Biodiversity and Conservation

, Volume 17, Issue 2, pp 261–276 | Cite as

“Missing” protists: a molecular prospective

  • Slava Epstein
  • Purificación López-GarcíaEmail author
Original Paper


Molecular ecology methods based on 18S rRNA amplification and sequencing have revealed an astounding diversity of microbial eukaryotes in every environment sampled so far. This is certainly true of new species and genera, as essentially every new survey discovers a wealth of novel diversity at this level. This is almost certain for taxa that are higher in taxonomic hierarchy, as many molecular surveys reported novel clades within established protistan phyla, with some of these clades repeatedly confirmed by subsequent studies. It may also be that the molecular approaches discovered several lineages of the highest taxonomic order, but this claim has not been vigorously verified as yet. Overall, the field of protistan diversity remains in its infancy. The true scale of this diversity is unknown, and so are the distribution of this diversity, its patterns, spatial and temporal dynamics, and ecological role. The sampled diversity appears to be just the tip of the iceberg, and this offers outstanding opportunities for microbial discovery for the purposes of both basic and applied research.


18S rRNA Cryptic species Eukaryotic phylogeny Molecular ecology Species richness 


  1. Amaral Zettler LA, Gomez F, Zettler E et al (2002) Microbiology: eukaryotic diversity in Spain’s River of Fire. Nature 417:137PubMedCrossRefGoogle Scholar
  2. Anderson IC, Cairney JW (2004) Diversity and ecology of soil fungal communities: increased understanding through the application of molecular techniques. Environ Microbiol 6:769–779PubMedCrossRefGoogle Scholar
  3. Atkins MS, Teske AP, Anderson OR (2000) A survey of flagellate diversity at four deep-sea hydrothermal vents in the Eastern Pacific Ocean using structural and molecular approaches. J Eukaryot Microbiol 47:400–411PubMedCrossRefGoogle Scholar
  4. Baldauf SL (2003) The deep roots of eukaryotes. Science 300:1703–1706PubMedCrossRefGoogle Scholar
  5. Bass D, Cavalier-Smith T (2004) Phylum-specific environmental DNA analysis reveals remarkably high global biodiversity of Cercozoa (Protozoa). Int J Syst Evol Microbiol 54:2393–2404PubMedCrossRefGoogle Scholar
  6. Behnke A, Bunge J, Barger K et al (2006) Microeukaryote community patterns along an O2/H2S gradient in a supersulfidic anoxic fjord (Framvaren, Norway). Appl Environ Microbiol 72:3626–3636PubMedCrossRefGoogle Scholar
  7. Beijerinck MW (1913) De infusies en de ontdekking der backteriën. Müller, Amsterdam, the NetherlandsGoogle Scholar
  8. Berney C, Fahrni J, Pawlowski J (2004) How many novel eukaryotic ‘kingdoms’? Pitfalls and limitations of environmental DNA surveys. BMC Biol 2:13PubMedCrossRefGoogle Scholar
  9. Boenigk J, Pfandl K, Stadler P et al (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 7:685–697PubMedCrossRefGoogle Scholar
  10. Brinkmann H, van der Giezen M, Zhou Y et al (2005) An empirical assessment of long-branch attraction artefacts in deep eukaryotic phylogenomics. Syst Biol 54:743–757PubMedCrossRefGoogle Scholar
  11. Brocks JJ, Logan GA, Buick R et al (1999) Archaean molecular fossils and the early rise of eukaryotes. Science 285:1025–1027CrossRefGoogle Scholar
  12. Bunge J, Fitzpatrick M (1993) Estimating the number of species—a review. J Am Stat Assoc 88:364–373CrossRefGoogle Scholar
  13. Cavalier-Smith T (2004) Only six kingdoms of life. Proc R Soc Lond B Biol Sci 271:1251–1262CrossRefGoogle Scholar
  14. Chao A (1984) Non-parametric estimation of the number of classes in a population. Scand J Statist 11:265–270Google Scholar
  15. Chao A (2005) Species richness estimation. In: Balakrishnan C, Read B, Vidakovic B (eds) Encyclopedia of statistical sciences. Wiley, New York, pp 7907–7916Google Scholar
