Éditeur(s) : HAL CCSD
Résumé : Ctenophores are one of the four animal phyla positioned outside from the Bilateria. Most ctenophore species are planktonic and gelatinous, and are easily recognisable by their eight comb rows used for swimming. Ctenophore systematics remains nowadays poorly understood. Anatomical characters do not help much in placing them within the animal tree, and similarly the grounds for establishment of their internal phylogeny as well as delimitation of species are notoriously weak due to a paucity of informative morpho-anatomical characters. Until now, the use of molecular data has failed to improve significantly this situation. The aim of this PhD thesis was to bring a contribution to our understanding of ctenophore evolution at different taxonomic scales. At the metazoan level, the position of ctenophores was addressed using a phylogenomic approach. Taxonomic sampling was significantly improved through sequencing and assembly of transcriptomes of 22 non-bilaterian species (belonging to ctenophores, cnidarians and sponges). Two independent datasets were analysed, one consisting in an update of an existing supermatrix of 128 genes, the other one (4235 genes) having been entirely built de novo, thanks to a newly-devised semi-automated protocol intended to eliminate all major potential causes of artefacts (contaminations, paralogies, missing data). Results clearly contradict recent phylogenomic studies which claimed Ctenophora to be the most early-diverging animal lineage, our analyses instead supporting Porifera as the sister-group to other metazoans. The exact position of ctenophores remains however uncertain at this stage, different conditions of analyses yielding three contradictory hypotheses as open possibilities. At the intra-phyletic level, analyses of a ADNr 18S and ITS (internal transcribed spacers) dataset, together with study of duplicated genes and the results of phylogenomic analyses, allowed resolving most phylogenetic relationships between ctenophore orders and families, including the placement of the ctenophore tree root. Finally, at smaller taxonomic scale, in-depth comparison between two species of the genus Pleurobrachia using immunohistochemistry demonstrates the potential of these techniques for uncovering new structural “micro-anatomical” characters useful for diagnosis and identification of ctenophore species. It is our hope that this work will contribute to improving the systematics of a poorly known phylum of great importance for understanding early animal evolution.
Les cténophores représentent l’un des quatre embranchements animaux extérieurs aux Bilateria. La majoritédes espèces sont planctoniques et gélatineuses, et sont reconnaissables à leurs huit rangées de peignes dont lebattement permet la nage. Leur systématique est encore de nos jours mal comprise. L'anatomie descténophores offre peu de caractères aussi bien pour placer la lignée dans l’arbre des métazoaires, que pourétablir les relations de parenté au sein de l’embranchement et délimiter les espèces, et jusqu’à présent lesdonnées moléculaires n’ont pas permis de résoudre ces problèmes de manière satisfaisante. L’objectif de cetravail de thèse est de contribuer à améliorer notre compréhension de l'évolution des cténophores àdifférentes échelles taxonomiques. A l’échelle des métazoaires, la position phylogénétique des cténophores aété abordée par une approche phylogénomique. Un effort significatif a été réalisé pour améliorerl’échantillonnage taxonomique à travers le séquençage et l’assemblage des transcriptomes de 22 espèces denon-Bilateria (cténophores, cnidaires, spongiaires). Deux jeux de données indépendants ont été analysés, lepremier représentant une mise à jour d’une supermatrice existante de 128 gènes ; le second (4235 gènes)ayant été entièrement construit de novo via un protocole original comportant la mise au point de nouvellesméthodes pour traiter de manière semi-automatisée les principales sources potentielles d’artéfact(contaminations, paralogies, données manquantes). Les résultats contredisent certaines études récentes enmontrant que les spongiaires et non les cténophores représentent le groupe-frère des autres métazoaires. Laposition exacte de ces derniers reste à ce stade incertaine (trois options se présentant suivant les analyses). Al’échelle intra-phylétique, l'analyse d'un jeu de données comprenant les marqueurs ADNr 18S et InternalTranscribed Spacers (ITS), associée à des analyses de gènes dupliqués chez les cténophores et aux analysesphylogénomiques précédentes, a permis de résoudre une grande partie des relations phylogénétiques entre lesordres et les familles de cténophores, tout en permettant de préciser la position de la racine. Enfin, à uneéchelle taxonomique plus fine, une comparaison approfondie entre deux espèces du genre Pleurobrachia aumoyen de marquages immunohistochimiques montre le potentiel de ces techniques comme source denouveaux caractères structuraux « micro-anatomiques » à valeur diagnostique pour la délimitation etl’identification des espèces de cténophores. En conclusion, ce travail se veut une contribution au progrès dela systématique d’un embranchement encore méconnu et d’une grande importance pour la compréhensiondes évènements anciens de l’évolution animale.
https://tel.archives-ouvertes.fr/tel-01165014

