Dissertations / Theses on the topic 'Mitogenomes'

Für eine umfassende Untersuchung der Phylogenie der Decapoda wurden von mir die mitochondrialen Genome von 13 Dekapoden sequenziert. Zusammen mit den in der GenBank verfügbaren Sequenzen von 31 Dekapoden und dem von der Universität Bonn zur Verfügung gestellten mitochondrialen Genom von Dromia personata deckt dieser Datensatz alle großen Teilgruppen der Decapoda ab. Maximum likelihood (ML)-Analysen und Bayesian inference (BI)-Analysen der Nucleotidsequenzen und Aminosäuresequenzen ergaben bezüglich der Verwandtschaft der hochrangigen Taxa ähnliche Topologien: (((((((Anomala, Brachyura), Thalassinida: Gebiidea) Thalassinida: Axiidea), Astacidea), Achelata), Stenopodidea), Caridea), Dendrobranchiata). Gleichwohl wurde mit den Polychelida ein problematisches Taxon mit ungewissen Verwandtschaftsbeziehungen identifiziert. Auf der Eben der Unterordnungen sind die Thalassinida paraphyletisch, was mit einigen morphologischen und einigen jüngeren molekularen Studien konsistent ist, alle anderen gebräuchlichen Taxa sind monophyletisch. Es handelt sich um eine Inversion, die sich vom S-E-F tRNA cluster bis zum I-Q-M tRNA cluster erstreckt und in Procambarus fallax f. virginalis und Homarus gammarus auftritt. Im Vergleich mit dem Genarrangement des Limulus polyphemus zeigen beide Astaciden in dieser Region exakt dieselbe Inversion wie der Priapulide Priapulus caudatus, die daher innerhalb der Ecdysozoa als konvergent angenommen werden muss. Auch neben dieser Inversion innerhalb der Astacidea zeigen die Genarrangements aller verfügbaren Dekapoden mehrere interessante Eigenschaften. Um die beobachteten einzigartigen genomischen Eigenschaften zu erklären, schlage ich mit dem „inversion triggered duplication“ Model ein neues Modell für Gen-Rearrangements vor.
For a comprehensive study of decapod phylogeny at the mitochondrial genome level, I have sequenced the mitochondrial genome of 13 decapods. Together with available sequences of 31 decapods from GenBank, and the mitochondrial genome of Dromia personata provided by the Bonn University, the dataset now cover all major decapod taxa. Maximum likelihood (ML) and Bayesian inference (BI) of the nucleotide and amino acid datasets reveal similar topologies at the higher level relationships: (((((((Anomala, Brachyura), Thalassinida: Gebiidea) Thalassinida: Axiidea), Astacidea), Achelata), Stenopodidea), Caridea), Dendrobranchiata). Nevertheless, one problematic taxon, Polychelida, with ambiguous affinities is recognized. At the lower level, most taxa are monophyletic, whereas the Thalassinida is paraphyletic, which is consistent with some morphological and molecular results. An inversion spanning from S-E-F tRNA cluster to the I-Q-M tRNA cluster occurred in Procambarus fallax f. virginalis, Homarus gammarus, and one priapulid Priapulus caudatus. Compared with the gene arrangement of the horseshoe crab Limulus polyphemus, both astacids and the priapulid exhibit the same inversion, which is therefore supposed to be a convergent event of the clade Astacidea and Priapulida among Ecdysozoa. Other than this notable feature observed in astacids, the gene arrangements in all available decapods show some interesting characters. To explain these unique genomic features observed here, a new gene rearrangement model is proposed, which is called the “inversion triggered duplication” model.

Chez les plantes, la dynamique structurelle et l'évolution concertée des génomes nucléaire et cytoplasmiques restent peu documentées. L'objectif de cette thèse était d'étudier la diversité structurelle et l'évolution des mitogénomes et des plastomes chez les Oleaceae à l'aide d'une approche de phylogénomique. Nous avons d'abord assemblé des mitogénomes à partir de données de séquençage de faible couverture, obtenues à partir de matériel frais et d'herbier. Une grande variation de structure du mitogénome a été observée chez l'olivier, et un gène chimérique potentiellement associé à un type de stérilité mâle a été identifié. Enfin, nous avons étudié l'évolution des plastomes et des gènes mitochondriaux chez les Oleaceae. Une accélération de l'évolution du plastome a été observée dans deux lignées indépendantes. Ce changement de trajectoire évolutive pourrait être la conséquence d'une transmission occasionnelle de plastes par le pollen, modifiant les pressions sélectives sur certains gènes
In plants, the structural dynamics and concerted evolution of nuclear and cytoplasmic genomes are poorly understood. The objective of this thesis was to study the structural diversity and evolution of mitogenomes and plastomes in the family Oleaceae with a phylogenomic approach. First, we assembled mitogenomes from low-coverage sequencing data obtained from live and herbarium material. Considerable structural variation of mitogenomes was observed in the olive, and a chimeric gene potentially associated to a type of male sterility was detected. Finally, we studied the evolution of plastomes and mitochondrial genes in the Oleaceae. Accelerated evolution of plastomes was observed in two independent lineages. This change of evolutionary rate could be the consequence of an occasional transmission of plastids with pollen, modifying selective pressures on some genes

As one of only two surviving outgroups to all jawed vertebrates, lampreys (Petromyzontiformes) can provide important information in our understanding of early vertebrate evolution. However, with many phylogenetic aspects of lamprey evolution still uncertain, the ability to use contemporary lampreys in this role depends on robust phylogenetic hypotheses regarding the interrelationship of the three lamprey families, as well as the relationship between lampreys and hagfishes. To achieve this, complete mitogenome data of Southern Hemisphere lampreys is required. Another contentious issue in lamprey taxonomy is the status of paired species. Whilst many studies have focused on Northern Hemisphere species pairs, this study is the first to compare Mordacia mordax and Mordacia praecox, two lamprey species endemic to Australia and the only species pair in the Southern Hemisphere. The complete mitochondrial genome of Mordacia mordax and Mordacia praecox was determined twice independently, in a single shotgun sequencing run on an Ion Torrent PGM, and using a combination of Sanger sequencing of short range PCR products and Roche 454 GS Junior pyrosequencing of long range PCR products. Both of the mitogenomes contain the 37 typical vertebrate genes. Their gene order and contents are identical to those of previously described lamprey mitogenomes, with the exception of a novel tandem repeat array located between Cyt b and tRNA proline. The tandem repeat array, referred to as NCIII, contains pseudogenes of tRNA proline and phenylalanine, indicating that it has arisen by tandem duplication of the tRNA proline – phenylalanine region. Characterisation of NCIII revealed that the number of repeat copies was polymorphic between individuals of both species, and was a source of both intra-individual and inter-individual variation. Consistent with other studies of lamprey species pairs, the mitogenome of M. mordax and M. praecox are nearly identical. Phylogenetic analyses were carried out using the newly determined mitogenomes, together with five additional lamprey species and two hagfishes. Most tree topologies obtained strongly support the hypothesis that Petromyzontidae plus Geotriidae are a clade whose sister group is Mordaciidae. Additionally, lamprey divergence times were estimated by a temporally-calibrated phylogenetic analysis that included 20 vertebrate mitogenomes and was done using nine well-established fossil calibration points. The recovered topology strongly supported the hypothesis that lampreys separated from hagfishes about 409 MYA, and that lamprey divergence involved the early radiation of Mordaciidae (about 132 MYA), followed by the monophyletic divergence of Geotriidae plus Petromyzontidae about 85 MYA. Taken together, the results in this study provide robust hypotheses regarding the interrelationship of lamprey families and the relationship between hagfish and lampreys, whilst providing an estimation of their divergence times.

