Made available in DSpace on 2017-07-10T14:17:13Z (GMT). No. of bitstreams: 1 Dissertacao PPRN Mariane_ Gavazzoni 2016.pdf: 1443094 bytes, checksum: 5fe99142fa5f6e62c58e029f07ceeebb (MD5) Previous issue date: 2016-02-22 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Astyanax is a polyphyletic taxon with richness of species and wide geographic distribution. Astyanax comprises species that are morphologically very similar with poorly detailed taxonomic delimitations, which makes it difficult to identify and to establish phylogenetic relationships. In order to provide data to contribute to cytogenetic, taxonomy and systematics of Astyanax, cytogenetics analyzes were carried out on ten Astyanax species from three river basins. Astyanax altiparanae and A. aff. fasciatus were collected on the Upper Paraná River basin; A. abramis and A. asuncionensis were collected on the Middle-low Paraná River basin; and A. cf. aramburui, A. eigenmanniorum, A. aff. fasciatus, A. jacuhiensis, A. aff. laticeps, A. cf. paris and Astyanax sp. were collected on the Upper-Middle Uruguai River basin. The results show interespecific variation in diploid number, 2n=46 chromosomes for A. cf. aramburui and A. aff. fasciatus, 2n=48 chromosomes for A. eigenmanniorum, and 2n=50 chromosomes for the remaining species. NORs (Ag-staining and 18S rDNA-FISH) showed species bearing single sites and species bearing multiple sites (up to 10 cistrons in Astyanax sp.), confirming the high variability reported for the genus. The species from A. bimaculatus complex (A. abramis, A. altiparanae, A. jacuhiensis e A. asuncionensis) showed single NORs, a plesiomorphic condition for the complex. FISH with 5S rDNA probes revealed a more conserved condition, with centromeric sites in at least one metacentric chromosome pair and one subtelocentric/acrocentric chromosome pair, however with interspecific variation, which proves it to be an important marker in the characterization and differentiation of these species. Heterochromatin distribution pattern was distinct for all species, except for A. cf. aramburui and A. aff. fasciatus (Ijuí River). This demonstrates that cytogenetic similarities may indicate closer relationship between each other than among to the other analyzed species; on the other hand, 5S rDNA genes showed to be important in differentiation of these cryptic species. The results reported the first cytogenetic data for A. cf. paris and reinforce their cytogenetic similarity with other congenus species, and we also report the occurrence of a Astyanax species that has not been taxonomically described yet. Thus, the results of this study provide data that assist taxonomy and systematic of Astyanax clade and Astyanax paris clade , reinforcing the need for extensive revisions, especially in A. bimaculatus and A. fasciatus complex, including markers such as 5S rRNA and heterochromatin distribution pattern, forthe better understanding of the phylogenetic relationships in Astyanax. / Astyanax é um taxon polifilético com grande riqueza de espécies e ampla distribuição geográfica. Compreende espécies com formas bastante semelhantes e delimitações taxonômicas pouco detalhadas que dificultam a identificação e o estabelecimento das relações filogenéticas. Com o objetivo de fornecer dados que contribuam com a citogenética, taxonomia e sistemática de Astyanax, foram realizadas análises citogenéticas em dez espécies de Astyanax de três bacias hidrográficas. Foram coletados exemplares de Astyanax altiparanae e A. aff. fasciatus na bacia do Alto rio Paraná; A. abramis e A. asuncionensis na bacia do Médio-Baixo rio Paraná; e A. cf. aramburui, A. eigenmanniorum, A. aff. fasciatus, A. jacuhiensis, A. aff. laticeps, A. cf. paris e Astyanax sp. na bacia do Alto-Médio rio Uruguai. Os resultados mostraram variação interespecífica no número diplóide, de 2n=46 cromossomos em A. cf. aramburui e A. aff. fasciatus, 2n=48 cromossomos em A. eigenmanniorum, e 2n=50 cromossomos nas demais espécies. As AgRONs, confirmadas pela 18S rDNA-FISH, evidenciaram espécies portando sítios simples e espécies portando sítios múltiplos (até 10 cístrons em Astyanax sp.), confirmando a alta variabilidade encontrada no gênero. Nas espécies do complexo A. bimaculatus (A. abramis, A. altiparanae, A. jacuhiensis e A. asuncionensis), foram evidenciadas RONs simples, característica plesiomórfica para o complexo. FISH com sonda de 5S rDNA evidenciou uma condição mais conservada, com cístrons centroméricos em pelo menos um par de cromossomos metacêntricos e em um par de cromossomos subtelocêntricos/acrocêntricos, porém com variação interespecífica, demostrando ser um importante marcador na caracterização e diferenciação destas espécies. O padrão de distribuição da heterocromatina mostrou-se distinto para as espécies, com exceção de A. cf. aramburui e A. aff. fasciatus (rio Ijuí), onde foi verificado semelhanças citogenéticas que podem indicar maior proximidade entre estas espécies quando comparadas com as demais analisadas, sendo que a distribuição dos genes 5S rDNA se mostrou importante na diferenciação destas espécies crípticas. Os resultados relatam os primeiros dados citogenéticos para A. cf. paris e reforçam sua semelhança citogenética com outras espécies congêneres, além de relatar a ocorrência de uma espécie de Astyanax ainda não descrita taxonomicamente. Em suma, os resultados do presente estudo fornecem dados que auxiliam na taxonomia e sistemática do clado Astyanax e clado Astyanax paris , reforçando a necessidade de revisões amplas, principalmente nos complexos A. bimaculatus e A. fasciatus que incluam marcadores como 5S rDNA e padrão de distribuição da heterocromatina, para melhor compreensão das relações filogenéticas em Astyanax.

Mesoclemmys vanderhaegei Bour, 1973 pertence à família Chelidae, que apresenta maior riqueza de espécies de Testudines no Brasil. Apesar de não se suspeitar do monofiletismo da família, há muitas dúvidas sobre as relações entre os táxons de Chelidae, com a possibilidade de vários grupos se revelarem parafiléticos. Mesoclemmys vanderhaegei tem uma ampla distribuição no centro-sul do continente sul-americano, havendo sido estudada mais especificamente sob uma temática ecológica. Seus aspectos taxonômicos e filogenéticos, porém, ainda necessitam de uma investigação mais detalhada, visto que o grupo pode representar um complexo de espécies. O presente trabalho visa, portanto, estudar as populações de M. vanderhaegei sob um ponto de vista taxonômico e filogenético, buscando identificar e caracterizar as populações através das divergências evolutivas que venham a ser reveladas, justificando assim um eventual desmembramento da espécie. Sendo assim, as populações de M. vanderhaegei foram investigadas por meio de técnicas de morfometria tradicional, morfometria geométrica, e uma filogenia molecular foi proposta para o gênero. Para a morfometria tradicional foram analisadas medidas lineares do plastrão, carapaça e cabeça; enquanto que para a morfometria geométria foram selecionados landmarks nos vértices dos escudos do casco e na linha média dorsal e ventral de cada espécime. Para o estudo de filogenia molecular, foram sequenciados três genes nucleares (RAG-2, R35 e c-mos), e dois mitocondriais (12S e NADH) de representantes da maioria das populações da espécie, e de todas as espécies do gênero. A análise discriminante tanto para os dados de medidas lineares quanto para os de landmarks separa a população do sul da Bacia do Paraguai (próxima à localidade tipo da espécie) das demais populações brasileiras. As análises morfométricas diferenciam outras duas populações: uma população composta por espécimes da Chapada dos Guimarães (MT), região de Cuiabá e um exemplar de Bonito (MS); enquanto a outra população é representada por espécimes de São João da Ponte (MG), na Bacia do São Francisco. A topologia resultante da análise de máxima verossimilhança aponta M. vanderhaegei como um grupo polifilético, com a possibilidade de ser dividida em quatro espécies: uma com os espécimes do sul da Bacia Amazônica; outra com os espécimes de Montes Claros (MG), na Bacia do São Francisco; uma terceira com os espécimes de São João da Lagoa e do interior de São Paulo (Bacia do Paraná, sub