, Volume 19, Issue 2, pp 283–295 | Cite as

Deep sequencing uncovers protistan plankton diversity in the Portuguese Ria Formosa solar saltern ponds

  • Sabine Filker
  • Anna Gimmler
  • Micah Dunthorn
  • Frédéric Mahé
  • Thorsten StoeckEmail author
Original Paper


We used high-throughput sequencing to unravel the genetic diversity of protistan (including fungal) plankton in hypersaline ponds of the Ria Formosa solar saltern works in Portugal. From three ponds of different salinity (4, 12 and 38 %), we obtained ca. 105,000 amplicons (V4 region of the SSU rDNA). The genetic diversity we found was higher than what has been described from solar saltern ponds thus far by microscopy or molecular studies. The obtained operational taxonomic units (OTUs) could be assigned to 14 high-rank taxonomic groups and blasted to 120 eukaryotic families. The novelty of this genetic diversity was extremely high, with 27 % of all OTUs having a sequence divergence of more than 10 % to deposited sequences of described taxa. The highest degree of novelty was found at intermediate salinity of 12 % within the ciliates, which traditionally are considered as the best known and described taxon group within the kingdom Protista. Further substantial novelty was detected within the stramenopiles and the chlorophytes. Analyses of community structures suggest a transition boundary for protistan plankton between 4 and 12 % salinity, suggesting different haloadaptation strategies in individual evolutionary lineages as a result of environmental filtering. Our study makes evident the gaps in our knowledge not only of protistan and fungal plankton diversity in hypersaline environments, but also in their ecology and their strategies to cope with these environmental conditions. It substantiates that specific future research needs to fill these gaps.


Hypersaline Novel diversity Protistan plankton Salt gradient Solar saltern 



We would like to thank A. M. Amaral, J. Reis and R. Costa from CCMAR for their help and support during the sampling period in Faro, Portugal. We appreciate the permission and support of the Ria Formosa salt works staff for sampling. We also thank R. Müller for writing the script to merge the output files from QIIME and JAguc, L. Bittner and D. Forster for help with QIIME and R and the anonymous reviewers for constructive comments. This research was funded by an ASSEMBLE grant to TS and SF and the Deutsche Forschungsgemeinschaft (DFG) grant STO414/3-2.

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical standards

All performed experiments comply with the current laws of our country.

Supplementary material

792_2014_713_MOESM1_ESM.pdf (200 kb)
Supplementary material 1 (PDF 200 kb)


