare. Phytopathology, 95, 1489–98.
Geisen, S., Laros, I., Vizcaíno, A., Bonkowski, M., de Groot, G. A. (2015a). Not all are free-living: high-throughput DNA metabarcoding reveals a diverse community of protists parasitizing soil metazoa. Molecular Ecology, 24(17), 4556–69.
Geisen, S., Tveit, A. T., Clark, I. M., et al. (2015b). Metatranscriptomic census of active protists in soils. The ISME Journal, 9(10), 2178–90.
Gilles, A., Meglécz, E., Pech, N., Ferreira, S., Malausa, T. and Martin, J-F. (2011). Accuracy and quality assessment of 454 GS-FLX Titanium pyrosequencing. BMC Genomics, 12, 245.
Glücksman, E., Bell, T., Griffiths, R. I. and Bass, D. (2010). Closely related protist strains have different grazing impacts on natural bacterial communities. Environmental Microbiology, 12, 3105–13.
Guillou, L., Bachar, D., Audic, S., et al. (2013). The Protist Ribosomal Reference database (PR2): a catalog of unicellular eukaryote small sub-unit rRNA sequences with curated taxonomy. Nucleic Acids Research, 41 (Database issue), D597–604.
Hartikainen, H., Stentiford, G. D., Bateman, K. S., et al. (2014). Mikrocytids are a broadly distributed and divergent radiation of parasites in aquatic invertebrates. Current Biology, 24, 807–12.
Heinrichs, G., de Hoog, G. S. and Haase, G. (2012). Barcode identifiers as a practical tool for reliable species assignment of medically important black yeast species. Journal of Clinical Microbiology, 50, 3023–30.
Hess, D. and Melkonian, M. (2013). The mystery of Clade X: Ociraptor gen. nov. and Viridiraptor gen. nov. are highly specialized, algivorous amoeba-flagellates (Glissomonadida, Cercozoa). Protist, 164, 706–47.
Heywood, J. L., Sieracki, M. E., Bellows, W., Poulton, N. J. and Stepanauskas, R. (2011). Capturing diversity of marine heterotrophic protists: one cell at a time. The ISME Journal, 5(4), 674–84.
Howe, A. T., Bass, D., Chao, E. E. and Cavalier-Smith, T. (2011a). New genera, species, and improved phylogeny of Glissomonadida (Cercozoa). Protist, 162, 710–22.
Howe, A. T., Bass, D., Scoble, J., et al. (2011b). Novel cultured protists identify deep-branching environmental DNA clades of Cercozoa: new genera Tremula, Micrometopion, Minimassisteria, Nudifila, Peregrinia. Protist, 162, 332–72.
Howe, A. T., Bass, D., Vickerman, K., Chao, E.E-Y. and Cavalier-Smith, T. (2009). Phylogeny, taxonomy, and astounding genetic diversity of Glissomonadida ord. nov., the dominant gliding zooflagellates in soil (Protozoa, Cercozoa). Protist, 160, 159–89.
Jeon, S., Bunge, J., Leslin, C., Stoeck, T., Hong, S. and Epstein, S. S. (2008). Environmental rRNA inventories miss over half of protistan diversity. BMC Microbiology, 8, 222.
Jones, M. D. M., Forn, I., Gadhela, C., et al. (2011). Discovery of novel intermediate forms redefines the fungal tree of life. Nature, 474, 200–3.
Jumpponen, A. and Jones, K. L. (2009). Massively parallel 454 sequencing indicates hyperdiverse fungal communities in temperate Quercus macrocarpa phyllosphere. New Phytologist, 184, 438–48.
Karpov, S. A., Bass, D., Mylnikov, A. P. and Cavalier-Smith, T. (2006). Molecular phylogeny of Cercomonadidae and kinetid patterns of Cercomonas and Eocercomonas gen. nov. (Cercomonadida, Cercozoa). Protist, 157, 125–58.