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Formigas cortadeiras do gênero Atta, popularmente conhecidas como saúvas, são as mais derivadas dentro da tribo Attini. Apresentam grande importância ecológica, porém, pelo hábito de cortarem folhas para manutenção do fungo simbionte e pelo enorme tamanho das colônias, causam muitos prejuízos às lavouras, pastagens e plantações, sendo consideradas pragas agrícolas. Atta laevigata Smith, 1858 apresenta vasta distribuição pelo Brasil e é responsável pela herbivoria de inúmeras plantas dicotiledôneas, gramíneas e espécies nativas de diferentes biomas. O presente trabalho teve como objetivos a caracterização parcial do transcriptoma e do genoma mitocondrial de A. laevigata. Foram caracterizadas 2006 sequências únicas do transcriptoma, a partir de uma biblioteca de cDNA preparada com indivíduos inteiros da formiga. Entre essas sequências, 16 provavelmente representam genes com grande número de transcritos. Esses 16 genes estão relacionados a três funções celulares: (i) conservação de energia através de reações redox na mitocôndria; (ii) estrutural, pelo citoesqueleto e músculos; (iii) regulação da expressão gênica e metabolismo. Considerando o estilo de vida e processos biológicos chaves para essas formigas, 146 sequências foram identificadas com base na sua utilização para o controle de cortadeiras pragas. A partir de dados da biblioteca de cDNA e procedimentos envolvendo primer walking, o genoma mitocondrial de A. laevigata foi parcialmente caracterizado, apresentandose com 17920 pb, maior, portanto, do que outros já descritos em Hymenoptera, mesmo considerando-se a impossibilidade de determinação da sequência de uma pequena porção do mtDNA, envolvendo a região controle, uma parte do 12S e os tRNAs S1, V e M. Como já descrito para outros mitogenomas, o de A. laevigata apresentou alto conteúdo AT, os mesmos 13 genes codificadores...
Leafcutter ants from Atta genus, popularly known as “saúvas”, are the most derived of the tribe Attini. They have major ecological importance, but, because of their habit of cutting leaves for the maintenance of the symbiotic fungus and the huge colony size, they impose severe economic damages to plantations, pastures, and agriculture, being considered as agriculture pests. Atta laevigata shows wide distribution in Brazil and it is responsible for the herbivory of many dicots, grass, and native species from different biomes. The present work aimed to characterize the transcriptome and the mitochondrial genome of A. laevigata. 2,006 unique sequences of the transcriptome were characterized from a cDNA library constructed with whole individuals. Among those sequences, 16 are likely from genes with high number of transcripts. Those 16 genes are related with three cellular functions: (i) energy conservation through redox reactions in mitochondria; (ii) cytoskeleton and muscle structuring; (iii) regulation of gene expression and metabolism. Based on lifestyle and key biological processes of these ants, 146 sequences were identified with potential use for controlling pest leafcutters. Using data from cDNA library and primer walking proceedings, the mitochondrial genome of A. laevigata was partially characterized with 17,920 bp, being larger than the others already described for Hymenoptera. A small part of the mtDNA was not sequenced, including the control region, a portion of 12S and tRNAs S1, V, and M. As described before for other mitogenomes, A. laevigata mtDNA displayed high AT contain, the same 13 proteincoding genes and the two ribosomal subunits with length and location according to the hypothetic ancestral mitogenome. Rearrangements were found for the tRNAs, but the most remarkable difference were the high number and longer length of intergenic regions presented in the mtDNA... (Complete abstract click electronic access below)

The New World Junonia butterflies include well-studied model organisms yet their phylogeny remains unresolved by traditional cox1 DNA barcodes. Sixteen Junonia mitochondrial genomes were sequenced using next generation MiSeq technology. Junonia lemonias, an Old World species, has mitochondrial genome features typical of Ditrysian Lepidoptera, and synteny is maintained throughout Junonia. Analysis of Junonia mitogenomes produced a robust phylogeny that was used with biogeographic information to infer that Junonia crossed the Pacific Ocean to invade the New World on 3 separate occasions. Junonia vestina, a high elevation species from the Andes Mountains, shows high altitude adaptation in the mitochondrial protein coding loci atp6, atp8, cox1, cob, nad1, and nad2, with the strongest effects seen in cox1 and nad1. There is some overlap between these genes with human loci that have disease associations with the same amino acid positions which could help elucidate the function of high elevation mutations in J. vestina.
February 2016

The basking shark, Cetorhinus maximus, has historically been a target of international fisheries, leading to well-documented declines in parts of its global distribution. Currently, the basking shark is listed as globally ‘Vulnerable’ and regionally ‘Endangered’ (North Pacific and Northeast Atlantic) on the IUCN Red List of Threatened Species, rendering the species an international conservation priority. Here, we assessed the global matrilineal genetic population structure and evolutionary history of the basking shark by completing the first whole mitochondrial genome sequence level survey of animals sampled from three globally widespread geographic regions: the western North Atlantic (n = 11), the eastern North Atlantic (n = 11), and within New Zealand territorial waters (n = 12). Despite the relatively large amount of sequence data assessed (~16,669 bp per individual), whole mitogenome analyses showed no evidence of population differentiation (ΦST = -0.047, P > 0.05) and very low nucleotide diversity (π = 0.0014 ± 0.000) across a global seascape. The absence of population structure across large distances and even between ocean basins is indicative of long-dispersal by this species, including an ability to cross known biogeographic barriers known to differentiate populations of other highly vagile pelagic fishes. Notably, evolutionary analyses of the mitogenome sequences revealed two globally sympatric but evolutionary divergent lineages, with a Bayesian framework estimated coalescence time of ~2.46 million years ago. Coalescent-based Bayesian skyline analysis uncovered subtle evidence of Pleistocene demographic flux for this species, including a potential decline in female effective population size. Thus, historical population changes may be responsible for the occurrence of the two highly divergent, yet sympatric lineages, as population declines may have resulted in the loss of intermediate haplotypes and resulted in an overall loss of genetic diversity. This work supports the recognition of basking sharks as a single matrilineal global population, and as such requires the application of a cooperative multiagency and international approach to fisheries management to conserve this highly vulnerable and ecologically unique species.

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FACEPE
A ordem Lepidoptera apresenta uma gama de espécimes de importância econômica, algumas são agentes polinizadores e outras são pragas. Vários estudos têm sido realizados com base na morfologia com base em dados moleculares para elucidar a evolução da ordem. O DNA mitocondrial é muito utilizado por proporcionar boa resolução filogenética. Com base em genes mitocondriais informativos, nós propusemos um conjunto de dados que pode ser utilizado em análise filogenética de Lepidoptera obtendo a mesma robustez que a análise com mtDNAs completos. Para isso, as sequências dos mitogenomas de Lepidoptera foram recuperadas no banco de dados do NCBI. Foi identificada a ordem gênica das sequências utilizando o programa MAUVE. As regiões de interesse em D. flavipennella foram sequenciadas para testar a eficiência dos marcadores moleculares em sequência nova. Foi realizada análise de entropia, teste de sinal filogenético e de saturação para verificar características de bons marcadores moleculares e foram realizadas análises filogenéticas nos programas PhyML e MrBayes. Foram realizados também testes com e sem a terceira posição dos códons para verificar a influência da terceira posição nas análises filogenéticas de Lepidoptera. As regiões estudadas foram concatenadas para aumentar os valores de confiança das árvores. Verificamos que com a concatenação dos genes COI, ATP6, COIII, ND3, ND5, CYTB, ND1 e 16S foi possível obter resultados com robustez semelhante a dos mitogenomas completos.