Bacia do Tietê); além daquela formada pelos espécimes próximos à localidade tipo. Sugere-se, portanto, (1) que a distribuição de Mesoclemmys vanderhaegei seja restringida ao norte da Argentina, Paraguai, e oeste do Mato Grosso do Sul até que novos estudos venham a elucidar o quanto a espécie adentra o território brasileiro; (2) que as populações da região da Chapada dos Guimarães e do interior de Minas Gerais sejam elevadas ao nível de espécie; (3) e que se façam novos estudos para elucidar a relação das populações da Bacia do Tocantins-Araguaia e Paraná com os outros grupos do complexo.! / Mesoclemmys vanderhaegei Bour, 1973 belongs to the family Chelidae, which presents the greatest variety of species of Testudines in Brazil. In spite of theories that suggest that the family might be monophyletic, there are still areas of doubt over the relationships between the taxa that belong to the Chelidae, and the possibility that various groups might prove to be paraphyletic. Mesoclemmys vanderhaegei has a large distribution range along the central and southern regions of the South American continent, and has been most specifically studied through an ecological perspective. A more detailed investigation of its taxonomic and phylogenetic characteristics is still both relevant and necessary, since the complex might represent multiple species. The present work aims, therefore, to study the populations of M. vanderhaegei from a phylogenetic and taxonomic perspective, looking to identify and characterize the populations in accordance with the evolutionary divergences that may be observed, thus justifying the subdivision of the complex into mutiple species. With this in mind, the populations of M. vanderhaegei were investigated using techniques of traditional and geometric morphometry, and a molecular phylogeny was proposed for the genus. The traditional morphometric data accounted for linear measurements of the plastron, carapace and head whilegeometric morphometric data were chosen as landmarks on the vertices of the scutes of the shells and on the dorsal and ventral midline of each specimen. Using samples of the majority of the populations of the species, and of all the species of the genus, three nuclear (RAG-2, R35 and c-mos) and two mitochondrial genes (12S and NADH) were sequenced for the study of the molecular phylogeny. Both the discriminant analysis of the linear measurements data and that of the landmarks separate the population found in the South of the Paraguay Basin (near the type locality of the species) from other Brazilian populations. The morphometric analyses show two other distinct populations: a population composed of specimens from Chapada dos Guimarães (MT), in the region of Cuiabá and one specimen from Bonito (MS); while the other population is represented by specimens taken from São João da Lagoa (MG), in the Basin of São Francisco. The resulting tree topology of theMaximum-Likelihood analysis derived from the molecular data suggests that M. vanderhaegei should be considered a polyphyletic group, that can be divided into four species: one species represented by the specimens from the South of the Amazonian Basin; another represented by the specimens from Montes Claros (MG), in the Basin of São Francisco; a third represented by the specimens from São João da Lagoa and the interior of São Paulo (Paraná Basin, sub Basin of the river Tietê); in addition to the one formed of the specimens close to the type locality. This would suggest, therefore, (1) that the distribution of Mesoclemmys vanderhaegei is restricted to the North of Argentina, Paraguay, and the West of Mato Grosso do Sul, until further studies are made to elucidate the extent to which the species has spread into Brazilian territory; (2) that the population of the Chapada dos Guimarães region and of the interior of Minas Gerais should be elevated to the category of species; (3) and that new studies must be carried out to determine the relationship between the populations of the Basin of Tocantins-Araguaia and Paraná with the other groups of the complex.!

No description available.