Kiontke, K. C., Félix, M. A., Ailion, M., et al. (2011). A phylogeny and molecular barcodes for Caenorhabditis, with numerous new species from rotting fruits. BMC Evolutionary Biology, 11, 339.
Koeppel, A., Perry, E. B., Sikorski, J., et al. (2008). Identifying the fundamental units of bacterial diversity: a paradigm shift to incorporate ecology into bacterial systematics. Proceedings of the National Academy of Sciences of the United States of America, 105, 2504–9.
Kubartová, A., Ottosson, E., Dahlberg, A. and Stenlid, J. (2012). Patterns of fungal communities among and within decaying logs, revealed by 454 sequencing. Molecular Ecology, 18, 4514–32.
Kunin, V., Engelbrektson, A., Ochmann, H. and Hugenholtz, P. (2009). Wrinkles in the rare biosphere: pyrosequencing errors can lead to artificial inflation of diversity estimates. Environmental Microbiology, 12, 118–23.
Lecroq, B., Lejzerowicz, F., Bachar, D., et al. (2011). Ultra-deep sequencing of foraminiferal microbarcodes unveils hidden richness of early monothalamous lineages in deep-sea sediments. Proceedings of the National Academy of Sciences of the United States of Americ, 108, 13177–82.
Levine, N. D., Corliss, J. O., Cox, F. E., et al. (1980). A newly revised classification of the protozoa. Journal of Protozoology, 27, 37–58.
Liu, L., Li, Y., Li, S., et al. (2012). Comparison of next-generation sequencing systems. Journal of Biomedicine and Biotechnology, 2012, Article ID 251364.
Logares, R., Audic, S., Bass, D., et al. (2014). Patterns of rare and abundant marine microbial eukaryotes. Current Biology, 24, 813–21.
Logares, R., Audic, S., Santini, S., Pernice, M. C., de Vargas, C. and Massana, R. (2012). Diversity patterns and activity of uncultured flagellates unveiled with pyrosequencing. The ISME Journal, 6, 1823–33.
Loman, N. J., Misra, R. V., Dallman, T. J., et al. (2012). Performance comparison of benchtop high-throughput sequencing platforms. Nature Biotechnology, 30, 434–41.
Marinho, M. A., Junqueira, A. C. and Azeredo-Espin, A. M. (2011). Evaluation of the internal transcribed spacer 2 (ITS2) as a molecular marker for phylogenetic inference using sequence and secondary structure information in blow flies (Diptera, Calliphoridae). Genetica, 139, 1189–207.
Massana, R., Unrein, F., Rodríguez-Martínez, R., et al. (2009). Grazing rates and functional diversity of uncultured heterotrophic flagellates. The ISME Journal, 3, 588–96.
Matsen, F. A., Hoffman, N. G., Gallagher, A. and Stamatakis, A. (2012). A format for phylogenetic placements. PLoS One, 7, e31009.
Matsen, F. A., Kodner, R. B. and Armbrust, E. V. (2010). pplacer, linear time maximum-likelihood and Bayesian phylogenetic placement of sequences onto a fixed reference tree. BMC Bioinformatics, 11, 538.
Medinger, R., Nolte, V., Vinay Pandey, R., et al. (2010). Diversity in a hidden world: potential and limitation of next-generation sequencing for surveys of molecular diversity of eukaryotic microorganisms. Molecular Ecology, 19, 32–40.
Merget, B., Koetschan, C., Hackl, T., et al. (2012). The ITS2 database. Journal of Visualised Experiments, 61, 3806.
Moreira, D. and López-García, P. (2002). The molecular ecology of microbial eukaryotes unveils a hidden world. Trends in Microbiology, 10, 31–8.
Müller, T., Philippi, N., Dandekar, T., Schultz, J. and Wolf, M. (2007). Distinguishing species. RNA, 13, 1469–72.
Nassonova, E., Smirnov, A., Fahrni, J. and Pawlowski, J. (2010). Barcoding amoebae: comparison of SSU, ITS and COI genes as tools for molecular identification of naked lobose amoebae. Protist, 161, 102–15.