NNT : 2014PA066448
tel-01165014
https://tel.archives-ouvertes.fr/tel-01165014
https://tel.archives-ouvertes.fr/tel-01165014/document
https://tel.archives-ouvertes.fr/tel-01165014/file/these_archivage_3158995o.pdf

Éditeur(s) : HAL CCSD
Résumé : International audience
Symbiosis is a widespread phenomenon in the biosphere. In marine benthic environments breakdown of symbiosis is responsible for coral bleaching and dramatically affects coral reef ecosystems functioning [1]. In the water column, planktonic organisms are key component of pelagic ecosystems and a number of species form mutualistic association with microalgae forming a photosymbiosis [2]. Here we intended to investigate the genetic basis of photosymbiosis, through a transcriptomic approach on marine plankton organisms. We focused more specifically on associations occurring between radiolarian host (protist, zooplankton) and dinoflagellates symbiont (protist, phytoplankton) living inside the host cell. It has recently been highlighted that these holobionts are widespread in oligotrophic open oceans and there are also evidences of their fundamental implication in biogeochemical carbon, silica and strontium cycles [3, 4, 5, 6]. RNA-seq technologies allow obtaining an unprecedented amount of data for unicellular organisms isolated from the environment [7]. The study of such non-model holobionts datasets requires de novo assembly, which implies considerable computational resources and the potential production of chimeric sequences [8, 9].We therefore developed an original strategy aiming at accelerating and improving de novo assembly for holobiont datasets. We chose SIMKA [10] a fast kmer-based method initially developed to estimate the similarity between numerous metagenomic datasets, and which has been recently adapted to extract their common sequences. As our symbionts are identified, we used SIMKA to compare our holobionts transcriptomes to publicly available dinoflagellates transcriptomes [11] generating two datasets, one composed of reads from the symbionts and another with reads from the host (for which no reference data are currently available). Independent assemblies were then performed in parallel, accelerating the study process, and minimizing the proportion of resulting chimeric sequences. Our strategy produced a unique and comprehensive genomic dataset for Radiolaria [12, 13], and offers a pragmatic, large scale, comparison strategy to assemble and study holobionts [9]. Our new sequences obtained from holobionts study will be used for phylogenomics investigation, as reference for environmental metagenomic studies and ultimately to understand and characterize the molecular basis of symbiotic relationships in the plankton.Bibliografic references :[1] Simon K. Davy, Denis Allemand, and Virginia M. Weis. “Cell Biology of Cnidarian-Dinoflagellate Symbiosis”. In: Microbiology and Molecular Biology Reviews 76.2 (June 1, 2012), pp. 229–261. issn: 1092-2172, 1098- 5557. doi: 10.1128/MMBR.05014-11.[2] Janouškovec, J. et al. “Major transitions in dinoflagellate evolution unveiled by phylotranscriptomics”. in: PNAS 201614842 (2016). doi:10.1073/pnas.1614842114[3] Tristan Biard et al. “In situ imaging reveals the biomass of giant protists in the global ocean”. In: Nature advance online publication (Apr. 20, 2016). issn: 0028-0836. doi: 10.1038/nature17652.[4] Lionel Guidi et al. “Plankton networks driving carbon export in the olig- otrophic ocean”. In: Nature 532.7600 (Apr. 28, 2016), pp. 465–470. issn: 0028-0836. doi: 10.1038/nature16942.[5] Diane K. Stoecker et al. “Acquired phototrophy in aquatic protists”. In: Aquatic Microbial Ecology 57.3 (Nov. 24, 2009), pp. 279–310. doi: 10.3354/ame01340.[6] Johan Decelle et al. “An original mode of symbiosis in open ocean plankton”. In: Proceedings of the National Academy of Sciences 109.44 (Oct. 30, 2012), pp. 18000–18005. issn: 0027-8424, 1091-6490. doi: 10. 1073/pnas.1212303109.[7] Sergio Balzano et al. “Transcriptome analyses to investigate symbiotic relationships between marine protists”. In: Frontiers in Microbiology 6 (Mar. 17, 2015). issn: 1664-302X. doi: 10.3389/fmicb.2015.00098.[8] Li, B. et al. “Evaluation of de novo transcriptome assemblies from RNA-Seq data”. In: Genome Biology 15, 553 (2014).[9] Sangwan, N., Xia, F. & Gilbert, J. A. “Recovering complete and draft population genomes from metagenome datasets”. In: Microbiome 4, 8 (2016).[10] Gaëtan Benoit et al. “Multiple comparative metagenomics using mul- tiset k -mer counting”. In: PeerJ Computer Science 2 (Nov. 14, 2016). doi: 10.7717/peerj-cs.94.[11] Patrick J. Keeling et al. “The Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP): Illuminating the Functional Diversity of Eukaryotic Life in the Oceans through Transcriptome Sequencing”. In: PLOS Biol 12.6 (June 2014), e1001889. issn: 1545-7885. doi: 10.1371/journal.pbio.1001889.[12] Burki, F. et al. “Evolution of Rhizaria: new insights from phylogenomic analysis of uncultured protists”. In BMC Evolutionary Biology 10, 377 (2010).
International Conference on Holobionts
Paris, France

