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Phylogénie, diversité et dynamique temporelle chez les ciliés tintinnidés marins

Abstract : The marine protistan diversity has been historically studied based on morphological characterization but has recently been the object of intense research using molecular approaches. Studies based on the amplification of molecular markers from environmental DNA revealed an outstanding diversity, partly new and uncharacterized. However, the actual extent of this diversity remains poorly known and highly debated. The main goal of this work was to improve our knowledge on protistan diversity to bridge the gap between molecular environmental surveys and classical protistology to better understand the ecology and evolution of unicellular eukaryotes. For this purpose, we used as a model the species-rich order of the tintinnid ciliates (Tintinnida, Ciliophora), which are easily distinguishable because of their secreted shell, the lorica, and commonly found in marine waters all around the globe. A two-year monitoring of the tintinnid populations in the Bay of Villefranche-sur- Mer (Mediterranean Sea, France), combining molecular analyses of the diversity based on single-cells and environmental DNA, gave us the opportunity to describe the tintinnid community composition and its temporal dynamics. In the first part of this work, we constructed a reference molecular phylogeny for the tintinnids including new sequences from 62 specimens of diverse morphologies, for which we amplified and sequenced the ribosomal coding genes (18S, 5.8S and 28S rRNA) and the corresponding intergenic spacers (ITS1 and ITS2). The taxonomic classification of the Tintinnida has been revised based on these molecular data. In the second part, in order to assess the accuracy of molecular-based approaches to describe the natural species assemblages of tintinnids, we compared the morphology-based diversity estimates with those derived from classical (amplification, cloning and Sanger sequencing of the 18S rRNA gene) and more recent (direct pyrosequencing of amplified 18S rRNA genes and ITS regions) molecular approaches. Even if there are still some disagreements between the different methods and/or molecular markers, the culture-independent approaches were efficient to describe the morphological diversity. However, a careful and rigorous analysis of pyrosequencing datasets, including sequence denoising and stringent sequence clustering in Operational Taxonomic Units (OTUs) with well-adjusted parameters, is necessary to avoid overestimating the species number. The third part of the thesis is dedicated to the study of the genetic diversity of tintinnids over a one-year survey in the Bay of Villefranche at five different depths by combining community fingerprinting analysis using denaturing gradient gel electrophoresis (DGGE) with direct PCR amplification and sequencing of 18S, 5.8S, and 28S rRNA genes and ITS regions. These analyses revealed marked seasonal changes, in particular in the sequence abundances of certain OTUs. In addition, despite an enriched phylogenetic reference sequence dataset for the tintinnids, we retrieved two abundant phylotypes without any closely related known species, highlighting the possible ecological relevance of unidentified species. Finally, we studied the intra-specific diversity of populations of the species based on 18S rDNA and ITS direct sequencing of single-cells collected over a period of two years. We detected signals of hybridization and sexual recombination among different genetic variants. We also found genetic structuring of the 18S rRNA gene data differentiating populations collected at different times. The implications of all these results are discussed in the framework of the diversity and ecology of tintinnid ciliates and, more generally, of marine protists
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Charles Bachy. Phylogénie, diversité et dynamique temporelle chez les ciliés tintinnidés marins. Sciences agricoles. Université Paris Sud - Paris XI, 2012. Français. ⟨NNT : 2012PA112105⟩. ⟨tel-00769949⟩

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