  16. Chao A, Bunge J (2002) Estimating the number of species in a stochastic abundance model. Biometrics 58:531–539PubMedCrossRefGoogle Scholar
  17. Chao A, Li PC, Agatha S et al (2006) A statistical approach to estimate soil ciliate diversity and distribution based on data from five continents. Oikos 114:479–493CrossRefGoogle Scholar
  18. Coleman AW (2002) Microbial eukaryote species. Science 297:337; author reply 337PubMedCrossRefGoogle Scholar
  19. Countway PD, Gast RJ, Savai P et al (2005) Protistan diversity estimates based on 18S rDNA from seawater incubations in the Western North Atlantic. J Eukaryot Microbiol 52:95–106PubMedCrossRefGoogle Scholar
  20. Courties C, Vaquer A, Troussellier M et al (1994) Smallest eukaryotic organisms. Nature 370:255CrossRefGoogle Scholar
  21. Dawson SC, Pace NR (2002) Novel kingdom-level eukaryotic diversity in anoxic environments. Proc Natl Acad Sci USA 99:8324–8329PubMedCrossRefGoogle Scholar
  22. Díez B, Pedrós-Alió C, Massana R (2001) Study of genetic diversity of eukaryotic picoplankton in different oceanic regions by small-subunit rRNA gene cloning and sequencing. Appl Environ Microbiol 67:2932–2941PubMedCrossRefGoogle Scholar
  23. Dolven JK, Lindqvist C, Albert VA et al (2007) Molecular diversity of alveolates associated with Neritic North Atlantic radiolarians. Protist 158:65–76PubMedCrossRefGoogle Scholar
  24. Edgcomb VP, Kysela DT, Teske A et al (2002) Benthic eukaryotic diversity in the Guaymas Basin hydrothermal vent environment. Proc Natl Acad Sci USA 99:7658–7662PubMedCrossRefGoogle Scholar
  25. Fenchel T, Finlay BJ (1995) Ecology and evolution in anoxic worlds. Oxford Univ Press, OxfordGoogle Scholar
  26. Fenchel T, Kristensen LD, Rasmussen L (1990) Water column anoxia: vertical zonation of planctonic protozoa. Mar Ecol Prog Ser 62:1–10CrossRefGoogle Scholar
  27. Fenchel T, Bernard C, Esteban G et al (1995) Microbial diversity and activity in a Danish fjord with anoxic deep water. Ophelia 43:45–100Google Scholar
  28. Finlay BJ (2002) Global dispersal of free-living microbial eukaryote species. Science 296:1061–1063PubMedCrossRefGoogle Scholar
  29. Finlay BJ, Fenchel T (1999) Divergent perspectives on protist species richness. Protist 150:229–233PubMedCrossRefGoogle Scholar
  30. Foissner W (1999) Protist diversity: estimates of the near-imponderable. Protist 150:363–368PubMedGoogle Scholar
  31. Foissner W (2006) Biogeography and dispersal of micro-organisms: a review emphasizing protists. Acta Protozool 45:111–136Google Scholar
  32. Guillou L, Chretiennot-Dinet MJ, Medlin LK et al (1999) Bolidomonas: a new genus with two species belonging to a new algal class, the Bolidophyceae (Heterokonta). J Phycol 35:368–381CrossRefGoogle Scholar
  33. Guillou L, Eikrem W, Chretiennot-Dinet MJ et al (2004) Diversity of picoplanktonic prasinophytes assessed by direct nuclear SSU rDNA sequencing of environmental samples and novel isolates retrieved from oceanic and coastal marine ecosystems. Protist 155:193–214PubMedCrossRefGoogle Scholar
  34. Gunderson JH, Goss SH, Coats DW (1999) The phylogenetic position of Amoebophrya sp. infecting Gymnodinium sanguineum. J Eukaryot Microbiol 46:194–197PubMedCrossRefGoogle Scholar
  35. Hong SH, Bunge J, Jeon SO, Epstein SS (2006) Predicting microbial species richness. Proc Natl Acad Sci USA 103:117–122PubMedCrossRefGoogle Scholar
  36. Janson S, Gisselson LA, Salomon PS et al (2000) Evidence for multiple species within the endoparasitic dinoflagellate Amoebophrya ceratii as based on 18S rRNA gene-sequence analysis. Parasitol Res 86:929–933PubMedCrossRefGoogle Scholar
  37. Jeon SO, Bunge J, Stoeck T et al (2006) Synthetic statistical approach reveals a high degree of richness of microbial eukaryotes in an anoxic water column. Appl Environ Microbiol 72:6578–6583PubMedCrossRefGoogle Scholar
