Extensive dinoflagellate phylogenies indicate infrequent marine-freshwater transitions.

We have constructed extensive 18S-28S rDNA dinoflagellate phylogenies (>200 sequences for each marker) using Maximum Likelihood and Bayesian Inference to study the evolutionary relationships among marine and freshwater species (43 new sequences). Our results indicated that (a) marine and freshwater species are usually not closely related, (b) several freshwater species cluster into monophyletic groups, (c) most marine-freshwater transitions do not seem to have occurred recently and, (d) only a small fraction of the marine lineages seem to have colonized fresh waters. Thus, it becomes apparent that the marine-freshwater boundary has acted as a barrier during the evolutionary diversification of dinoflagellates. Our results also shed light on the phylogenetic positions of several freshwater dinoflagellates which, to date, were uncertain.

[1]  J. Pawlowski,et al.  Freshwater Foraminiferans Revealed by Analysis of Environmental DNA Samples , 2003, The Journal of eukaryotic microbiology.

[2]  M. Gottschling,et al.  Phylogeny of calcareous dinoflagellates as inferred from ITS and ribosomal sequence data. , 2005, Molecular phylogenetics and evolution.

[3]  C. Labandeira,et al.  Slow Crawl Across the Salinity Divide: Delayed Colonization of Freshwater Ecosystems by Invertebrates , 2002 .

[4]  S. Muse.  Evolutionary analyses of DNA sequences subject to constraints of secondary structure. , 1995, Genetics.

[5]  Wei Qian,et al.  Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. , 2000, Molecular biology and evolution.

[6]  Y. Sako,et al.  RESTRICTION FRAGMENT LENGTH POLYMORPHISM OF RIBOSOMAL DNA INTERNAL TRANSCRIBED SPACER AND 5.8S REGIONS IN JAPANESE ALEXANDRIUM SPECIES (DINOPHYCEAE) 1 , 1994 .

[7]  Michael Melkonian,et al.  Nuclear and Nucleomorph SSU rDNA Phylogeny in the Cryptophyta and the Evolution of Cryptophyte Diversity , 2002, Journal of Molecular Evolution.

[8]  D. Bhattacharya,et al.  Development of a Dinoflagellate-Oriented PCR Primer Set Leads to Detection of Picoplanktonic Dinoflagellates from Long Island Sound , 2006, Applied and Environmental Microbiology.

[9]  David G. Mann,et al.  Evolution of the diatoms: insights from fossil, biological and molecular data , 2006 .

[10]  K. Jakobsen,et al.  GENETIC VARIABILITY AND MOLECULAR PHYLOGENY OF DINOPHYSIS SPECIES ( DINOPHYCEAE ) FROM NORWEGIAN WATERS INFERRED FROM SINGLE CELL ANALYSES OF rDNA , 2003 .

[11]  F. Ayala,et al.  Evolutionary origin of Plasmodium and other Apicomplexa based on rRNA genes. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[12]  R. Amann,et al.  Actinobacterial 16S rRNA genes from freshwater habitats cluster in four distinct lineages. , 2004, Environmental microbiology.

[13]  D. Bhattacharya,et al.  PHYLOGENY OF DINOFLAGELLATES BASED ON MITOCHONDRIAL CYTOCHROME B AND NUCLEAR SMALL SUBUNIT RDNA SEQUENCE COMPARISONS 1 , 2005 .

[14]  T. Tengs,et al.  Phenotypically Different Microalgal Morphospecies with Identical Ribosomal DNA: A Case of Rapid Adaptive Evolution? , 2007, Microbial Ecology.

[15]  Edward J. Noga,et al.  New 'phantom' dinoflagellate is the causative agent of major estuarine fish kills , 1992, Nature.

[16]  G. Hallegraeff.  A review of harmful algal blooms and their apparent global increase , 1993 .

[17]  Ø. Moestrup,et al.  Studies on woloszynskioid dinoflagellates I: Woloszynskia coronata re-examined using light and electron microscopy and partial LSU rDNA sequences, with description of Tovellia gen. nov. and Jadwigia gen. nov. (Tovelliaceae fam. nov.) , 2005 .