  1. Aguilera A, Manrubia SC, Gómez F, Rodríguez N, Amils R (2006) Eukaryotic community distribution and its relationship to water physicochemical parameters in an extreme acidic environment, Río Tinto (Southwestern Spain). Appl Environ Microb 72:5325–5330CrossRefGoogle Scholar
  2. Alexander E, Stock A, Breiner HW, Behnke A, Bunge J, Yakimov MM, Stoeck T (2009) Microbial eukaryotes in the hypersaline anoxic L’Atalante deep-sea basin. Environ Microbiol 11:360–381CrossRefPubMedGoogle Scholar
  3. Amaral-Zettler LA, Gómez F, Zettler E, Keenan BG, Amils R, Sogin ML (2002) Microbiology: eukaryotic diversity in Spain’s River of Fire. Nature 417:137CrossRefPubMedGoogle Scholar
  4. 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. doi: 10.1371/journal.pone.0006372 PubMedCentralCrossRefPubMedGoogle Scholar
  5. Amaral-Zettler LA, Zettler ER, Theroux SM, Palacios C, Aguilera A, Amils R (2011) Microbial community structure across the tree of life in the extreme Río Tinto. ISME J 5:42–50PubMedCentralCrossRefPubMedGoogle Scholar
  6. Amend AS, Seifert KA, Bruns TD (2010) Quantifying microbial communities with 454 pyrosequencing: does read abundance count? Mol Ecol 19:5555–5565CrossRefPubMedGoogle Scholar
  7. Bastian M, Heymann S, Jacomy M (2009) Gephi: an open source software for exploring and manipulating networks. ICWSM 8:361–362Google Scholar
  8. Behnke A, Engel M, Christen R, Nebel M, Klein R, Stoeck T (2011) Depicting more accurate pictures of protistan community complexity using pyrosequencing of hypervariable SSU rRNA gene regions. Env Microbiol 13:340–349CrossRefGoogle Scholar
  9. Berney C, Pawlowski J (2006) A molecular time-scale for eukaryote evolution recalibrated with the continuous microfossil record. Proc Roy Soc B Biol Sci 273:1867–1872CrossRefGoogle Scholar
  10. Bickford D, Lohman DJ, Sodhi NS et al (2007) Cryptic species as a window on diversity and conservation. Trends Ecol Evol 22:148–155CrossRefPubMedGoogle Scholar
  11. Bragg L, Stone G, Imelfort M, Hugenholtz P, Tyson GW (2012) Fast, accurate error-correction of amplicon pyrosequences using Acacia. Nat Methods 9:425–426CrossRefPubMedGoogle Scholar
  12. Bråte J, Logares R, Berney C, Ree DK, Klaveness D, Jakobsen KS, Shalchian-Tabrizi K (2010) Freshwater Perkinsea and marine-freshwater colonizations revealed by pyrosequencing and phylogeny of environmental rDNA. ISME J 4:1144–1153CrossRefPubMedGoogle Scholar
  13. Caporaso JG, Kuczynski J, Stombaugh J et al (2010) QIIME allows analysis of high-throughput community sequencing data. Nat Methods 7:335PubMedCentralCrossRefPubMedGoogle Scholar
  14. Caron DA, Countway PD, Jones AC, Kim DY, Schnetzer A (2012) Marine protistan diversity. Ann Rev Mar Sci 4:467–493CrossRefPubMedGoogle Scholar
  15. Caron DA, Countway PD, Savai P et al (2009) Defining DNA-based operational taxonomic units for microbial-eukaryote ecology. Appl Environ Microbiol 75:5797–5808PubMedCentralCrossRefPubMedGoogle Scholar
  16. Casamayor EO, Massana R, Benlloch S et al (2002) Changes in archaeal, bacterial and eukaryal assemblages along a salinity gradient by comparison of genetic fingerprinting methods in a multipond solar saltern. Environ Microbiol 4:338–348CrossRefPubMedGoogle Scholar
  17. Casamayor EO, Triadó-Margarit X, Castañeda C (2013) Microbial biodiversity in saline shallow lakes of the Monegros Desert, Spain. FEMS Microbiol Ecol 85:503–518CrossRefPubMedGoogle Scholar