  38. Johnson MD, Tengs T, Oldach DW et al (2004) Highly divergent SSU rRNA genes found in the marine ciliates Myrionecta rubra and Mesodinium pulex. Protist 155:347–359PubMedCrossRefGoogle Scholar
  39. Katz LA, McManus GB, Snoeyenbos-West OLO et al (2005) Reframing the ‘Everything is everywhere’ debate: evidence for high gene flow and diversity in ciliate morphospecies. Aquat Microbiol Ecol 41:55–65CrossRefGoogle Scholar
  40. Katz LA, Snoeyenbos-West O, Doerder FP (2006) Patterns of protein evolution in Tetrahymena thermophila: implications for estimates of effective population size. Mol Biol Evol 23:608–614PubMedCrossRefGoogle Scholar
  41. Kolodziej K, Stoeck T (2007) Cellular identification of a novel uncultured marine stramenopile (MAST-12 Clade) small-subunit rRNA gene sequence from a norwegian estuary by use of fluorescence in situ hybridization-scanning electron microscopy. Appl Environ Microbiol 73:2718–2726PubMedCrossRefGoogle Scholar
  42. Kunitomo Y, Sarashina I, Iijima M et al (2006) Molecular phylogeny of acantharian and polycystine radiolarians based on ribosomal DNA sequences, and some comparisons with data from the fossil record. Eur J Protistol 42:143–153PubMedCrossRefGoogle Scholar
  43. Lefèvre E, Bardot C, Noël C et al (2007) Unveiling fungal zooflagellates as members of freshwater picoeukaryotes: evidence from a molecular diversity study in a deep meromictic lake. Environ Microbiol 9:61–71PubMedCrossRefGoogle Scholar
  44. Lefranc M, Thenot A, Lepere C et al (2005) Genetic diversity of small eukaryotes in lakes differing by their trophic status. Appl Environ Microbiol 71:5935–5942PubMedCrossRefGoogle Scholar
  45. López-García P, Rodríguez-Valera F, Pedrós-Alió C et al (2001) Unexpected diversity of small eukaryotes in deep-sea Antarctic plankton. Nature 409:603–607PubMedCrossRefGoogle Scholar
  46. López-García P, Rodríguez-Valera F, Moreira D (2002) Towards the monophyly of Haeckel’s Radiolaria: 18S rRNA environmental data support the sisterhood of Polycystinea and Acantharea. Mol Biol Evol 19:118–121PubMedGoogle Scholar
  47. López-García P, Philippe H, Gaill F et al (2003) Autochthonous eukaryotic diversity in hydrothermal sediment and experimental micro-colonizers at the Mid-Atlantic Ridge. Proc Natl Acad Sci USA 100:697–702PubMedCrossRefGoogle Scholar
  48. López-García P, Vereshchaka A, Moreira D (2007) Eukaryotic diversity associated with carbonates and fluid-seawater interface in Lost City hydrothermal field. Environ Microbiol 9:546–554PubMedCrossRefGoogle Scholar
  49. Lovejoy C, Massana R, Pedros-Alio C (2006) Diversity and distribution of marine microbial eukaryotes in the Arctic Ocean and adjacent seas. Appl Environ Microbiol 72:3085–3095PubMedCrossRefGoogle Scholar
  50. Lowe CD, Day A, Kemp SJ et al (2005) There are high levels of functional and genetic diversity in Oxyrrhis marina. J Eukaryot Microbiol 52:250–257PubMedCrossRefGoogle Scholar
  51. Luo Q, Krumholz LR, Najar FZ et al (2005) Diversity of the microeukaryotic community in sulfide-rich Zodletone Spring (Oklahoma). Appl Environ Microbiol 71:6175–6184PubMedCrossRefGoogle Scholar
  52. Maidak BL, Cole JR, Lilburn TG et al (2001) The RDP-II (Ribosomal database project). Nucleic Acids Res 29:173–174PubMedCrossRefGoogle Scholar
  53. Massana R, Guillou L, Diez B et al (2002) Unveiling the organisms behind novel eukaryotic ribosomal DNA sequences from the ocean. Appl Environ Microbiol 68:4554–4558PubMedCrossRefGoogle Scholar
  54. Massana R, Balagué V, Guillou L et al (2004a) Picoeukaryotic diversity in an oligotrophic coastal site studied by molecular and culturing approaches. FEMS Microbiol Ecol 50:231–243CrossRefPubMedGoogle Scholar