Parmi les grillons de la sous-famille Eneopterinae, Cardiodactylus Saussure, 1878 (Orthoptera : Eneopterinae : Lebinthini) est le genre avec le plus grand nombre d’espèces et le plus largement distribué. Il comprend 82 espèces organisées en deux groupes d'espèces (groupes Noveaguineae et Efordi). L’aire de distribution des espèces englobe des centaines d'îles et de territoires allant des côtes de l'Asie du Sud-Est aux îles Samoa. Les espèces de Cardiodactylus présentent des patrons de distribution contrastés, certaines étant fortement endémiques tandis que d'autres sont distribuées dans toute la région du Pacifique occidental. Cette diversité, conjuguée au riche contexte géologique des régions de l'Asie du Sud-Est et du Pacifique, offre de grandes opportunités pour aborder des questions biogéographiques et étudier la dynamique de la diversification insulaire. Malgré les efforts récents pour améliorer les connaissances taxonomiques de Cardiodactylus, la diversité du genre est telle que de nombreuses nouvelles espèces restent encore à découvrir. Dans un premier temps, des études taxonomiques fondées sur des données morphologiques, moléculaires et acoustiques ont permis de décrire cinq nouvelles espèces de Nouvelle-Guinée orientale et de redécrire deux espèces. Dans un second temps, la phylogénie moléculaire de Cardiodactylus a été reconstruite. Les résultats soutiennent la monophylie du genre, des espèces testées et du groupe d'espèces Novaeguineae. Le groupe d'espèces Efordi est clairement paraphylétique, ce qui tend à confirmer la présence d'un troisième groupe d'espèces, comme le suggère l'étude taxonomique. Selon l'analyse de biogéographie historique, l'origine de Cardiodactylus se situe dans le Pacifique Ouest au cours de l'Éocène moyen (environ 42 Ma). Cardiodactylus a ensuite colonisé l'Asie du Sud-Est depuis l'Est vers l’Ouest via une zone de transition correspondant à la Nouvelle-Guinée, et ce en empruntant trois chemins indépendants : Java, Sulawesi et les Philippines. Ce travail montre que la grande diversité de Cardiodactylus en Asie du Sud-Est s’est mise en place au Miocène à travers l'accumulation d’événements de diversification in situ et d'événements d’immigration. Dans un troisième temps, un protocole et des jeux d'amorces ont été mis au point pour amplifier les mitogénomes de grillons. La méthode a été illustrée par l'amplification du mitogénome de l'espèce Cardiodactylus muiri Otte, 2007 par une approche de PCR longue associée au séquençage de nouvelle génération. Le but de ce travail est d'obtenir des données moléculaires plus informatives à faible coût, mais avec une couverture taxonomique élevée pour de futures reconstructions phylogénétiques
Among the crickets of the subfamily Eneopterinae, Cardiodactylus Saussure, 1878 (Orthoptera: Eneopterinae: Lebinthini) is the most speciose and widely distributed genus. It consists of 82 species organized in two species groups (Noveaguineae and Efordi species groups). The species distributions encompass hundreds of islands and territories ranging from the coasts of Southeast Asia to the Samoan islands. Cardiodactylus species show contrastive distribution patterns, some being highly endemic while others are distributed in the whole Western Pacific region. This diversity coupled with the rich geological context of the Southeast Asian and Pacific regions, offers great opportunities to address biogeographical questions and investigate the dynamics of diversification in islands.Despite recent efforts to improve the taxonomical knowledge of Cardiodactylus, its diversity is so high that many new species still continue to be discovered. In a first stage, taxonomic studies based on morphological, molecular and acoustic data led to the description of five new species from Eastern New Guinea and to the redescription of two species. In a second stage, the molecular phylogeny of Cardiodactylus was reconstructed. The results support the monophyly of the genus, of the tested species, and of the Novaeguineae species group. The Efordi species group is clearly found paraphyletic, which tends to confirm the presence of a third species group, as suggested by the taxonomical study. According to the historical biogeographic analysis, the origin of Cardiodactylus was recovered in the Western Pacific during the Mid-Eocene (ca. 42 Ma). Through the transition zone of New Guinea, Cardiodactylus colonized Southeast Asia from East to West through three independent passageways: Java, Sulawesi and the Philippines. This work showed that the high diversity of Southeast Asian Cardiodactylus took place during the Miocene, as a result of accumulation of in situ diversification and immigration events. In a third stage, a protocol and sets of primers were designed to amplify mitogenomes in crickets. The method was exemplified by amplifying the mitogenome of the species Cardiodactylus muiri Otte, 2007 with a long-PCR approach combined with Next-Generation Sequencing. The purpose of this work is to obtain more informative molecular data with relatively low-cost but high taxonomic coverage for future phylogenetic reconstructions

Resumo: Formigas cortadeiras do gênero Atta, popularmente conhecidas como saúvas, são as mais derivadas dentro da tribo Attini. Apresentam grande importância ecológica, porém, pelo hábito de cortarem folhas para manutenção do fungo simbionte e pelo enorme tamanho das colônias, causam muitos prejuízos às lavouras, pastagens e plantações, sendo consideradas pragas agrícolas. Atta laevigata Smith, 1858 apresenta vasta distribuição pelo Brasil e é responsável pela herbivoria de inúmeras plantas dicotiledôneas, gramíneas e espécies nativas de diferentes biomas. O presente trabalho teve como objetivos a caracterização parcial do transcriptoma e do genoma mitocondrial de A. laevigata. Foram caracterizadas 2006 sequências únicas do transcriptoma, a partir de uma biblioteca de cDNA preparada com indivíduos inteiros da formiga. Entre essas sequências, 16 provavelmente representam genes com grande número de transcritos. Esses 16 genes estão relacionados a três funções celulares: (i) conservação de energia através de reações redox na mitocôndria; (ii) estrutural, pelo citoesqueleto e músculos; (iii) regulação da expressão gênica e metabolismo. Considerando o estilo de vida e processos biológicos chaves para essas formigas, 146 sequências foram identificadas com base na sua utilização para o controle de cortadeiras pragas. A partir de dados da biblioteca de cDNA e procedimentos envolvendo primer walking, o genoma mitocondrial de A. laevigata foi parcialmente caracterizado, apresentandose com 17920 pb, maior, portanto, do que outros já descritos em Hymenoptera, mesmo considerando-se a impossibilidade de determinação da sequência de uma pequena porção do mtDNA, envolvendo a região controle, uma parte do 12S e os tRNAs S1, V e M. Como já descrito para outros mitogenomas, o de A. laevigata apresentou alto conteúdo AT, os mesmos 13 genes codificadores... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Leafcutter ants from Atta genus, popularly known as "saúvas", are the most derived of the tribe Attini. They have major ecological importance, but, because of their habit of cutting leaves for the maintenance of the symbiotic fungus and the huge colony size, they impose severe economic damages to plantations, pastures, and agriculture, being considered as agriculture pests. Atta laevigata shows wide distribution in Brazil and it is responsible for the herbivory of many dicots, grass, and native species from different biomes. The present work aimed to characterize the transcriptome and the mitochondrial genome of A. laevigata. 2,006 unique sequences of the transcriptome were characterized from a cDNA library constructed with whole individuals. Among those sequences, 16 are likely from genes with high number of transcripts. Those 16 genes are related with three cellular functions: (i) energy conservation through redox reactions in mitochondria; (ii) cytoskeleton and muscle structuring; (iii) regulation of gene expression and metabolism. Based on lifestyle and key biological processes of these ants, 146 sequences were identified with potential use for controlling pest leafcutters. Using data from cDNA library and primer walking proceedings, the mitochondrial genome of A. laevigata was partially characterized with 17,920 bp, being larger than the others already described for Hymenoptera. A small part of the mtDNA was not sequenced, including the control region, a portion of 12S and tRNAs S1, V, and M. As described before for other mitogenomes, A. laevigata mtDNA displayed high AT contain, the same 13 proteincoding genes and the two ribosomal subunits with length and location according to the hypothetic ancestral mitogenome. Rearrangements were found for the tRNAs, but the most remarkable difference were the high number and longer length of intergenic regions presented in the mtDNA... (Complete abstract click electronic access below)
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Doutor