[18]  S. Ho,et al.  Improving the analysis of dinoflagellate phylogeny based on rDNA. , 2005, Protist.

[19]  J. Felsenstein.  CONFIDENCE LIMITS ON PHYLOGENIES: AN APPROACH USING THE BOOTSTRAP , 1985, Evolution; international journal of organic evolution.

[20]  Roderic D. M. Page,et al.  TreeView: an application to display phylogenetic trees on personal computers , 1996, Comput. Appl. Biosci..

[21]  D. Hillis,et al.  Resolution of phylogenetic conflict in large data sets by increased taxon sampling. , 2006, Systematic biology.

[22]  C. Lee,et al.  Causes and consequences of recent freshwater invasions by saltwater animals. , 1999, Trends in ecology & evolution.

[23]  D. Vaulot,et al.  Telonemia, a new protist phylum with affinity to chromist lineages , 2006, Proceedings of the Royal Society B: Biological Sciences.

[24]  Derrick J. Zwickl.  Genetic algorithm approaches for the phylogenetic analysis of large biological sequence datasets under the maximum likelihood criterion , 2006 .

[25]  H. Akaike.  A new look at the statistical model identification , 1974 .

[26]  M. Miyamoto,et al.  Testing the covarion hypothesis of molecular evolution. , 1995, Molecular biology and evolution.

[27]  M. Newton.  Approximate Bayesian-inference With the Weighted Likelihood Bootstrap , 1994 .

[28]  Mark P. Simmons,et al.  How meaningful are Bayesian support values? , 2004, Molecular biology and evolution.

[29]  F. Hagen,et al.  Typical freshwater bacteria: an analysis of available 16S rRNA gene sequences from plankton of lakes and rivers , 2002 .

[30]  Andrew J. Alverson,et al.  Bridging the Rubicon: phylogenetic analysis reveals repeated colonizations of marine and fresh waters by thalassiosiroid diatoms. , 2007, Molecular phylogenetics and evolution.

[31]  H. Ettl,et al.  Süsswasserflora von Mitteleuropa , 1985 .

[32]  D. Anderson,et al.  Phylogeny, biogeography, and species boundaries within the Alexandrium minutum group. , 2005 .

[33]  L. Meester,et al.  The Monopolization Hypothesis and the dispersal–gene flow paradox in aquatic organisms , 2002 .

[34]  Sudhir Kumar,et al.  MEGA3: Integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment , 2004, Briefings Bioinform..

[35]  Vivek Gowri-Shankar,et al.  Consideration of RNA secondary structure significantly improves likelihood-based estimates of phylogeny: examples from the bilateria. , 2005, Molecular biology and evolution.

[36]  M. Weitere,et al.  Molecular Comparisons of Freshwater and Marine Isolates of the Same Morphospecies of Heterotrophic Flagellates , 2006, Applied and Environmental Microbiology.

[37]  D. Anderson,et al.  Dinoflagellates: a remarkable evolutionary experiment. , 2004, American journal of botany.

[38]  M. Lynch,et al.  Large global effective population sizes in Paramecium. , 2006, Molecular biology and evolution.

[39]  F. Tailor.  The biology of Dinoflagellates , 1987 .

[40]  J. Huelsenbeck.  Testing a covariotide model of DNA substitution. , 2002, Molecular biology and evolution.

[41]  Sandhya Dwarkadas,et al.  Parallel Metropolis coupled Markov chain Monte Carlo for Bayesian phylogenetic inference , 2002, Bioinform..

[42]  D. Wall,et al.  A Comparison of the Modern Genus Ceratium Schrank, 1793, with Certain Cretaceous Marine Dinoflagellates , 1975 .

[43]  B. Haq,et al.  Chronology of Fluctuating Sea Levels Since the Triassic , 1987, Science.

[44]  T. Cavalier-smith,et al.  Cell evolution and Earth history: stasis and revolution , 2006, Philosophical Transactions of the Royal Society B: Biological Sciences.

[45]  T. Richards,et al.  The molecular diversity of freshwater picoeukaryotes from an oligotrophic lake reveals diverse, distinctive and globally dispersed lineages. , 2005, Environmental microbiology.