Dans les îles du sud-ouest de l'océan Indien (SOOI) (Comores, Maurice, Mayotte, Réunion, Rodrigues et Seychelles), le flétrissement bactérien causé par le complexe d'espèces Ralstonia solanacearum (ceRs) est une phytobactériose considérée comme l'une des plus nuisibles pour les productions vivrières ou d'exportation. Les travaux de thèse présentés dans ce manuscrit avaient pour principal objectif l'exploration du niveau et de la distribution de la diversité génétique du ce Rs et de la structure génétique de ses populations dans le SOOI. Nous avons mené de vastes campagnes d'échantillonnage qui ont permis de constituer une large collection de 1704 isolats, principalement à partir de Solanacées (tomate, pomme de terre, piment, aubergine, poivron) et de géranium rosat. L'assignation phylogénétique des isolats a montré une très forte prévalence du phylotype I (88 %), qui est distribué dans chaque île du SOOI, tandis que les phylotypes II (9 %) et III (3 %) ne sont trouvés qu'à La Réunion. Deux souches de phylotype IV ont par ailleurs été signalées à l'île Maurice, représentant le premier rapport de ce groupe phylogénétique dans le SOOI. Une approche phylogénétique et de génotypage (MLSA/MLST) basée sur l'analyse de séquences de 6 gènes de ménage et 1 gène associé à la virulence (egl) a permis de révéler les relations génétiques entre 145 souches représentatives (diversité géographique + hôte d'isolement) du SOOI et 90 souches mondiales de référence. Le développement et l'application d'un schéma MLVA basé sur 17 séquences répétées en tandem (VNTR) sur près de 1300 souches a permis de révéler que les populations de phylotype I sont organisées en complexes clonaux dans le SOOI et que le niveau de diversité génétique est très contrasté selon les îles, Maurice présentant la plus forte diversité génétique. Un résultat majeur de cette thèse est la mise en évidence du déploiement d’une lignée génétique (sequevar I-31 ; STI-13 ; MT-035), surreprésentée dans les îles du SOOI, qui pourrait avoir été introduite via du matériel végétal contaminé depuis l'Afrique de Sud ou l’Afrique de l'Ouest. Nos études préliminaires montrent que l'haplotype majoritaire MT-035 (i) est le probable haplotype fondateur du complexe clonal le plus prévalent dans le SOOI, (ii) présente un pouvoir pathogène élevé (large gamme d'hôtes comprenant des plantes cultivées et des adventices, et forte agressivité sur Solanacées) et (iii) possède une forte aptitude à la compétition dans l'environnement via la production de bactériocines. Ces travaux permettront in fine de renforcer l'épidémiosurveillance et orienter les stratégies de lutte vis-à-vis de cet agent phytopathogène, notamment via le déploiement de cultivars résistants. / In the southwest Indian Ocean (SWIO) islands (Comoros, Mauritius, Mayotte, Réunion, Rodrigues and Seychelles), bacterial wilt caused by the Ralstonia solanacearum species complex (Rssc) is considered one of the most harmful plant disease for food crops or export. The main objective of this work presented in this manuscript was to explore the level and the distribution of the genetic diversity of Rssc and the genetic structure of its populations in SWIO. We conducted extensive sampling campaigns that resulted in a large collection of 1704 isolates, mainly from Solanaceae (tomato, potato, chilli, eggplant, pepper) and geranium rosat. The phylogenetic assignment of the isolates showed a very high prevalence of phylotype I (88 %), which is distributed in each island of the SWIO, while phylotypes II (9 %) and III (3 %) are found only in Réunion. Two phylotype IV strains have also been reported in Mauritius, representing the first report of this phylogenetic group in SWIO. A phylogenetic and genotyping approach (MLSA/MLST) based on sequence analysis of 6 housekeeping genes and 1 gene associated with virulence (egl) revealed the genetic