L'acquisition des données moléculaires a été bouleversée par le développement des techniques de séquençage haut-débit. Celles-ci ont augmenté la quantité des données et ont fait diminuer le coût de manière considérable. Ces nouvelles approches ont également redonné accès à des sources de matériel biologique qui étaient auparavant inutilisables en raison de la faible quantité et la forte dégradation de l'ADN qu'elles fournissent, notamment des tissus anciens, des échantillons de musée voire du matériel fossile. Un avantage supplémentaire c'est la possibilité de multiplexer les échantillons, c'est-à-dire, les mélanger et les séquencer simultanément grâce à l'utilisation de « tags » ou étiquettes permettant de les séparer après avec des outils bioinformatiques. Un marqueur qui a grandement bénéficié de ces technologies est le génome mitochondrial. En effet, nous montrons que grâce au ratio élevé entre l'ADN mitochondrial et l'ADN nucléaire par cellule, il est possible l'obtention de mitogénomes entiers, avec de couvertures adéquates, sans qu'un enrichissement préalable de l'échantillon soit nécessaire. Ceci permet d'envisager la réalisation de projets de mitogénomique comparative pour de groupes riches en espèces, requérant un échantillonnage taxonomique exhaustif et dont les divergences génétiques rendrait difficile l'usage du séquençage classique. C'est dans ce contexte que cette thèse aborde la systématique, la phylogénie et l'évolution moléculaires d'une famille de chauves-souris néotropicales : les Phyllostomidae. Cette famille est riche en espèces, avec plus de 160 taxons mais aussi en traits d'histoire de vie contrastés notamment le régime alimentaire. Cette diversité résulte en des morphologies convergentes dont les caractères sont en conséquence peu appropriés pour reconstruire l'histoire évolutive de ce groupe. La mitogénomique a prouvé être un outil efficace dans ce dessein, mais à présent aucune étude de ce type a été conduite pour cette famille. Nous avons d'abord réussi à séquencer les mitogénomes de représentants de toutes les lignées majeures couvrant également la diversité de traits d'histoire de vie. Nous montrons ensuite que l'utilisation de ces mitogénomes permet de résoudre les relations de parenté au niveau intrafamilial avec une résolution similaire à celle d'une concaténation de marqueurs mitochondriaux et nucléaires avec un soutien statistique robuste pour la plupart des nœuds de la phylogénie. Ceci nous a permis de clarifier plusieurs relations qui restaient controversées dans des études précédentes et confirmer plusieurs des clades proposés par celles-ci. Ensuite, nous abordons l'évolution du mitogénome en relation avec les traits d'histoire de vie en utilisant comme exemple le clade des vampires, les seules Mammifères hématophages, dont le génome mitochondrial semble avoir été touché par une accélération du taux d'évolution comme conséquence de l'action combinée de forces neutres et sélectives pour répondre aux contraintes imposées par ce régime alimentaire. Finalement, le cadre phylogénétique robuste proportionné par les 100 mitogénomes que nous avons séquencés pourra être utilisé comme référence pour étudier la diversification des Phyllostomidae
New sequencing technologies have revolutionized the acquisition of molecular data by increasing the amount of sequences at a considerably lower cost. These new technologies have also given access to samples previously neglected because they resulted in low-quantity and degraded DNA yields, as for example, old tissues, museum specimens and even fossil rests. An additional advantage comes from the possibility of multiplexing; this is, mixing several taxa in a single sample thanks to the use of tags or labels allowing late separating the sequences using bioinformatic tools. A molecular marker that has greatly benefited from these technologies is the mitochondrial genome. Indeed, we show that, thanks to the high per-cell ratio of mitochondrial to nuclear DNA, it's possible to obtain whole well-covered mitochondrial genomes without previous sample enrichment. This allows the accomplishment of projects of comparative mitogenomics for species-rich groups needing exhaustive taxon sampling and for which strong genetic divergences would difficult the use of classical sequencing.It is in this context that this thesis tackles the molecular systematics, phylogenetics and evolution of a Neotropical family of bats: the Phyllostomidae. This species-rich family, accounting for more than 160 species, is also the family of Mammals with the highest diversity of life history traits, for example, feeding on almost every possible source of food. This diversity results in convergent morphologies that make this kind of characters inadequate for reconstructing the evolutionary history of this group. Mitogenomics has proven useful in similar cases but no study of this kind has been conducted for this family. We got to sequence whole mitogenomes for representatives of all major lineages and covering the diversity of life history traits. We then show that using these mitogenomes allows solving intrafamilial relationships with a resolution similar to that resulting from a concatenation of mitochondrial and nuclear markers and with solid statistical support for most of the nodes of the phylogeny. This allowed clarifying several controversial relationships and confirming several clades proposed in previous studies. Next, we illustrate the evolution of mitogenomes and the influence of life history traits using the clade of vampire bats, the only hematophagous Mammals, whose mitogenome seem to have undergone an acceleration of evolutionary rate as a consequence of the combined action of neutral and selective forces in order to counter the constraints imposed by this feeding habit. Finally, the robust phylogenetic frame provided by the 100 mitogenomes that we sequenced, will be used for future studies about, for exemple, the diversification process of Phyllostomids

During the last decades, mitochondria and their DNA have become a hot topic of research due to their essential roles which are necessary for cells survival and pathology. In this study, multiple methods have been developed to help with the understanding of mitochondrial DNA and its evolution. These methods tackle two essential problems in this area: the accurate annotation of protein-coding genes and mitochondrial genome rearrangements. Mitochondrial genome sequences are published nowadays with increasing pace, which creates the need for accurate and fast annotation tools that do not require manual intervention. In this work, an automated pipeline for fast de-novo annotation of mitochondrial protein-coding genes is implemented. The pipeline includes methods for enhancing multiple sequence alignment, detecting frameshifts and building protein profiles guided by phylogeny. The methods are tested on animal mitogenomes available in RefSeq, the comparison with reference annotations highlights the high quality of the produced annotations. Furthermore, the frameshift method predicted a large number of frameshifts, many of which were unknown. Additionally, an eficient partially-local alignment method to investigate genomic rearrangements in mitochondrial genomes is presented in this study. The method is novel and introduces a partially-local dynamic programming algorithm on three sequences around the breakpoint region. Unlike the existing methods which study the rearrangement at the genes order level, this method allows to investigate the rearrangement on the molecular level with nucleotides precision. The algorithm is tested on both artificial data and real mitochondrial genomic sequences. Surprisingly, a large fraction of rearrangements involve the duplication of local sequences. Since the implemented approach only requires relatively short parts of genomic sequence around a breakpoint, it should be applicable to non-mitochondrial studies as well.