Nekrutenko, A. and Taylor, J. (2012). Next-generation sequencing data interpretation: enhancing reproducibility and accessibility. Nature Reviews Genetics, 13, 667–62.
Nesnidal, M. P., Helmkampf, M., Bruchhaus, I., El-Matbouli, M. and Hausdorf, B. (2013). Agent of whirling disease meets orphan worm: phylogenomic analyses firmly place myxozoa in cnidaria. PLoS One, 8, e54576.
Nikolaev, S. I., Berney, C., Fahrni, J. F., et al. (2004). The twilight of Heliozoa and rise of Rhizaria, an emerging supergroup of amoeboid eukaryotes. Proceedings of the National Academy of Sciences of the United States of America, 101, 8066–71.
Nilsson, R. H., Kristiansson, E., Ryberg, M., Hallenberg, N. and Larsson, K. H. (2008). Intraspecific ITS variability in the Kingdom Fungi as expressed in the international sequence databases and its implications for molecular species identification. Evolutionary Bioinformatics, 4, 193–201.
Pawlowski, J., Audic, S., Adl, S., et al. (2012). CBOL Protist Working Group: barcoding eukaryotic richness beyond the animal, plant and fungal kingdoms. PLoS Biology, 10, e1001419.
Quail, M. A., Smith, M., Coupland, P., et al. (2012). A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosicences and Ilumina MiSeq sequencers. BMC Genomics, 13, 341.
Richards, T. A. and Bass, D. (2005). Molecular screening of free-living microbial eukaryotes: diversity and distribution using a meta-analysis. Current Opinions in Microbiology, 8, 240–52.
Richards, T. A., Soanes, D. M., Jones, M. D., et al. (2011). Horizontal gene transfer facilitated the evolution of plant parasitic mechanisms in the oomycetes. Proceedings of the National Academy of Sciences of the United States of America, 108, 15258–63.
Richards, T. A. and Talbot, N. J. (2007). Plant parasitic oomycetes such as phytophthora species contain genes derived from three eukaryotic lineages. Plant Signaling and Behavior, 2, 112–14.
Shokralla, S., Spall, J. L., Gibson, J. F. and Hajibabaei, M. (2012). Next-generation sequencing technologies for environmental DNA research. Molecular Ecology, 21, 1794–805.
Song, J., Shi, L., Li, D., et al. (2012). Extensive pyrosequencing reveals frequent intra-genomic variations of internal transcribed spacer regions of nuclear ribosomal DNA. PLoS One, 7, e43971.
Stoeck, T., Bass, D., Nebel, M., et al. (2010). Multiple marker parallel tag environmental DNA sequencing reveals a highly complex eukaryotic community in marine anoxic water. Molecular Ecology, 19 (Supp. 1), 21–31.
Stoeck, T., Zuendorf, A., Breiner, H. W. and Behnke, A. (2007). A molecular approach to identify active microbes in environmental eukaryote clone libraries. Microbial Ecology, 53, 328–39.
Tarcz, S. (2013). Intraspecific differentiation of Paramecium novaurelia strains (Ciliophora, Protozoa) inferred from phylogenetic analysis of ribosomal and mitochondrial DNA variation. European Journal of Protistology, 49, 50–61.
Walker, G., Dorrell, R. A., Schlacht, A. and Dacks, J. B. (2011). Eukaryotic systematic: a 2011 user's guide for cell biologists and parasitologists. Parasitology, 138, 1638–63.
Whiteley, A. S., Jenkins, S., Waite, I., et al. (2012). Microbial 16S rRNA Ion Tag and community metagenome sequencing using the Ion Torrent (PGM) platform. Journal of Microbiological Methods, 91, 80–8.
Wuyts, J., Van de Peer, Y., Winkelmans, T. and De Wachter, R. (2002). The European database on small subunit ribosomal RNA. Nucleic Acids Research, 30, 183–5.