  18. Cho BC, Park JS, Xu K, Choi JK (2008) Morphology and molecular phylogeny of Trimyema koreanum n. sp., a ciliate from the hypersaline water of a solar saltern. J Eukaryot Microbiol 55:417–426CrossRefPubMedGoogle Scholar
  19. Csárdi G, Nepusz T (2006) The igraph software package for complex network research. Inter J Complex Syst 1695Google Scholar
  20. Dunthorn M, Klier J, Bunge J, Stoeck T (2012) Comparing the hyper-variable V4 and V9 regions of the small subunit rDNA for assessment of ciliate environmental diversity. J Eukaryot Microbiol 59:185–187CrossRefPubMedGoogle Scholar
  21. Dunthorn M, Otto J, Berger SA et al (2014a) Placing environmental next-generation sequencing amplicons from microbial eukaryotes into a phylogenetic context. Mol Biol Evol 31:993–1009CrossRefPubMedGoogle Scholar
  22. Dunthorn M, Stoeck T, Clamp J, Warren A, Mahé F (2014b) Ciliates and the rare biopshere: a review. J Eukaryot Microbiol 61:404–409CrossRefPubMedGoogle Scholar
  23. Edgar RC (2010) Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26:2460–2461CrossRefPubMedGoogle Scholar
  24. Edgar RC (2011) Usearch user guide 5.2Google Scholar
  25. Edgcomb V, Orsi W, Leslin C et al (2009) Protistan community patterns within the brine and halocline of deep hypersaline anoxic basins in the eastern Mediterranean Sea. Extremophiles 13:151–167CrossRefPubMedGoogle Scholar
  26. Elloumi J, Carrias J-F, Ayadi H, Sime-Ngando T, Boukhris M, Bouain A (2006) Composition and distribution of planktonic ciliates from ponds of different salinity in the solar saltwork of Sfax, Tunisia. Estuar Coast Shelf Sci 67:21–29CrossRefGoogle Scholar
  27. Elloumi J, Carrias J-F, Ayadi H, Sime-Ngando T, Bouaïn A (2009a) Communities structure of the planktonic halophiles in the solar saltern of Sfax, Tunisia. Estuar Coast Shelf Sci 81:19–26CrossRefGoogle Scholar
  28. Elloumi J, Guermazi W, Ayadi H, Bouain A, Aleya L (2009b) Abundance and biomass of prokaryotic and eukaryotic microorganisms coupled with environmental factors in an arid multi-pond solar saltern (Sfax, Tunisia). J Mar Biol Assoc UK 89:243–253CrossRefGoogle Scholar
  29. Engelbrektson A, Kunin V, Wrighton KC, Zvenigorodsky N, Chen F, Ochman H et al (2010) Experimental factors affecting PCR-based estimates of microbial species richness and evenness. ISME J 4:642–647CrossRefPubMedGoogle Scholar
  30. Epstein S, López-García P (2008) “Missing” protists: a molecular prospective. Biodivers Conserv 17:261–276CrossRefGoogle Scholar
  31. Estrada M, Henriksen P, Gasol JM, Casamayor EO, Pedrós-Alió C (2004) Diversity of planktonic photoautotrophic microorganisms along a salinity gradient as depicted by microscopy, flow cytometry, pigment analysis and DNA-based methods. FEMS Microbiol Ecol 49:281–293CrossRefPubMedGoogle Scholar
  32. Foissner W, Jung JH, Filker S, Rudolph J, Stoeck T (2014a) Morphology, ontogenesis and molecular phylogeny of Platynematum salinarum nov. spec., a new scuticociliate (Ciliophora, Scuticociliatia) from a solar saltern. Eur J Protistol 50:174–184CrossRefPubMedGoogle Scholar
  33. Foissner W, Filker S, Stoeck T (2014b) Schmidingerothrix salinarum nov. spec. is the molecular sister of the large oxytrichid clade (Ciliophora, Hypotricha). J Eukaryot Microbiol 61:61–74CrossRefPubMedGoogle Scholar
  34. Forster D, Behnke A, Stoeck T (2012) Meta-analyses of environmental sequence data identify anoxia and salinity as parameters shaping ciliate communities. Syst Biodivers 10:277–288CrossRefGoogle Scholar