  55. Massana R, Castresana J, Balague V et al (2004b) Phylogenetic and ecological analysis of novel marine stramenopiles. Appl Environ Microbiol 70:3528–3534PubMedCrossRefGoogle Scholar
  56. Massana R, Terrado R, Forn I et al (2006) Distribution and abundance of uncultured heterotrophic flagellates in the world oceans. Environ Microbiol 8:1515–1522PubMedCrossRefGoogle Scholar
  57. Medlin LK, Metfies K, Mehl H et al (2006) Picoeukaryotic plankton diversity at the Helgoland time series site as assessed by three molecular methods. Microb Ecol 52:53–71PubMedCrossRefGoogle Scholar
  58. Moon-van der Staay A-Y, van der Staay GWM, Guillou L et al (2000) Abundance and diversity of prymnesiophytes in the picoplankton community from the equatorial Pacific Ocean inferred from 18S rDNA sequences. Limnol Oceanogr 45:98–109CrossRefGoogle Scholar
  59. Moon-van der Staay SY, De Wachter R, Vaulot D (2001) Oceanic 18S rDNA sequences from picoplankton reveal unsuspected eukaryotic diversity. Nature 409:607–610CrossRefGoogle Scholar
  60. Moon-van der Staay SY, Tzeneva VA, van der Staay GW et al (2006) Eukaryotic diversity in historical soil samples. FEMS Microbiol Ecol 57:420–428CrossRefGoogle Scholar
  61. Moreira D, López-García P (2002) Molecular ecology of microbial eukaryotes unveils a hidden world. Trends Microbiol 10:31–38PubMedCrossRefGoogle Scholar
  62. Moreira D, López-García P (2003) Are hydrothermal vents oases for parasitic protists? Trends Parasitol 19:556–558PubMedCrossRefGoogle Scholar
  63. Moreira D, von der Heyden S, Bass D et al (2007) Global eukaryote phylogeny: Combined small- and large-subunit ribosomal DNA trees support monophyly of Rhizaria, Retaria and Excavata. Mol Phylogenet Evol:  doi:10.1016/j.ympev.2006.11.001
  64. Nanney DL, Park C, Preparata R et al (1998) Comparison of sequence differences in a variable 23S rRNA domain among sets of cryptic species of ciliated protozoa. J Eukaryot Microbiol 45:91–100PubMedCrossRefGoogle Scholar
  65. Nikolaev SI, Berney C, Fahrni JF et al (2004) The twilight of Heliozoa and rise of Rhizaria, an emerging supergroup of amoeboid eukaryotes. Proc Natl Acad Sci USA 101:8066–8071PubMedCrossRefGoogle Scholar
  66. O’Brien HE, Parrent JL, Jackson JA et al (2005) Fungal community analysis by large-scale sequencing of environmental samples. Appl Environ Microbiol 71:5544–5550PubMedCrossRefGoogle Scholar
  67. O’Brien EA, Koski LB, Zhang Y et al (2007) TBestDB: a taxonomically broad database of expressed sequence tags (ESTs). Nucleic Acids Res 35:D445–D451PubMedCrossRefGoogle Scholar
  68. Okamoto N, Inouye I (2005) The katablepharids are a distant sister group of the Cryptophyta: a proposal for Katablepharidophyta divisio nova/Kathablepharida phylum novum based on SSU rDNA and beta-tubulin phylogeny. Protist 156:163–179PubMedCrossRefGoogle Scholar
  69. Olsen GJ, Lane DJ, Giovannoni SJ et al (1986) Microbial ecology and evolution: a ribosomal RNA approach. Annu Revue Microbiol 40:337–365CrossRefGoogle Scholar
  70. Pace NR (1997) A molecular view of microbial diversity and the biosphere. Science 276:734–740PubMedCrossRefGoogle Scholar
  71. Philippe H, Lopez P, Brinkmann H et al (2000) Early-branching or fast-evolving eukaryotes? An answer based on slowly evolving positions. Proc R Soc Lond B Biol Sci 267:1213–1221CrossRefGoogle Scholar
  72. Reysenbach AL, Cady SL (2001) Microbiology of ancient and modern hydrothermal systems. Trends Microbiol 9:79–86PubMedCrossRefGoogle Scholar
  73. Richards TA, Bass D (2005) Molecular screening of free-living microbial eukaryotes: diversity and distribution using a meta-analysis. Curr Opin Microbiol 8:240–252PubMedCrossRefGoogle Scholar
  74. Richards TA, Vepritskiy AA, Gouliamova DE, Nierzwicki-Bauer SA (2005) The molecular diversity of freshwater picoeukaryotes from an oligotrophic lake reveals diverse, distinctive and globally dispersed lineages. Environ Microbiol 7:1413–1425PubMedCrossRefGoogle Scholar