[46]  K. Jakobsen,et al.  GENETIC VARIABILITY AND MOLECULAR PHYLOGENY OF DINOPHYSIS SPECIES (DINOPHYCEAE) FROM NORWEGIAN WATERS INFERRED FROM SINGLE CELL ANALYSES OF rDNA 1 , 2003 .

[47]  Emilie Lefèvre,et al.  Unveiling fungal zooflagellates as members of freshwater picoeukaryotes: evidence from a molecular diversity study in a deep meromictic lake. , 2007, Environmental microbiology.

[48]  A. von Haeseler,et al.  A stochastic model for the evolution of autocorrelated DNA sequences. , 1994, Molecular phylogenetics and evolution.

[49]  R. Wachter,et al.  Oceanic 18S rDNA sequences from picoplankton reveal unsuspected eukaryotic diversity , 2001, Nature.

[50]  G. Hallegraeff,et al.  TAKAYAMA GEN. NOV. (GYMNODINIALES, DINOPHYCEAE), A NEW GENUS OF UNARMORED DINOFLAGELLATES WITH SIGMOID APICAL GROOVES, INCLUDING THE DESCRIPTION OF TWO NEW SPECIES 1 , 2003 .

[51]  G. Procaccini,et al.  Intraspecific diversity in Scrippsiella trochoidea (Dinopbyceae): evidence for cryptic species , 2003 .

[52]  T. Cavalier-smith,et al.  Combined Heat Shock Protein 90 and Ribosomal RNA Sequence Phylogeny Supports Multiple Replacements of Dinoflagellate Plastids , 2006, The Journal of eukaryotic microbiology.

[53]  David Posada,et al.  MODELTEST: testing the model of DNA substitution , 1998, Bioinform..

[54]  G. Vermeij,et al.  NEOGASTROPOD MOLLUSCS FROM THE MIOCENE OF WESTERN AMAZONIA, WITH COMMENTS ON MARINE TO FRESHWATER TRANSITIONS IN MOLLUSCS , 2002 .

[55]  Derrick J. Zwickl,et al.  Increased taxon sampling is advantageous for phylogenetic inference. , 2002, Systematic biology.

[56]  F. M. Taylor,et al.  Illumination or confusion? Dinoflagellate molecular phylogenetic data viewed from a primarily morphological standpoint , 2004 .

[57]  Ramakant Sharma,et al.  Phylogeny Estimation and Hypothesis Testing using Maximum Likelihood , 2003 .

[58]  N. Galtier,et al.  Maximum-likelihood phylogenetic analysis under a covarion-like model. , 2001, Molecular biology and evolution.

[59]  J. Thompson,et al.  The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. , 1997, Nucleic acids research.

[60]  Michael Zuker,et al.  Algorithms and Thermodynamics for RNA Secondary Structure Prediction: A Practical Guide , 1999 .

[61]  C. Pedrós-Alió,et al.  Unexpected diversity of small eukaryotes in deep-sea Antarctic plankton , 2001, Nature.

[62]  John P. Huelsenbeck,et al.  MRBAYES: Bayesian inference of phylogenetic trees , 2001, Bioinform..

[63]  G. Williams,et al.  The early Mesozoic radiation of dinoflagellates , 1996, Paleobiology.

[64]  T. A. Hall,et al.  BIOEDIT: A USER-FRIENDLY BIOLOGICAL SEQUENCE ALIGNMENT EDITOR AND ANALYSIS PROGRAM FOR WINDOWS 95/98/ NT , 1999 .

[65]  H. Stosch.  Observations on vegetative reproduction and sexual life cycles of two freshwater dinoflagellates, Gymondinium pseudopalustre Schiller and Woloszynskia apiculata sp. nov. , 1973 .

[66]  L. Katz,et al.  Reframing the Everything is everywhere debate: evidence for high gene flow and diversity in ciliate morphospecies , 2005 .

[67]  R. Figueroa,et al.  LIFE CYLE AND SEXUALITY OF THE FRESHWATER RAPHIDOPHYTE GONYOSTOMUM SEMEN (RAPHIDOPHYCEAE) 1 , 2006 .