  35. Galtier N, Gouy M, Gautier C (1996) SEAVIEW and PHYLO_WIN: two graphic tools for sequence alignment and molecular phylogeny. Comput Appl Biosci 12:543–548PubMedGoogle Scholar
  36. Giovannoni SJ, DeLong EF, Olsen GJ, Pace NR (1988) Phylogenetic group-specific oligodeoxynucleotide probes for identification of single microbial cells. J Bacteriol 170:720–726PubMedCentralPubMedGoogle Scholar
  37. Gostinčar C, Lenassi M, Gunde-Cimerman N, Plemenitaš A (2011) Fungal adaptation to extremely high salt concentrations. Adv Appl Microbiol 77:71–96CrossRefPubMedGoogle Scholar
  38. Hauer G, Rogerson A (2005) Heterotrophic protozoa from hypersaline environments. In: Gunde-Cimerman N, Oren A, Plemenitas A (eds) Adaptation to life at high salt concentrations in archaea, bacteria, and eukarya. Cellular origin, life in extreme habitats and astrobiology, vol 9. Springer, Dordrecht, pp 519–540CrossRefGoogle Scholar
  39. Heger TJ, Mitchell EAD, Todorov M, Golemansky V, Lara E, Leander BS, Pawlowski J (2010) Molecular phylogeny of euglyphid testate amoebae (Cercozoa: Euglyphida) suggests transitions between marine supralittoral and freshwater/terrestrial environments are infrequent. Mol Phylogenet Evol 55:113–122CrossRefPubMedGoogle Scholar
  40. Heidelberg KB, Nelson WC, Holm JB, Eisenkolb N, Andrade K, Emerson JB (2013) Characterization of eukaryotic microbial diversity in hypersaline Lake Tyrrell, Australia. Front Microbiol 4Google Scholar
  41. Jumpponen A (2007) Soil fungal communities underneath willow canopies on a primary successional glacier forefront: rDNA sequence results can be affected by primer selection and chimeric data. Microbial Ecol 53:233–246CrossRefGoogle Scholar
  42. Ki JS (2012) Hypervariable regions (V1–V9) of the dinoflagellate 18S rRNA using a large dataset for marker considerations. J Appl Phycol 24:1035–1043CrossRefGoogle Scholar
  43. Kis-Papo T, Weig AR, Riley R, Peršoh D, Salamov A, Sun H, Lipzen A, Wasser SP, et al. (2014) Genomic adaptations of the halophilic Dead Sea filamentous fungus Eurotium rubrum. Nat Commun 5Google Scholar
  44. Knoll AH, Walter MR, Narbonne GM, Christi-Blick N (2004) A new period for the geologic time scale. Science 305:621–622CrossRefPubMedGoogle Scholar
  45. Kunin V, Engelbrektson A, Ochman H, Hugenholtz P (2010) Wrinkles in the rare biosphere: pyrosequencing errors lead to artificial inflation of diversity estimates. Environ Microbiol 12:118–123CrossRefPubMedGoogle Scholar
  46. Lanyi JK (1974) Salt-dependent properties of proteins from extremely halophilic bacteria. Bacteriol Rev 38:272–290PubMedCentralPubMedGoogle Scholar
  47. Lara E, Berney C, Harms H, Chatzinotas A (2007) Cultivation-independent analysis reveals a shift in ciliate 18S rRNA gene diversity in a polycyclic aromatic hydrocarbon-polluted soil. FEMS Microbiol Ecol 62:365–373CrossRefPubMedGoogle Scholar
  48. Lee CE, Bell MA (1999) Causes and consequences of recent freshwater invasions by saltwater animals. Trends Ecol Evol 14:282–288Google Scholar
  49. Lei Y, Xu K, Choi JK, Hong HP, Wickham SA (2009) Community structure and seasonal dynamics of planktonic ciliates along salinity gradients. Eur J Protistol 45:305–319CrossRefPubMedGoogle Scholar
  50. Logares R, Bråte J, Bertilsson S, Clasen JL, Shalchian-Tabrizi K, Rengefors K (2009) Infrequent marine-freshwater transitions in the microbial world. Trends Microbiol 17:414–422CrossRefPubMedGoogle Scholar
  51. Logares R, Bråte J, Heinrich F, Shalchian-Tabrizi K, Bertilsson S (2010) Infrequent transitions between saline and fresh waters in one of the most abundant microbial lineages (SAR11). Mol Biol Evol 27:347–357CrossRefPubMedGoogle Scholar