Dissertação de Mestrado em Bioinformática e Aplicações às Ciências da Vida
Freshwater mussels (Bivalvia: Unionida) play key ecological roles and provide important services to humans. However, they are among the most threatened faunal groups in the world. Most freshwater mussel species present two divergent mitochondrial DNA molecules (F-type or M-type) in males as a result of Doubly Uniparental Inheritance. The number of available freshwater mussel mitogenomes is low, particularly for M-type genomes. A further shortcoming is that published mitogenomes are restricted to only a few families within this order, with no mitogenomes being available for the others. In the present dissertation, 149 samples of freshwater mussels were newly sequenced by NextGeneration Sequencing (NGS) using MiSeq Illumina runs. Quality control of the obtained reads, assembly, and annotation was carried out using multiple bioinformatics tools. Comparative mitogenomic analyses were then performed using all obtained (F-type) mitogenomes with different evaluation metrics, e.g., mitogenome length, GC content, start and stop codon frequency, AT/GC skews, and Ka/Ks ratio on protein-coding genes. The results were compared with previous analyses on molluscan mitogenomes revealing interesting patterns that can be explored in future studies. A case study was carried out in a subset of the 149 mitogenomes, focusing on the subfamily Ambleminae, for which two out of five tribes lacked published complete mitogenomes. Phylogenetic analyses were carried out on these mitogenomes using standard methods, e.g., Maximum Likelihood and Bayesian Inference. The phylogeny on the newly analysed mitogenomes, i.e., Pleurobema oviforme, Amblema plicata, and Popenaias popeii, confirm the most recent tribal classification within Ambleminae. Results also suggest that all Ambelminae share the same gene arrangement (UF1).
Os bivalves de água doce (Bivalvia: Unionida) desempenham papéis ecológicos essenciais e fornecem serviços importantes para os seres humanos. No entanto, eles são um dos grupos faunísticos mais ameaçados do mundo. A maioria dos bivalves de água doce apresentam duas moléculas de DNA mitocondrial divergentes nos machos (Fêmea ou tipo F e Macho ou Tipo M) como resultado de Herança Duplamente Uniparental. O número de mitogenomas de bivalves de água doce é baixo, particularmente para genomas tipo M. Uma outra limitação é que os mitogenomas publicados são restritos a apenas algumas famílias dentro desta ordem, sem mitogenomas disponíveis para as outras. Na presente dissertação, 149 amostras de bivalves de água doce foram sequenciadas por Sequenciação de Nova Geração (NGS) usando Illumina MiSeq. O controle de qualidade das reads, assembly e anotação foram realizadas usando várias ferramentas bioinformáticas. As análises mitogenômicas comparativas foram realizadas usando todos os mitogenomas do tipo F com diferentes métricas de avaliação, como comprimento do mitogenoma, conteúdo de GC, frequência de codões de iniciação e terminação, AT/GC skews e rácio Ka/Ks em genes codificadores de proteínas. Os resultados foram comparados com análises anteriores em mitogenomas de moluscos, revelando padrões interessantes que podem ser explorados em estudos futuros. Um estudo foi realizado num subconjunto dos 149 mitogenomas, com foco na subfamília Ambleminae, na qual duas entre cinco tribos não tinham mitogenomas completos publicados. Análises filogenéticas foram realizadas nestes mitogenomas usando métodos como Máxima Verossimilhança e Inferência Bayesiana. A filogenia nos mitogenomas analisados Pleurobema oviforme, Amblema plicata, e Popenaias popeii, confirmam a classificação tribal mais recente em Ambleminae. Os resultados revelam também que todos os mitogenomas partilham a mesma ordem dos genes (UF1).

碩士
國立中央大學
生命科學系
106
Microhyla fissipes was first recognized in Tainan, Taiwan (type locality), however it is not highly accepted. Herein, author used the sample from Yi-Lan, Taiwan as a representative of Taiwan samples and obtained the complete mitochondrial genome sequence (accession number: MF673131) in order to study the phylogeny of Microhyla fissipes and its relevants, especially in the comparison with the China sample (NC_009422). Although the genome of our sample (16,729 base pairs) contains genes and gene order almost identical to other amphibians’, some variations have been accumulated between Taiwan and China samples. Interestingly, we noticed that ND5 and ND6 genes evolved very differently from other genes. Also there were strongly codon usage bias with high thymine frequency at the second codon and very low guanine frequency at the third codon.

碩士
國立臺灣海洋大學
環境生物與漁業科學學系
103
There are 8 genus and around 90 species of Pomacanthidae in the world. The Royal angelfish or regal angelfish, Pygoplites diacanthus, is a species of marine angelfish of the family Pomacanthidae, and the monotypic genus Pygoplites. It was found in tropical Indo-Pacific oceans, ranged from East Africa, Red Sea, east to French Polynesia Tuamotu Archipelago, north to Japan and south to Australia. This species is popular in aquarium trade because of its colorful body patterns. The Royal angelfish in Indian and Pacific Oceans have has different color morph, The Indian Royal angelfish has an orange face; while Pacific one has a grey face. Whether the Royal angelfish in different Ocean represent different populations or different species is still unclear. The establishment of mitogenome and the analysis of DNA barcode may help to resolve the questions of population genetics, molecular phylogenetics, and evolution for Royal angelfishes. Firstly, we used Next Generation Sequencing (NGS) for the short reads sequencing to decode the mitogenome of Royal angelfish from Pacific (Indonesia) and Indian Ocean (Maldives). The mitogenome of Pacific Royal angelfish was totally 16,784 bp, including 13 protein coding genes, 22 transfer RNAs, 2 ribosomal RNAs and one control region; while the mitogenome of Indian Ocean Royal angelfish was 16,767 bp.The nucleotides composition is similar between two geminate species, including 28.5% A, 28.9%C, 16.3%G and 26.3%T. The mitogenome difference between these two morph types is about 1.55%. The most different region was 10% for control region, then 1.73% for ND6. The analyses of life barcode COI suggested that Royal angelfishes can be divided was into two groups. The estimated divergence time based on the molecular clock concept was about 0.5 My, it could because of the low sea level during Pleistocene ice ages, the emerge of the Indo-Australian Archipelago may form vicariance which direct to the divergence of Royal angelfish in different Oceans and led to the formation of many geminated species in Indo-Pacific.