relationships between 145 representative SWIO strains (geographic diversity + host) and 90 global reference strains. The development and application of MLVA scheme based on 17 variable number of tandem repeat sequences (VNTR) on nearly 1300 strains revealed that phylotype I populations are organized into clonal complexes in SWIO and that the level of genetic diversity is highly contrasted according to the islands, with Mauritius having the highest genetic diversity. This work highlights the deployment of a genetic lineage (Sequevar I-31, STI-13, MT-035), overrepresented in SWIO islands, which could have been introduced via contaminated plant material from South Africa or West Africa. Our preliminary studies show that the main haplotype MT-035 (i) is the probable founding haplotype of the most prevalent clonal complex in SWIO, (ii) has high pathogenicity (wide range of hosts including cultivated plants and weeds, and high aggressiveness on Solanaceae) and (iii) has a strong ability to compete in the environment via the production of bacteriocins. This work will ultimately strengthen epidemiosurveillance and guide control strategies of this plant pathogen, including the deployment of resistant cultivars.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2016-07-28T11:13:37Z No. of bitstreams: 2 Dissertação - Carolina Martins Treméa - 2015.pdf: 3039510 bytes, checksum: 5e06579e2f9fb745276cc6cc6cd1866e (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-07-28T11:15:34Z (GMT) No. of bitstreams: 2 Dissertação - Carolina Martins Treméa - 2015.pdf: 3039510 bytes, checksum: 5e06579e2f9fb745276cc6cc6cd1866e (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-07-28T11:15:34Z (GMT). No. of bitstreams: 2 Dissertação - Carolina Martins Treméa - 2015.pdf: 3039510 bytes, checksum: 5e06579e2f9fb745276cc6cc6cd1866e (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2015-04-13 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Cryptococcosis is a fungal infection caused by species of the complex Cryptococcus neoformans (C. neoformans and C. gattii) with high incidence in patients with acquired immunodeficiency syndrome (AIDS). Antifungal drugs used in therapy of this infection are scarce and, in addition, have evident toxicity and low effectiveness, which motivates the search for new candidates to antifungals. Essential oils (EOs) extracted from the fruit of Litsea cubeba (Litsea cubeba (Lour.) Pers.) and leaves of Palmarosa (Cymbopogon martini (Roxb.) Will. Watson) and its major constituents, citral and geraniol, were investigated as antifungal sources. The aims of this work were to determine the isolated or associated antifungal activity of EOs and major constituents with amphotericin B (AMB) or fluconazole (FLU) on 15 isolates and two standard strains of C. neoformans complex, evaluate the time-kill curve, the action on the biosynthesis of ergosterol and on cell membrane, in addition to verify the cytotoxicity of EOs and major constituents on human erythrocytes. By in vitro susceptibility testing and minimum fungicidal concentration (MFC), the best activities were to L. cubeba and citral. The EOs and major constituents exhibited minimum inhibitory concentration (MIC) between 16 and 512 μg/mL and 64 and 2048 μg/mL, with a MIC90 of 128 μg/mL for L. cubeba and citral, and 512 μg/mL for C. martini and geraniol. To evaluate the association of EOs and major constituents with antifungal agents was performed the Chequerboard method, that showed indifference in the association of AMB and L. cubeba (94.1%) or citral (70.6%). In association with FLU, L. cubeba was antagonistic (76.5%) and indifferent (94%) for citral. C. martini and geraniol showed synergism associated with AMB, respectively, in 52.9 and 70.6% of isolates, and showed indifference when associated with the FLU in 64.7% and 88.2%. C. gattii 24065 time-kill curve