[68]  J. Burkholder,et al.  New ‘phantom’ dinoflagellate is the causative agent of major estuarine fish kills , 1992, Nature.

[69]  A. Boltovskoy.  Peridinium gatunense Nygaard. Estructura y estéreoultraestructura tecal (Dinoflagellida) , 1973 .

[70]  Antonis Rokas,et al.  Comparing bootstrap and posterior probability values in the four-taxon case. , 2003, Systematic biology.

[71]  D. Hillis,et al.  Ribosomal DNA: Molecular Evolution and Phylogenetic Inference , 1991, The Quarterly Review of Biology.

[72]  P. Henriksen,et al.  Baldinia anauniensis gen. et sp. nov.: a ‘new’ dinoflagellate from Lake Tovel, N. Italy , 2007 .

[73]  D. Hillis,et al.  Ribosomal RNA secondary structure: compensatory mutations and implications for phylogenetic analysis. , 1993, Molecular biology and evolution.

[74]  G. Flaim,et al.  Studies on woloszynskioid dinoflagellates II: On Tovellia sanguinea sp. nov., the dinoflagellate responsible for the reddening of Lake Tovel, N. Italy , 2006 .

[75]  R. Fensome,et al.  Dinoflagellate phylogeny revisited: reconciling morphological and molecular based phylogenies , 1999 .

[76]  D. Anderson,et al.  IDENTIFICATION OF GROUP‐ AND STRAIN‐SPECIFIC GENETIC MARKERS FOR GLOBALLY DISTRIBUTED ALEXANDRIUM (DINOPHYCEAE). II. SEQUENCE ANALYSIS OF A FRAGMENT OF THE LSU rRNA GENE 1 , 1994 .

[77]  J. Albert,et al.  Miocene marine incursions and marine/freshwater transitions: Evidence from Neotropical fishes , 2006 .

[78]  T. Cavalier-smith,et al.  Genetic diversity of goniomonads: an ancient divergence between marine and freshwater species , 2004 .

[79]  Ø. Moestrup,et al.  ULTRASTRUCTURE AND LSU RDNA‐BASED PHYLOGENY OF ESOPTRODINIUM GEMMA (DINOPHYCEAE), WITH NOTES ON FEEDING BEHAVIOR AND THE DESCRIPTION OF THE FLAGELLAR BASE AREA OF A PLANOZYGOTE 1 , 2006 .

[80]  G. Procaccini,et al.  POLARELLA GLACIALIS, GEN. NOV., SP. NOV. (DINOPHYCEAE): SUESSIACEAE ARE STILL ALIVE! , 1999 .

[81]  L. Graham,et al.  Genetically Distinct Populations of the Dinoflagellate Peridinium limbatum in Neighboring Northern Wisconsin Lakes , 2004, Microbial Ecology.

[82]  T. Cavalier-smith,et al.  Culturing and environmental DNA sequencing uncover hidden kinetoplastid biodiversity and a major marine clade within ancestrally freshwater Neobodo designis. , 2005, International journal of systematic and evolutionary microbiology.

[83]  B. Methé,et al.  Contrasts between marine and freshwater bacterial community composition: Analyses of communities in Lake George and six other Adirondack lakes , 1998 .

[84]  Susanne Menden-Deuer,et al.  Molecular data and the evolutionary history of dinoflagellates , 2004 .

[85]  B. Methé,et al.  Nearly identical 16S rRNA sequences recovered from lakes in North America and Europe indicate the existence of clades of globally distributed freshwater bacteria. , 1998, Systematic and applied microbiology.

[86]  Ilha Lee,et al.  Stoeckeria algicida n. gen., n. sp. (Dinophyceae) from the Coastal Waters off Southern Korea: Morphology and Small Subunit Ribosomal DNA Gene Sequence , 2005, The Journal of eukaryotic microbiology.

[87]  B. Finlay.  Global Dispersal of Free-Living Microbial Eukaryote Species , 2002, Science.

[88]  M. Holzmann,et al.  Molecular phylogeny of Foraminifera a review , 2002 .