The evolutionary origin and the phylogeny of higher metazoan taxa is still under debate although considerable progress has been made in the past 20 years. Metazoa represents a monophyletic group of highly diverse animals including Bilateria, Cnidaria, Porifera, Ctenophores, and Placozoa. Bilateria comprises the majority of metazoans and consists of three major clades: Deuterostomia, Spiralia (= Lophotrochozoa sensu lato), and Ecdysozoa, whereas the sister group taxa Spiralia and Ecdyzozoa form the monophyletic clade Protostomia. Molecular data have profoundly changed the view of the bilaterian tree of life. One of the main questions concerning bilaterian phylogeny is the on-going debate about the evolution of complexity in Bilateria. It was assumed that the last common ancestor of Deuterostomia, Ecdysozoa and Spiralia had a segmented and coelomate body organization resembling that of an annelid. On the contrary, the traditional view is the evolution of Bilateria from a simple body organization towards more complex forms, assuming that the last common ancestor of Bilateria resembles a platyhelminth-like animal without coelomic cavities and segmentation. To resolve this question, it is necessary to unravel the phylogenetic relationships within Bilateria. By using mitogenomic and phylogenomic data, this thesis had a major contribution to clarify phylogenetic relationships within problematic metazoan taxa: (1) the phylogeny of Deuterostomia, (2) the questionable monophyly of Platyzoa, and first assumptions concerning the phylogeny of Gnathostomulida, Gastrotricha and Polycladida, (3) phylogenetic relationships within annelid taxa, especially Terebelliformia, Diurodrilidae, and Syllidae, with new insights into the evolution of mitochondrial gene order, and (4) new insights into the evolution of annelids, especially the interstitial ones, as well as the colonization of the interstitial realm.

碩士
國立臺灣大學
海洋研究所
94
Animal mitochondrial genome was well studied in past decades with a closed circular structure containing 13 protein coding regions, 2 rRNAs and 22 tRNAs. Nevertheless, mitochondrial genomes of the Anthozoa are different from the typical animal mt genome in several characteristics including, losing most of the tRNAs, possessing group I introns, incompact gene arrangement, and slow evolution. Even within the major lineages of the Anthozoa, the gene containing group I intron and gene arrangement could be very different. Thus, more information should be obtained in order to address the molecular evolution of mitochondrial genome in the Anthozoa. In this study, I determined 3 mt genomes of Siderastrea corals and compared to the published mt genomes of the scleractinian corals. Mt genome gene arrangement of Siderastrea is nearly identical to the published scleractinian mt genomes except the existence of cox1 intron. Mt genomes are longer (19387-19619 bp) than those of other scleractinian corals (16138-18338 bp). Comparing the phylogenetic relationship of these corals, the loosen mt genome of Siderastrea is probably an ancestral character of the mitochondrial evolution in the Scleractinia. Cox1 group I introns were detected in Siderastrea species with a LAGLI-DGEG homing endonuclease to perform intron translocation function. Cox1 group I introns were also found in Actiniaria (Metridium senile) with the same insertion site and higher similarity than that of fungi’s cox1 intron, suggesting that the insertion event was ancient probably prior to the split of the common ancestor of Scleractinia and Actiniaria. The group I intron was probably lost several times during the course of evolution in most of the scleractinian lineages. The finding of coxI group I introns in the three Siderastrea species from the Caribbean, Indian Ocean, and Pacific Ocean suggested that this intron existed before the divergence of the common ancestor of Siderastrea. The genetic divergence was accumulated in both intron as well as the other mitochondrial regions after these species become relict and endemic in these Oceans. The result also demonstrated that the deep divergence within genus might not be caused by obscuring of conventional taxonomy. Siderastrea represents an old and reliable taxonomic group and may provide a new perspective view for evolution of the Scleractinia.