  75. Rodriguez F, Derelle E, Guillou L et al (2005) Ecotype diversity in the marine picoeukaryote Ostreococcus (Chlorophyta, Prasinophyceae). Environ Microbiol 7:853–859PubMedCrossRefGoogle Scholar
  76. Savin MC, Martin JL, LeGresley M et al (2004) Plankton diversity in the Bay of Fundy as measured by morphological and molecular methods. Microb Ecol 48:51–65PubMedCrossRefGoogle Scholar
  77. Slapeta J, López-García P, Moreira D (2006a) Global dispersal and ancient cryptic species in the smallest marine eukaryotes. Mol Biol Evol 23:23–29PubMedCrossRefGoogle Scholar
  78. Slapeta J, López-García P, Moreira D (2006b) Present status of the molecular ecology of kathablepharids. Protist 157:7–11PubMedCrossRefGoogle Scholar
  79. Slapeta J, Moreira D, López-García P (2005) The extent of protist diversity: insights from molecular ecology of freshwater eukaryotes. Proc Biol Sci 272:2073–2081PubMedCrossRefGoogle Scholar
  80. Sogin ML, Gunderson JH, Elwood HJ et al (1989) Phylogenetic meaning of the kingdom concept: an unusual ribosomal RNA from Giardia lamblia. Science 243:75–77PubMedCrossRefGoogle Scholar
  81. Stoeck T, Epstein S (2003) Novel eukaryotic lineages inferred from small-subunit rRNA analyses of oxygen-depleted marine environments. Appl Environ Microbiol 69:2657–2663PubMedCrossRefGoogle Scholar
  82. Stoeck T, Taylor GT, Epstein SS (2003) Novel eukaryotes from the permanently anoxic Cariaco Basin (Caribbean Sea). Appl Environ Microbiol 69:5656–5663PubMedCrossRefGoogle Scholar
  83. Stoeck T, Hayward B, Taylor GT et al (2006) A multiple PCR-primer approach to access the microeukaryotic diversity in environmental samples. Protist 157:31–43PubMedCrossRefGoogle Scholar
  84. Stoeck T, Kasper J, Bunge J, Leslin C, Ilyin V, Epstein S (2007a) Protistan diversity in the arctic: a case of paleoclimate shaping modern biodiversity? PLoS ONE 2:e728PubMedCrossRefGoogle Scholar
  85. Stoeck T, Zuendorf A, Breiner HW, Behnke A (2007b) A molecular approach to identify active microbes in environmental eukaryote clone libraries. Microb Ecol 53:328–339PubMedCrossRefGoogle Scholar
  86. Takishita K, Miyake H, Kawato M et al (2005) Genetic diversity of microbial eukaryotes in anoxic sediment around fumaroles on a submarine caldera floor based on the small-subunit rDNA phylogeny. Extremophiles 9:185–196PubMedCrossRefGoogle Scholar
  87. Takishita K, Tsuchiya M, Kawato M et al (2006) Genetic diversity of microbial eukaryotes in anoxic sediment of the saline meromictic lake Namako-Ike (Japan): on the detection of anaerobic or anoxic-tolerant lineages of eukaryotes. Protist 2006 Sep 1; [Epub ahead of print]Google Scholar
  88. Woese CR (1987) Bacterial evolution. Microbiol Rev 51:221–271PubMedGoogle Scholar
  89. Worden AZ (2006) Picoeukaryote diversity in coastal waters of the Pacific Ocean. Aquat Microbiol Ecol 43:165–175CrossRefGoogle Scholar
  90. Worden AZ, Nolan JK, Palenik B (2004) Assessing the dynamics and ecology of marine picophytoplankton: The importance of the eukaryotic component. Limnol Oceanogr 49:168–179CrossRefGoogle Scholar
  91. Yuan J, Chen MY, Shao P et al (2004) Genetic diversity of small eukaryotes from the coastal waters of Nansha Islands in China. FEMS Microbiol Lett 240:163–170PubMedCrossRefGoogle Scholar
  92. Zuendorf A, Bunge J, Behnke A et al (2006) Diversity estimates of microeukaryotes below the chemocline of the anoxic Mariager Fjord, Denmark. FEMS Microbiol Ecol 58:476–491PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  1. 1.Marine Science CenterNortheastern UniversityNahantUSA
  2. 2.Unité d’Ecologie, Systématique et EvolutionCNRS UMR8079, Université Paris-SudOrsay CedexFrance

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