under the action of EOs and major constituents showed a rapid fungicidal activity, which occurred after one hour incubation for L. cubeba, C. martini and geraniol, and after two hours for citral. The amount of ergosterol was quantified after contact with the EOs and major constituents and showed that L. cubeba and citral inhibited 100% of sterol biosynthesis in MIC and 64% for C. martini. This is the probably mechanism of action of these agents, except for geraniol, which reduced only 12% in MIC. The major compounds of EOs showed no ability to injure the plasma membrane of C. gattii, since an average of 2.7% propidium iodide labeled cells were detected by flow cytometry. The in vitro cytotoxicity assay by hemolysis showed that EOs and major constituents were not toxic in the MIC and MFCs. Taken together, our results showed that the EOs of L. cubeba and C. martini and their major constituents, citral e geraniol are promising as candidates for new antifungal agents. / A criptococose é uma infecção fúngica causada por espécies do complexo Cryptococcus neoformans (C. neoformans e C. gattii) com alta incidência em indivíduos com a síndrome da imunodeficiência adquirida (aids). Os antifúngicos utilizados na terapia dessa infecção são escassos e, além disso, possuem toxicidade evidente e baixa eficácia, o que motiva a busca por novos candidatos a antifúngicos. Os óleos essenciais (OEs) extraídos do fruto de Litsea cubeba (Litsea cubeba (Lour.) Pers.) e das folhas de Palmarosa (Cymbopogon martini (Roxb.) Will. Watson) e seus constituintes majoritários, citral e geraniol, foram investigados como fontes de ação antifúngica. Os objetivos foram determinar a atividade antifúngica isolada e associada à anfotericina B (AMB) ou fluconazol (FLU) sobre 15 isolados e duas cepas padrão do complexo C. neoformans, além de avaliar a curva de morte, a ação sobre a biossíntese do ergosterol e sobre a membrana celular do fungo e a citotoxicidade dos OEs e constituintes majoritários sobre hemácias humanas. Por meio de testes de suscetibilidade in vitro e de concentração fungicida mínima (CFM), verificou-se melhor atividade para L. cubeba e citral. Os OEs e constituintes majoritários avaliados exibiram concentração inibitória mínima (CIM) entre 16 e 512 μg/mL e CFM entre 64 e 2048 μg/mL, com CIM90 de 128 μg/mL para L. cubeba e citral e de 512 μg/mL para C. martini e geraniol. Para avaliar a associação dos OEs com antifúngicos foi realizado o método de Chequerboard, onde houve indiferença na associação de AMB a L. cubeba (94,1%) e citral (70,6%), e na associação com FLU, L. cubeba foi antagônico (76,5%) e citral indiferente (94,%). C. martini e geraniol apresentaram sinergismo associados a AMB, respectivamente, em 52,9 e 70,6% dos isolados, e indiferença associado ao FLU em 64,7% e 88,2%. A curva de morte de C. gattii 24065 sob ação dos OEs e constituintes majoritários mostrou uma rápida ação fungicida, que ocorreu após uma hora de incubação para L. cubeba, C. martini e geraniol, e em duas horas para o citral. A quantificação do ergosterol após contato com os OEs e constituintes majoritários mostrou que L. cubeba e citral inibiram 100% da biossíntese desse esterol na CIM e 64% para C. martini, o que provavelmente é um mecanismo de ação destes agentes, exceto para o geraniol, que reduziu apenas 12% na CIM. Os OEs e compostos majoritários não apresentaram capacidade de lesionar a membrana plasmática de C. gattii, visto que em média de 2,7% das células foram marcadas com iodeto de propídio e detectadas por citometria de fluxo. O ensaio de citotoxicidade in vitro por hemólise mostrou que os OEs e constituintes majoritários não foram tóxicos nas CIMs e CFMs. Os resultados observados no presente trabalho mostram que os OEs de L. cubeba e C. martini e seus constituintes majoritários, citral e geraniol, são promissores candidatos a antifúngicos.