Les systèmes sexuels et les mécanismes responsables de la détermination du sexe chez les animaux sont issus de stratégies diverses. Cette incroyable diversité se reflète notamment chez les bivalves, où autant les facteurs génétiques qu’environnementaux y jouent un rôle, avec des espèces utilisant divers modes de reproduction, tels que le gonochorisme ou l’hermaphroditisme simultané ou séquentiel. La découverte la plus notable est celle d’un système de déterminisme sexuel unique qui impliquerait les mitochondries. Spécifiquement, un système de transmission sexe-spécifique de l’ADN mitochondrial, connu sous le nom de DUI (« Double Uniparental Inheritance » ou double transmission uniparentale), serait lié au maintien du gonochorisme chez certaines espèces de bivalves. La DUI implique un ADN mitochondrial qui est transmis de façon maternelle (ADNmt F) aux femelles et aux mâles, et l’autre transmis de façon paternelle (ADNmt M) aux mâles seulement. Les ADNmt F et M chez les espèces à DUI sont caractérisés par des traits uniques, comme une modification du gène cox2, ou encore la présence de nouveaux gènes associés à chacun des génomes mitochondriaux (des gènes sexe-spécifiques) qui ont une fonction autre que la production d’énergie contrairement aux autres gènes mitochondriaux typiques. Le lien entre la DUI et la détermination du sexe étant encore flou, trois approches ont été proposées pour aider à le démystifier, chacune des approches constituant un chapitre de cette thèse. Les deux premiers chapitres se sont concentrés sur des espèces de moules d’eau douce de l’ordre des Unionida, où une corrélation entre gonochorisme et DUI et hermaphroditisme et SMI (« Strictly Maternally Inheritance » ou transmission strictement maternelle) a été décrite. La première approche consistait à produire une analyse transcriptomique comparative entre les gonades mâles et femelles de deux espèces à DUI gonochoriques, Venustaconcha ellipsiformis et Utterbackia peninsularis (famille Unionidae), pour mieux comprendre les mécanismes sous-jacents à la détermination du sexe et à la DUI chez ces bivalves. Cette étude a révélé 12 000 gènes orthologues, avec 2 583 gènes différentiellement exprimés chez les deux espèces, dont les gènes Sry, Dmrt1 et Foxl2 connus pour être des éléments clés dans la détermination du sexe chez les vertébrés et d’autres bivalves. Nos résultats ont aussi été comparés avec d’autres espèces à DUI, notamment avec la palourde marine Ruditapes philippinarum, pour identifier des éléments partagés entre des espèces éloignées qui pourraient être responsables de la régulation de la DUI. Globalement, ces résultats corroborent l'hypothèse selon laquelle un mécanisme d'ubiquitination modifié pourrait être responsable de la rétention de l'ADNmt paternel chez les bivalves mâles. Les analyses ont aussi révélé que la méthylation de l'ADN pourrait être impliquée dans la régulation de la DUI. Une deuxième analyse transcriptomique comparative a été réalisée afin de discerner les mécanismes sous-jacents à la détermination du sexe et à la DUI, mais cette fois-ci entre l’espèce à DUI gonochorique U. peninsularis et l’espèce proche parente à SMI hermaphrodite U. imbecillis. Cette étude a permis de supporter l’hypothèse d’une implication des mécanismes d’ubiquitination et de méthylation dans la régulation de la DUI, ainsi que de confirmer un rôle des gènes conservés liés à la détermination du sexe également chez les bivalves hermaphrodites. Nos résultats ont également révélé de nouveaux gènes candidats ayant des rôles potentiels dans la DUI, y compris des nucléases et des facteurs impliqués dans l’autophagie / mitophagie. Finalement, afin d’identifier des éléments génétiques mitochondriaux qui pourraient faire partie des mécanismes sous-jacents à la DUI et la détermination du sexe chez les bivalves, nous avons séquencé les ADNmt F et M complets de deux nouvelles espèces à DUI de deux familles de l’ordre des Venerida, Scrobicularia plana (famille Semelidae) et Limecola balthica (famille Tellinidae). En effet, la description complète des ADNmt chez les espèces à DUI a été effectuée chez plusieurs espèces de moules d’eau douce (ordre Unionoida), mais peu d’espèces l’ont été pour les ordres Mytilida et Venerida. Ces études sont essentielles pour retracer des signatures génétiques mitochondriales partagées par différentes espèces à DUI. Nos résultats ont révélé les plus grosses différences de taille (>10kb) et de divergence nucléotidique (jusqu’à 50% de divergence) entre les ADNmt M et F, parmi toutes les espèces à DUI. Ces différences de taille sont principalement dues à une immense insertion (>3.5kb) dans la séquence du gène cox2 du génome mitochondrial M, chez nos deux espèces, un trait précédemment décrit chez les moules d’eau douce. Le gène cox2 des mâles de S. plana est la plus longue séquence à travers le règne animal. Une autre fonctionnalité importante portés par les ADNmt F et M est la présence de nouveaux gènes spécifiques au sexe, comme reportée chez toutes les autres espèces à DUI jsuqu’à maintenant. Les résultats combinés de cette thèse soutiennent le partage de plusieurs éléments génétiques clés entre les espèces à DUI. De plus, un parallèle avec le système CMS (« Cytoplasmic Male Sterility » ou stérilité cytoplasmique mâle) chez les plantes, les seuls autres organismes possédant un déterminisme sexuel qui implique les mitochondries, est proposé pour expliquer le rôle de l’ADNmt dans la détermination du sexe chez les espèces de bivalves à DUI.
Sexual systems and sex determining mechanisms described among animals are extraordinarily diverses. This amazing diversity is present in bivalves where both environment and genetic factors occur, leading to, among others, gonochoric and simultaneous or sequential hermaphroditic species. The most impressive discovery is a sex-determining system that would involve mitochondria. Specifically, a unique mitochondrial DNA inheritance system, known as Doubly Uniparental Inheritance (DUI), would be related to the maintenance of gonochorism in some bivalve species. DUI involves two mitochondrial DNA lineages, one that is maternally transmitted (F mtDNA) to females and males, and the other that is transmitted paternally (M mtDNA) to males only. The F and M mtDNAs, in DUI species, are characterized by unique traits, such as a modification of the cox2 gene, or the presence of new genes associated with each of the mitochondrial genomes (sex-specific genes) that have a function other than energy production, unlike other typical mitochondrial genes. Since the link between DUI and sex determination is still unclear, three approaches have been proposed to help demystify it, with each of the approaches constituting a chapter of this thesis. The first two chapters focused on freshwater mussel species of the order Unionida, where a correlation between gonochorism and DUI and hermaphroditism and SMI (Strictly Maternally Inheritance) was described. The first approach was to produce a comparative transcriptomic analysis between the male and female gonads of two gonochoric DUI species; Venustaconcha ellipsiformis and Utterbackia peninsularis (Unionidae family), to better understand the mechanisms underlying sex determination and DUI in these bivalves. This study revealed 12,000 orthologous genes, with 2 583 genes differentially expressed in both species, including Sry, Dmrt1, and Foxl2 known to be key sex-determining genes in vertebrates and other bivalve species. Our results were also compared with other DUI species, including the marine clam Ruditapes philippinarum, to identify shared elements between distant species that may be responsible for DUI regulation. Overall, these results support the hypothesis that a modified ubiquitination mechanism may be responsible for the retention of paternal mtDNA in male bivalves. The analyzes also revealed that DNA methylation could be involved in DUI regulation. 7 A second comparative transcriptomic analysis was performed to discern the mechanisms underlying sex determination and DUI between the gonochoric DUI species, U. peninsularis, and the closely related SMI hermaphroditic species, U. imbecillis. This study supported the hypothesis of an involvement of ubiquitination and methylation mechanisms in DUI regulation, as well as confirmed a role of conserved genes related to sex determination in hermaphroditic bivalves. Our results also revealed novel candidate genes with potential roles in DUI, including nucleases and factors involved in autophagy / mitophagy mechanisms. Finally, to identify mitochondrial genetic elements that could be part of the mechanisms underlying DUI and sex determination in bivalves, we sequenced the complete F and M mtDNAs of two new DUI species, from two families of the order Venerida; Scrobicularia plana (Semelidae family) and Limecola balthica (Tellinidae family). The complete description of mtDNAs in DUI species has been carried out for several species of freshwater mussels (Unionoida order), but very few species have been described for the orders Mytilida and Venerida. Such studies are essential for tracing mitochondrial genetic signatures shared by different DUI species. Our results revealed the largest differences in size (>10kb) and nucleotide divergence (up to 50% divergence) between M and F mtDNAs, among all DUI species. These differences in size are mainly due to a huge insertion (> 3.5kb) in the cox2 gene of the M mtDNA from both species, a trait previously described in freshwater mussels. The cox2 gene in S. plana males represents the longest cox2 sequence across the animal kingdom. Another important feature of F and M mtDNAs is the presence of new sex-specific genes, as reported in all other DUI species so far. The combined results of this thesis support the sharing of several key genetic elements among DUI species. In addition, a parallel with the Cytoplasmic Male Sterility (CMS) system in plants, the only other organisms with a sex determination system that involves mitochondria, is proposed to explain the role of mtDNA in sex determination in DUI bivalve species.

