Abstract
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.
Similar content being viewed by others
References
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 72:5325–5330
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 11:360–381
Amaral-Zettler LA, Gómez F, Zettler E, Keenan BG, Amils R, Sogin ML (2002) Microbiology: eukaryotic diversity in Spain’s River of Fire. Nature 417:137
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
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 5:42–50
Amend AS, Seifert KA, Bruns TD (2010) Quantifying microbial communities with 454 pyrosequencing: does read abundance count? Mol Ecol 19:5555–5565
Bastian M, Heymann S, Jacomy M (2009) Gephi: an open source software for exploring and manipulating networks. ICWSM 8:361–362
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 13:340–349
Berney C, Pawlowski J (2006) A molecular time-scale for eukaryote evolution recalibrated with the continuous microfossil record. Proc Roy Soc B Biol Sci 273:1867–1872
Bickford D, Lohman DJ, Sodhi NS et al (2007) Cryptic species as a window on diversity and conservation. Trends Ecol Evol 22:148–155
Bragg L, Stone G, Imelfort M, Hugenholtz P, Tyson GW (2012) Fast, accurate error-correction of amplicon pyrosequences using Acacia. Nat Methods 9:425–426
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 4:1144–1153
Caporaso JG, Kuczynski J, Stombaugh J et al (2010) QIIME allows analysis of high-throughput community sequencing data. Nat Methods 7:335
Caron DA, Countway PD, Jones AC, Kim DY, Schnetzer A (2012) Marine protistan diversity. Ann Rev Mar Sci 4:467–493
Caron DA, Countway PD, Savai P et al (2009) Defining DNA-based operational taxonomic units for microbial-eukaryote ecology. Appl Environ Microbiol 75:5797–5808
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 4:338–348
Casamayor EO, Triadó-Margarit X, Castañeda C (2013) Microbial biodiversity in saline shallow lakes of the Monegros Desert, Spain. FEMS Microbiol Ecol 85:503–518
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 55:417–426
Csárdi G, Nepusz T (2006) The igraph software package for complex network research. Inter J Complex Syst 1695
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 59:185–187
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 31:993–1009
Dunthorn M, Stoeck T, Clamp J, Warren A, Mahé F (2014b) Ciliates and the rare biopshere: a review. J Eukaryot Microbiol 61:404–409
Edgar RC (2010) Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26:2460–2461
Edgar RC (2011) Usearch user guide 5.2
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 13:151–167
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 67:21–29
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 81:19–26
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 89:243–253
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 4:642–647
Epstein S, López-García P (2008) “Missing” protists: a molecular prospective. Biodivers Conserv 17:261–276
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 49:281–293
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 50:174–184
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 61:61–74
Forster D, Behnke A, Stoeck T (2012) Meta-analyses of environmental sequence data identify anoxia and salinity as parameters shaping ciliate communities. Syst Biodivers 10:277–288
Galtier N, Gouy M, Gautier C (1996) SEAVIEW and PHYLO_WIN: two graphic tools for sequence alignment and molecular phylogeny. Comput Appl Biosci 12:543–548
Giovannoni SJ, DeLong EF, Olsen GJ, Pace NR (1988) Phylogenetic group-specific oligodeoxynucleotide probes for identification of single microbial cells. J Bacteriol 170:720–726
Gostinčar C, Lenassi M, Gunde-Cimerman N, Plemenitaš A (2011) Fungal adaptation to extremely high salt concentrations. Adv Appl Microbiol 77:71–96
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 519–540
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 55:113–122
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 4
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 53:233–246
Ki JS (2012) Hypervariable regions (V1–V9) of the dinoflagellate 18S rRNA using a large dataset for marker considerations. J Appl Phycol 24:1035–1043
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 5
Knoll AH, Walter MR, Narbonne GM, Christi-Blick N (2004) A new period for the geologic time scale. Science 305:621–622
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 12:118–123
Lanyi JK (1974) Salt-dependent properties of proteins from extremely halophilic bacteria. Bacteriol Rev 38:272–290
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 62:365–373
Lee CE, Bell MA (1999) Causes and consequences of recent freshwater invasions by saltwater animals. Trends Ecol Evol 14:282–288
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 45:305–319
Logares R, Bråte J, Bertilsson S, Clasen JL, Shalchian-Tabrizi K, Rengefors K (2009) Infrequent marine-freshwater transitions in the microbial world. Trends Microbiol 17:414–422
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 27:347–357
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 6:1823–1833
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 36:193–202
Lozupone C, Knight R (2007) Global patterns in bacterial diversity. Proc Natl Acad Sci USA 104:11436–11440
Lynch MD, Bartram AK, Neufeld JD (2012) Targeted recovery of novel phylogenetic diversity from next-generation sequence data. ISME J 6:2067–2077
Lynn DH (2008) The Ciliated Protozoa, vol 3. Springer, New York
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:e593
McGenity TJ, Oren A (2012) Hypersaline Environments. In: Bell EM (ed) Life at extremes: environments, organisms and strategies for survival. CABI, UK, USA, pp 402–437
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 19:32–40
Moon-van der Staay SY, De Wachter R, Vaulot D (2001) Oceanic 18S rDNA sequences from picoplankton reveal unsuspected eukaryotic diversity. Nature 409:607–610
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 3:154–158
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 9:749–773
Oren A (2008) Microbial life at high salt concentrations: phylogenetic and metabolic diversity. Saline Systems 4:13
Pandey B, Yeragi S (2004) Preliminary and mass culture experiments on a heterotrichous ciliate, Fabrea salina. Aquacult 232:241–254
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 12:1173–1184
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 33:173–179
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 158:397–413
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 160:265–283
Patterson DJ, Simpson AGB (1996) Heterotrophic flagellates from coastal marine and hypersaline sediments in Western Australia. Eur J Protistol 32:423–448
Pedrós-Alió C (2004) Trophic ecology of solar salterns. In: Ventosa A (ed) Halophilic microorganisms. Springer, Berlin, pp 33–48
Post FJ, Borowitzka LJ, Borowitzka MA, Mackay B, Moulton T (1983) The protozoa of a western Australian hypersaline lagoon. Hydrobiologica 105:95–113
R Development Core Team (2008) R. A language and environment for statistical computing. In: R Foundation for Statistical Computing. Vienna, Austria
Ruinen J (1938) Notizen über Salzflagellaten II. Über die Verbreitung der Salzflagellaten. Arch f Protistenkd 90:161–177
Sáez AG, Lozano E (2005) Body doubles. Nature 433:111
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 61:371–380
Stock A, Breiner HW, Pachiadaki M et al (2012) Microbial eukaryote life in the new hypersaline deep-sea basin Thetis. Extremophiles 16:21–34
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 157:31–43
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 19:21–31
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 16:430–444
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 85:27–36
Vermeij GJ, Dudely R (2000) Why are there so few evolutionary transitions between aquatic and terrestrial ecosystems? Biol J Linn Soc 70:541–554
Weber AP, Horst RJ, Barbier GG, Oesterhelt C (2007) Metabolism and metabolomics of eukaryotes living under extreme conditions. Int Rev Cytol 256:1–34
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 28:4698–4708
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 52:79–92
Acknowledgments
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.
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by A. Oren.
Accession numbers
Pyro-reads are deposited in NCBI´s SRA under the accession number SRP050177.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Filker, S., Gimmler, A., Dunthorn, M. et al. Deep sequencing uncovers protistan plankton diversity in the Portuguese Ria Formosa solar saltern ponds. Extremophiles 19, 283–295 (2015). https://doi.org/10.1007/s00792-014-0713-2
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00792-014-0713-2