Le genre Ectocarpus Lyngbye (Ectocarpales, Phaeophyceae) regroupe des algues marines filamenteuses caractérisées par un cycle haploïde-diploïde. L'objectif de la thèse était de délimiter les espèces et d'étudier la spéciation dans ce genre. Nous avons commencé par clarifier le nombre d'espèces cryptiques en utilisant deux loci indépendants et une approche intégrative associant une analyse de détection de " barcode gap " avec des reconstructions phylogénétiques. Nos résultats montrent l'existence d'au moins 15 espèces qui se répartissent en un groupe monophylétique composé d'E. crouaniorum (Ecro) et de deux espèces proches ainsi que d'un mélange paraphylétique composé des 12 autres espèces incluant E. siliculosus (Esil). Deuxièmement, les analyses de séquençage Rad et de phylogénomique ont permis de résoudre les relations au sein du groupe paraphylétique. Les espèces se regroupent maintenant en deux clades divergents (Ecro and Esil). Des niveaux de divergence variables entre espèces sont révélés au sein du clade Esil. Des phénomènes d'hybridation entre les espèces les plus apparentées, et trouvées en sympatrie, sont suspectés. Finalement, l'importance de l'isolement reproducteur a été étudié entre les espèces Esil et Ecro, les plus communes, mais les plus divergentes, en utilisant des marqueurs spécifiques de chacune des espèces. Nos résultats indiquent que la méiose agit comme une forte barrière reproductive entre ces espèces et démontrent que les espèces du genre Ectocarpus sont d'excellents systèmes pour étudier les conséquences évolutives de l'hybridation et de l'introgression pour le maintien ou la divergence des espèces grâce à leur cycle haploïde-diploïde. / The genus Ectocarpus Lyngbye (Ectocarpales, Phaeophyceae) comprises marine filamentous algae characterized by an alternation between two independent multicellular organisms of different ploidy. The general objective of the thesis was to study species delineation and speciation within this genus. We started clarifying the number of cryptic species using two unlinked loci (COI-5P and ITS1) and an integrative approach associating barcode gap detection analyses with phylogenetic reconstructions. We showed the presence of at least 15 species partitioned within a monophyletic group composed of E. crouaniorum (Ecro) and two closely related species and a paraphyletic assemblage composed of the remaining 12 other species including E. siliculosus (Esil). Second, Rad sequencing and phylogenomics analyses allowed to resolve the relationships within the paraphyletic assemblage. The different species becomes well separated into two divergent clades (Ecro and Esil). A diversity of taxa with various levels of divergence was revealed within the clade Esil and hybridization between the closest and sympatric species was suggested. Finally, the importance of reproductive isolation among the two commonest but most divergent species Esil and Ecro was studied using species-specific nuclear and cytoplasmic markers jointly with 9 microsatellites. We showed that meiosis acts as a strong reproductive barrier among these two species and demonstrates that the species of the genus Ectocarpus are excellent systems to study evolutionary consequences of hybridization and introgression for the maintenance or breakdown of species because of their haploid diploid life cycle.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has revolutionized the field of bacterial diagnostics and is also used for routine analysis in smaller clinical laboratories. For identification of moulds, pre-analytical steps are more complicated and time consuming than for bacteria, and the choice of reference library has a big impact on the utility of MALDI-TOF MS. The aim of this study was to investigate if MALDI-TOF MS is applicable for identification of moulds belonging to the genus Aspergillus at the hospital laboratory in Gävle. Therefore, strains belonging to the genus Aspergillus and Penicillium, were analysed with MALDI-TOF MS after 2, 4 and 7 days of incubation. Two different extraction protocols were used and compared. Mass spectra were compared to reference spectra in two different databases: MSI-2 and RUO Compass library/BDAL (Bruker). Of the strains included, 97 % were correctly identified to species complex level with MSI-2. Only 25 % were identified to species level with RUO Compass library/BDAL (Bruker). However, totally 56 % were correctly identified to species complex level if a lower score value limit than recommended for identification, were applied. Significantly raised score values were observed with one of the protein extraction protocols used. Although, in most cases, the strains were considered identified to species complex level with either method. This pilot study conveys the feasibility of MALDI-TOF MS for identification of Aspergillus species in a clinical laboratory. While there are still issues to address, applying MALDI-TOF MS has the potential to allow for quicker and more precise identification, also in this specific clinical setting.