  52. Logares R, Audic S, Santini S, Pernice MC, de Vargas C, Massana R (2012) Diversity patterns and activity of uncultured marine heterotrophic flagellates unveiled with pyrosequencing. ISME J 6:1823–1833PubMedCentralCrossRefPubMedGoogle Scholar
  53. López-Garcia P, Lopez-Lopez A, Moreira D, Rodriguez-Valera F (2001) Diversity of free-living prokaryotes from a deep-sea site at the Antarctic Polar Front Fems microbiology. Ecology 36:193–202Google Scholar
  54. Lozupone C, Knight R (2007) Global patterns in bacterial diversity. Proc Natl Acad Sci USA 104:11436–11440PubMedCentralCrossRefPubMedGoogle Scholar
  55. Lynch MD, Bartram AK, Neufeld JD (2012) Targeted recovery of novel phylogenetic diversity from next-generation sequence data. ISME J 6:2067–2077PubMedCentralCrossRefPubMedGoogle Scholar
  56. Lynn DH (2008) The Ciliated Protozoa, vol 3. Springer, New YorkGoogle Scholar
  57. Mahé F, Rognes T, Quince C, DeVargas C, Dunthorn M (2014) Swarm: a fast and robust clustering method for amplicon-based studies. Peer J 2:e593PubMedCentralCrossRefPubMedGoogle Scholar
  58. McGenity TJ, Oren A (2012) Hypersaline Environments. In: Bell EM (ed) Life at extremes: environments, organisms and strategies for survival. CABI, UK, USA, pp 402–437CrossRefGoogle Scholar
  59. Medinger R, Nolte V, Pandey RV, Jost S, Ottenwalder B, Schlotterer C, Boenigk J (2010) Diversity in a hidden world: potential and limitation of next-generation sequencing for surveys of molecular diversity of eukaryotic microorganisms. Mol Ecol 19:32–40PubMedCentralCrossRefPubMedGoogle Scholar
  60. Moon-van der Staay SY, De Wachter R, Vaulot D (2001) Oceanic 18S rDNA sequences from picoplankton reveal unsuspected eukaryotic diversity. Nature 409:607–610CrossRefPubMedGoogle Scholar
  61. Nebel M, Pfabel C, Stock A, Dunthorn M, Stoeck T (2010) Delimiting operational taxonomic units for assessing ciliate environmental diversity using small-subunit rRNA gene sequences. Environ Microbiol Rep 3:154–158CrossRefGoogle Scholar
  62. Nebel ME, Wild S, Holzhauser M, Huttenberger L, Reitzig R, Sperber M, Stoeck T (2011) JAGUC: a software package for environmental diversity analyses. J Bioinform Comput Biol 9:749–773CrossRefPubMedGoogle Scholar
  63. Oren A (2008) Microbial life at high salt concentrations: phylogenetic and metabolic diversity. Saline Systems 4:13CrossRefGoogle Scholar
  64. Pandey B, Yeragi S (2004) Preliminary and mass culture experiments on a heterotrichous ciliate, Fabrea salina. Aquacult 232:241–254CrossRefGoogle Scholar
  65. Park JS, Simpson AGB (2010) Characterization of halotolerant Bicosoecida and Placididea (Stramenopila) that are distinct from marine forms, and the phylogenetic pattern of salinity preferences in heterotrophic stramenopiles. Environ Microbiol 12:1173–1184CrossRefPubMedGoogle Scholar
  66. Park JS, Kim H, Choi DH, Cho BC (2003) Active flagellates grazing on prokaryotes in high salinity waters of a solar saltern. Aquat Microb Ecol 33:173–179CrossRefGoogle Scholar
  67. Park JS, Simpson AGB, Lee WJ, Cho BC (2007) Ultrastructure and phylogenetic placement within Heterolobosea of the previously unclassified, extremely halophilic heterotrophic flagellate Pleurostomum flabellatum (Ruinen 1938). Protist 158:397–413CrossRefPubMedGoogle Scholar