hal-01575069
https://hal.inria.fr/hal-01575069

Éditeur(s) : HAL CCSD Wiley
Résumé : International audience
Analysis of microbial biodiversity is hampered by a lack of reference genomes from most bacteria, viruses, and algae. This necessitates either the cultivation of a restricted number of species for standard sequencing projects or the analysis of highly complex environmental DNA metagenome data. Single-cell genomics (SCG) offers a solution to this problem by constraining the studied DNA sample to an individual cell and its associated symbionts, prey, and pathogens. We used SCG to study marine heterotrophic amoebae related to Paulinella ovalis (A. Wulff) P.W. Johnson, P.E. Hargraves & J.M. Sieburth (Rhizaria). The genus Paulinella is best known for its photosynthetic members such as P.chromatophora Lauterborn that is the only case of plastid primary endosymbiosis known outside of algae and plants. Here, we studied the phagotrophic sister taxa of P.chromatophora that are related to P.ovalis and found one SCG assembly to contain -cyanobacterial DNA. These cyanobacterial contigs are presumably derived from prey. We also uncovered an associated cyanophage lineage (provisionally named phage PoL_MC2). Phylogenomic analysis of the fragmented genome assembly suggested a minimum genome size of 200Kbp for phage PoL_MC2 that encodes 179 proteins and is most closely related to Synechococcus phage S-SM2. For this phage, gene network analysis demonstrates a highly modular genome structure typical of other cyanophages. Our work demonstrates that SCG is a powerful approach for discovering algal and protist biodiversity and for elucidating biotic interactions in natural samples.
ISSN: 0022-3646

hal-01544797
https://hal.archives-ouvertes.fr/hal-01544797
DOI : 10.1111/jpy.12028

Éditeur(s) : HAL CCSD Elsevier
Résumé : International audience
Resolving the early diversification of animal lineages has proven difficult, even using genome-scale datasets. Several phylogenomic studies have supported the classical scenario in which sponges (Porifera) are the sister group to all other animals (''Porifera-sister''3 hypothesis), consistent with a single origin of the gut, nerve cells, and muscle cells in the stem lineage of eumetazoans (bilaterians + ctenophores + cnidarians). In contrast, several other studies have recovered an alternative topology in which ctenophores are the sister group to all other animals (including sponges). The ``Ctenophora-sister'' hypothesis implies that eumetazoan-specific traits, such as neurons and muscle cells, either evolved once along the metazoan stem lineage and were then lost in sponges and placozoans or evolved at least twice independently in Ctenophora and in Cnidaria + Bilateria. Here, we report on our reconstruction of deep metazoan relationships using a 1,719-gene dataset with dense taxonomic sampling of non-bilaterian animals that was assembled using a semi-automated procedure, designed to reduce known error sources. Our dataset outperforms previous metazoan gene superalignments in terms of data quality and quantity. Analyses with a best-fitting site-heterogeneous evolutionary model provide strong statistical support for placing sponges as the sister-group to all other metazoans, with ctenophores emerging as the second-earliest branching animal lineage. Only those methodological settings that exacerbated long-branch attraction artifacts yielded Ctenophora-sister. These results show that methodological issues must be carefully addressed to tackle difficult phylogenetic questions and pave the road to a better understanding of how fundamental features of animal body plans have emerged.
ISSN: 0960-9822

hal-01681528
https://hal.archives-ouvertes.fr/hal-01681528
DOI : 10.1016/j.cub.2017.02.031