Les champignons mycorhiziens arbusculaires (CMA) sont très répandus dans le sol où ils forment des associations symbiotiques avec la majorité des plantes appelées mycorhizes arbusculaires. Le développement des CMA dépend fortement de la plante hôte, de telle sorte qu'ils ne peuvent vivre à l'état saprotrophique, par conséquent ils sont considérés comme des biotrophes obligatoires. Les CMA forment une lignée évolutive basale des champignons et ils appartiennent au phylum Glomeromycota. Leurs mycélia sont formés d’un réseau d’hyphes cénocytiques dans lesquelles les noyaux et les organites cellulaires peuvent se déplacer librement d’un compartiment à l’autre. Les CMA permettent à la plante hôte de bénéficier d'une meilleure nutrition minérale, grâce au réseau d'hyphes extraradiculaires, qui s'étend au-delà de la zone du sol explorée par les racines. Ces hyphes possèdent une grande capacité d'absorption d’éléments nutritifs qui vont être transportés par ceux-ci jusqu’aux racines. De ce fait, les CMA améliorent la croissance des plantes tout en les protégeant des stresses biotiques et abiotiques. Malgré l’importance des CMA, leurs génétique et évolution demeurent peu connues. Leurs études sont ardues à cause de leur mode de vie qui empêche leur culture en absence des plantes hôtes. En plus leur diversité génétique intra-isolat des génomes nucléaires, complique d’avantage ces études, en particulier le développement des marqueurs moléculaires pour des études biologiques, écologiques ainsi que les fonctions des CMA. C’est pour ces raisons que les génomes mitochondriaux offrent des opportunités et alternatives intéressantes pour étudier les CMA. En effet, les génomes mitochondriaux (mt) publiés à date, ne montrent pas de polymorphismes génétique intra-isolats. Cependant, des exceptions peuvent exister. Pour aller de l’avant avec la génomique mitochondriale, nous avons besoin de générer beaucoup de données de séquençages de l’ADN mitochondrial (ADNmt) afin d’étudier les méchanismes évolutifs, la génétique des population, l’écologie des communautés et la fonction des CMA. Dans ce contexte, l’objectif de mon projet de doctorat consiste à: 1) étudier l’évolution des génomes mt en utilisant l’approche de la génomique comparative au niveau des espèces proches, des isolats ainsi que des espèces phylogénétiquement éloignées chez les CMA; 2) étudier l’hérédité génétique des génomes mt au sein des isolats de l’espèce modèle Rhizophagus irregularis par le biais des anastomoses ; 3) étudier l’organisation des ADNmt et les gènes mt pour le développement des marqueurs moléculaires pour des études phylogénétiques. Nous avons utilisé l’approche dite ‘whole genome shotgun’ en pyroséquençage 454 et Illumina HiSeq pour séquencer plusieurs taxons de CMA sélectionnés selon leur importance et leur disponibilité. Les assemblages de novo, le séquençage conventionnel Sanger, l’annotation et la génomique comparative ont été réalisés pour caractériser des ADNmt complets. Nous avons découvert plusieurs mécanismes évolutifs intéressant chez l’espèce Gigaspora rosea dans laquelle le génome mt est complètement remanié en comparaison avec Rhizophagus irregularis isolat DAOM 197198. En plus nous avons mis en évidence que deux gènes cox1 et rns sont fragmentés en deux morceaux. Nous avons démontré que les ARN transcrits les deux fragments de cox1 se relient entre eux par épissage en trans ‘Trans-splicing’ à l’aide de l’ARN du gene nad5 I3 qui met ensemble les deux ARN cox1.1 et cox1.2 en formant un ARN complet et fonctionnel. Nous avons aussi trouvé une organisation de l’ADNmt très particulière chez l’espèce Rhizophagus sp. Isolat DAOM 213198 dont le génome mt est constitué par deux chromosomes circulaires. En plus nous avons trouvé une quantité considérable des séquences apparentées aux plasmides ‘plasmid-related sequences’ chez les Glomeraceae par rapport aux Gigasporaceae, contribuant ainsi à une évolution rapide des ADNmt chez les Glomeromycota. Nous avons aussi séquencé plusieurs isolats de l’espèces R. irregularis et Rhizophagus sp. pour décortiquer leur position phylogénéque et inférer des relations évolutives entre celles-ci. La comparaison génomique mt nous montré l’existence de plusieurs éléments mobiles comme : des cadres de lecture ‘open reading frames (mORFs)’, des séquences courtes inversées ‘short inverted repeats (SIRs)’, et des séquences apparentées aux plasimdes ‘plasmid-related sequences (dpo)’ qui impactent l’ordre des gènes mt et permettent le remaniement chromosomiques des ADNmt. Tous ces divers mécanismes évolutifs observés au niveau des isolats, nous permettent de développer des marqueurs moléculaires spécifiques à chaque isolat ou espèce de CMA. Les données générées dans mon projet de doctorat ont permis d’avancer les connaissances fondamentales des génomes mitochondriaux non seulement chez les Glomeromycètes, mais aussi de chez le règne des Fungi et les eucaryotes en général. Les trousses moléculaires développées dans ce projet peuvent servir à des études de la génétique des populations, des échanges génétiques et l’écologie des CMA ce qui va contribuer à la compréhension du rôle primorial des CMA en agriculture et environnement.
Arbuscular mycorrhizal fungi (AMF) are the most widespread eukaryotic symbionts, forming mutualistic associations known as Arbuscular Mycorrhizae with the majority of plantroots. AMF are obligate biotrophs belonging to an ancient fungal lineage of phylum Glomeromycota. Their mycelia are formed by a complex network made up of coenocytic hyphae, where nuclei and cell organelles can freely move from one compartment to another. AMF are commonly acknowledged to improve plant growth by enhancing mineral nutrient uptake, in particular phosphate and nitrate, and they confer tolerance to abiotic and biotic stressors for plants. Despite their significant roles in ecosystems, their genetics and evolution are not well understood. Studying AMF is challenging due to their obligate biotrophy, their slow growth, and their limited morphological criteria. In addition, intra-isolate genetic polymorphism of nuclear DNA brings another level of complexity to the investigation of the biology, ecology and function of AMF. Genetic polymorphism of nuclear DNA within a single isolate limits the development of efficient molecular markers mainly at lower taxonomic levels (i.e. the inter-isolate level). Instead, mitochondrial (mt) genomics have been used as an attractive alternative to study AMF. In AMF, mt genomes have been shown to be homogeneous, or at least much less polymorphic than nuclear DNA. However, by generating large mt sequence datasets we can investigate the efficiency and usefulness of developing molecular marker toolkits in order to study the dynamic and evolutionary mechanisms of AMF. This approach also elucidates the population genetics, community ecology and functions of Glomeromycota. Therefore, the objectives of my Ph.D. project were: 1) To investigate mitochondrial genome evolution using comparative mitogenomic analyses of closely related species and isolates as well as phylogenetically distant taxa of AMF; 2) To explore mt genome inheritance among compatible isolates of the model AMF Rhizophagus irregularis through anastomosis formation; and 3) To assess mtDNA and mt genes for marker development and phylogenetic analyses. We used whole genome shotgun, 454 pyrosequencing and HiSeq Illimina to sequence AMF taxa selected according to their importance and availability in our lab collections. De novo assemblies, Sanger sequencing, annotation and comparative genomics were then performed to characterize complete mtDNAs. We discovered interesting evolutionary mechanisms in Gigaspora rosea: 1) we found a fully reshuffled mt genome synteny compared to Rhizaphagus irregularis DAOM 197198; and 2) we discovered the presence of fragmented cox1 and rns genes. We demonstrated that two cox1 transcripts are joined by trans-splicing. We also reported an unusual mtDNA organization in Rhizophagus sp. DAOM 213198, whose mt genome consisted of two circular mtDNAs. In addition, we observed a considerably higher number of mt plasmidrelated sequences in Glomeraceae compared with Gigasporaceae, contributing a mechanism for faster evolution of mtDNA in Glomeromycota. We also sequenced other isolates of R. irregularis and Rhizophagus sp. in order to unravel their evolutionary relationships and to develop molecular toolkits for their discrimination. Comparative mitogenomic analyses of these mtDNAs revealed the occurrence of many mobile elements such as mobile open reading frames (mORFs), short inverted repeats (SIRs), and plasmid-related sequences (dpo) that impact mt genome synteny and mtDNA alteration. All together, these evolutionary mechanisms among closely related AMF isolates give us clues for designing reliable and efficient intra- and inter-specific markers to discriminate closely related AMF taxa and isolates. Data generated in my Ph.D. project advances our knowledge of mitochondrial genomes evolution not only in Glomeromycota, but also in the larger framework of the Fungal kingdom and Eukaryotes in general. Molecular toolkits developed in this project will offer new opportunities to study population genetics, genetic exchanges and ecology of AMF. In turn, this work will contribute to understanding the role of these fungi in nature, with potential applications in both agriculture and environmental protection.