  68. Park JS, Simpson AGB, Brown S, Cho BC (2009) Ultrastructure and molecular phlyogeny of two heterolobosean amoebae, Euplaesiobystra hypersalinica gen. et sp. nov. and Tulamoeba peronaphora gen. et sp. nov., isolated from an extremely hypersaline habitat. Protist 160:265–283CrossRefPubMedGoogle Scholar
  69. Patterson DJ, Simpson AGB (1996) Heterotrophic flagellates from coastal marine and hypersaline sediments in Western Australia. Eur J Protistol 32:423–448CrossRefGoogle Scholar
  70. Pedrós-Alió C (2004) Trophic ecology of solar salterns. In: Ventosa A (ed) Halophilic microorganisms. Springer, Berlin, pp 33–48CrossRefGoogle Scholar
  71. Post FJ, Borowitzka LJ, Borowitzka MA, Mackay B, Moulton T (1983) The protozoa of a western Australian hypersaline lagoon. Hydrobiologica 105:95–113CrossRefGoogle Scholar
  72. R Development Core Team (2008) R. A language and environment for statistical computing. In: R Foundation for Statistical Computing. Vienna, AustriaGoogle Scholar
  73. Ruinen J (1938) Notizen über Salzflagellaten II. Über die Verbreitung der Salzflagellaten. Arch f Protistenkd 90:161–177Google Scholar
  74. Sáez AG, Lozano E (2005) Body doubles. Nature 433:111CrossRefPubMedGoogle Scholar
  75. Shao C, Li L, Zhang Q, Song W, Berger H (2014) Molecular phylogeny and ontogeny of a new ciliate genus, Paracladotricha salina n. g., g. sp. (Ciliophora, Hypotrichia). J Eukaryot Microbiol 61:371–380PubMedCentralCrossRefPubMedGoogle Scholar
  76. Stock A, Breiner HW, Pachiadaki M et al (2012) Microbial eukaryote life in the new hypersaline deep-sea basin Thetis. Extremophiles 16:21–34CrossRefPubMedGoogle Scholar
  77. Stoeck T, Hayward B, Taylor GT, Varela R, Epstein SS (2006) A multiple PCR-primer approach to access the microeukaryotic diversity in environmental samples. Protist 157:31–43CrossRefPubMedGoogle Scholar
  78. Stoeck T, Bass D, Nebel M, Christen R, Jones MD, Breiner HW, Richards TA (2010) Multiple marker parallel tag environmental DNA sequencing reveals a highly complex eukaryotic community in marine anoxic water. Mol Ecol 19:21–31CrossRefPubMedGoogle Scholar
  79. Stoeck T, Breiner HW, 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 16:430–444PubMedCentralCrossRefPubMedGoogle Scholar
  80. Triadó-Margarit X, Casamayor EO (2013) High genetic diversity and novelty in planktonic protists inhabiting inland and coastal high salinity water bodies. FEMS Microbiol Ecol 85:27–36CrossRefPubMedGoogle Scholar
  81. Vermeij GJ, Dudely R (2000) Why are there so few evolutionary transitions between aquatic and terrestrial ecosystems? Biol J Linn Soc 70:541–554CrossRefGoogle Scholar
  82. Weber AP, Horst RJ, Barbier GG, Oesterhelt C (2007) Metabolism and metabolomics of eukaryotes living under extreme conditions. Int Rev Cytol 256:1–34CrossRefPubMedGoogle Scholar
  83. Wuyts J, De Rijk P, Van de Peer Y, Pison G, Rousseeuw P, De Wachter R (2000) Comparative analysis of more than 3000 sequences reveals the existence of two pseudoknots in area V4 of eukaryotic small subunit ribosomal RNA. Nucleic Acids Res 28:4698–4708PubMedCentralCrossRefPubMedGoogle Scholar
  84. Zhu F, Massana R, Not F, Marie D, Vaulot D (2005) Mapping of picoeucaryotes in marine ecosystems with quantitative PCR of the 18S rRNA gene. FEMS Microbiol Ecol 52:79–92CrossRefPubMedGoogle Scholar

Copyright information

© Springer Japan 2014

Authors and Affiliations

  • Sabine Filker
    • 1
  • Anna Gimmler
    • 1
  • Micah Dunthorn
    • 1
  • Frédéric Mahé
    • 1
  • Thorsten Stoeck
    • 1
    Email author
  1. 1.School of BiologyUniversity of KaiserslauternKaiserslauternGermany

Personalised recommendations