Éditeur(s) : HAL CCSD Wiley/Blackwell
Résumé : Whole genome sequencing is helping generate robust phylogenetic hypotheses for a range of taxonomic groups that were previously recalcitrant to classical molecular phylogenetic approaches. As a case study, we performed a shallow shotgun sequencing of eight species in the tropical tree family Chrysobalanaceae to retrieve large fragments of high-copy number DNA regions and test the potential of these regions for phylogeny reconstruction. We were able to assemble the nuclear ribosomal cluster (nrDNA), the complete plastid genome (ptDNA) and a large fraction of the mitochondrial genome (mtDNA) with approximately 1000x, 450x and 120x sequencing depth respectively. The phylogenetic tree obtained with ptDNA resolved five of the seven internal nodes. In contrast, the tree obtained with mtDNA and nrDNA data were largely unresolved. This study demonstrates that genome skimming is a cost-effective approach and shows potential in plant molecular systematics within Chrysobalanaceae and other under-studied groups.
ISSN: 1755-098X

hal-01204217
https://hal.archives-ouvertes.fr/hal-01204217
DOI : 10.1111/1755-0998.12246
PRODINRA : 283530

Éditeur(s) : HAL CCSD
Résumé : Due to their phylogenetic position outside from Bilateria, cnidarians and ctenophores are key phyla to understanding the origin and early evolution of molecular mechanisms, cell types, or body plan features at the level of Metazoa. Unlike other non-bilaterian phyla (sponges and placozoans), they possess neurons and muscle cells. The first part of my work relates to the problem of the origin of the metazoan nervous system. Recent studies have suggested a dual origin for nerve cells, with ctenophore neurons having emerged independently from those of cnidarians and bilaterians. This hypothesis relies on phylogenomic analyses supporting ctenophores as sister-group to all other metazoans, and on the purported absence in ctenophores of almost all well-known neuro-transmission systems, including the cholinergic system. Refuting this claim, I present substantial evidence in favour of acetylcholine (Ach) being used in the ctenophore Pleurobrachia pileus as an extra-cellular messenger, including within the nervous system. This conclusion arises from the conjunction of multiple approaches at the molecular scale (orthology analyses, Ach immuno-localisation, gene in situ hybridisation), at the cellular scale (calcium imaging on muscle cells exposed to Ach) and at the organism scale (pharmacological treatments with an Ach analogue and Ach antagonists). In the second part of my thesis, I address the conservation of molecular mechanisms involved in the control of cellular proliferation. Notably, the Hippo pathway is known for its role in bilaterians in the negative control of cellular proliferation. I show that components of this pathway are conserved in Pleurobrachia pileus and in Clytia hemisphaerica and that the nuclear vs. cytoplasmic localisation of the transcriptional co-factor Yorkie is correlated to the extent of proliferation within C. hemisphaerica tissues. Finally, RNAseq differential gene expression analyses along a cellular conveyor belt of the C. hemisphaerica medusa suggests a tendency for conservation of function among a set of genes whose mammalian orthologues have documented roles in the arrest of cellular proliferation.
Les Cnidaires et les Cténaires sont deux embranchements extérieurs aux Bilatériens qui, de par leur position phylogénétique, constituent des modèles d'étude cruciaux pour comprendre l'origine et l'évolution de mécanismes moléculaires ou encore de types cellulaires à grande échelle chez les Métazoaires. Ils sont les deux seuls embranchements non-Bilatériens à posséder des cellules nerveuses ainsi que des cellules musculaires. La première partie de mon travail de thèse s'inscrit dans le contexte de la compréhension de l'origine du système nerveux des Métazoaires. Des études récentes suggèrent une origine double du système nerveux ; ainsi celui des Cténaires aurait été acquis de manière convergente par rapport à celui des Cnidaires et des Bilatériens. Cela repose sur des données récentes de phylogénomique proposant les Cténaires en tant que groupe-frère de tous les autres Métazoaires et sur l'absence supposée chez les Cténaires de quasiment tous les systèmes de neurotransmission connus y compris le système cholinergique. A l'encontre de ces hypothèses, j'ai pu mettre en évidence l'existence d'un système cholinergique chez le Cténaire Pleurobrachia pileus ainsi que son étroite association avec le système nerveux. Cette conclusion repose sur une multiplicité d'approches à l'échelle moléculaire (analyses d'orthologie, immunolocalisation, hybridations in situ), cellulaire (imagerie calcique) et de l'organisme (approche pharmacologique). Dans la seconde partie de ma thèse, j'aborde la question de la conservation des mécanismes moléculaires impliqués dans le contrôle de la prolifération cellulaire à l'échelle des Métazoaires. J'ai pu mettre en évidence la conservation des acteurs.
https://tel.archives-ouvertes.fr/tel-01633223

NNT : 2015PA066629
tel-01633223
https://tel.archives-ouvertes.fr/tel-01633223
https://tel.archives-ouvertes.fr/tel-01633223v2/document
https://tel.archives-ouvertes.fr/tel-01633223/file/2015PA066629.pdf