Une forte structure génétique a été décrite dans la littérature parmi les populations européennes de la coque de lagune Cerastoderma glaucum. Cependant, les limites géographiques entre les grandes divisions génétiques varient d'un marqueur à l'autre et leur faible nombre ne permettait pas de tester réellement les échanges génétiques entre ces groupes. Le but principal de cette thèse était donc d'estimer la structure des populations de C. glaucum à l'aide de marqueurs, en considérant les rôles respectifs de la divergence passée et de la fragmentation de l'habitat. Pour atteindre ses objectifs, des populations des côtes atlantiques et méditerranéennes ont été étudiées en utilisant : i) des marqueurs génétiques classiques (un EPIC et des marqueurs microsatellites déjà publiés); ii) des séquences associées à des sites de restriction (“restriction associated DNA sequences”) ou RADseq. Alors que les marqueurs génétiques classiques ont révélé le caractère divergent des populations de Méditerranée orientale, l'approche RADseq a permis de reconstruire les relations phylogénétiques entre groupes au sein de l'espèce avec une résolution jamais encore ateinte. Trois lignées profondément divergentes ont ainsi été identifiée au sein de C. glaucum: une en Mer Egée – Mer Noire, une en Mer Ionienne et la dernière largement distribuée de la Méditerranée à la Baltique. Cette dernière lignée c'est par ailleurs sous divisée en entités isolées en Méditerranée Occidentale, en Atlantique, en Mer du Nord et an Baltique. Comparée à l'espèce soeur C. edule, qui profite d'un habitat moins fragmenté, C. glaucum présente une beaucoup plus forte différenciation génétique entre populations. Il semble donc que des incompatibilités génétiques, conséquences de l'isolement géographique et de l'adaptation locale, aient pu être à l'origine d'un complexe d'espèces. Ces résultats soulignent par ailleurs le rôle évolutif particulier de l'habitat lagunaire, accélérateur de la diversification génétique. / Previous studies found a high level of genetic structuring among the European populations of the lagoon cockle Cerastoderma glaucum. However, the geographic locations of the major genetic divisions differed among markers and the small number of genetic markers used lacked the power to test for gene flow between divergent clusters. Therefore, the main aim of this thesis was to assess the population structure of C. glaucum using new genetic markers in the light of the respective role of past divergence and habitat fragmentation. To reach these objectives, populations from the Atlantic and Mediterranean coasts have been investigated using: i) classical genetic markers (EPIC together with previously published microsatellites); ii) restriction-site associated DNA sequencing (RADseq). When the classical genetic markers revealed the divergent character of Eastern Mediterranean populations, the RADseq approach allowed inferring phylogenetic relationships with an unprecedented resolution. Three deeply divergent lineages were described within C. glaucum: one in the Aegean-Black Sea region, one in the Ionian Sea, and the last one widely distributed from the Western Mediterranean to the Baltic Sea. This last lineage underwent further diversification with isolated entities detected in Western Mediterranean, Atlantic, North Sea and Baltic Sea. Compared to the sister species C. edule, which thrives in a less fragmented habitat, C. glaucum displayed a much stronger genetic differentiation among populations. Consequently, it seems that genetic incompatibilities, which emerged as a by-product of geographic isolation together with local adaptation, led to the origin of a species complex. These results highlight the evolutionary role of lagoon habitats, where genetic diversification can proceed very quickly.

(in English) Pipistrellus pipistrellus species complex contains two sympatric species inhabiting Europe and part of West and Central Asia (Pipistrellus pipistrellus s. str, Pipistrellus pygmaeus s. str) and several other lines, isolated in the Mediterranean (North Africa, islands and peninsulas of the Mediterranean Sea). This taxon is a part of the extensive radiation within the genus Pipistrellus, which in today's concept includes about 30 species. Mosaic line of P. pipistrellus complex, located at different stages of diversification and secondary contacts in the Mediterranean biodiversity hotspot, is a suitable model for research on speciation. In this thesis we focused on analyses of distribution, phylogeography, population structure and demography based on mitochondrial data from 323 individuals, representing almost the entire range. Control region of mitochondrial DNA was chosen as a genetic marker. Variability in the 378 pb long fragment acknowledged the existence of several genetically distinct lines whose species status is discussed. Observed fylogeografic pattern confirms the existence of groups of radiation centers in the Mediterranean region. An allopatric speciation was there, two of the lines (P. pipistrellus s. str and P. pygmaeus s. str.) later expanded into Europe and their ranges...

Dans la mucoviscidose, le tableau clinique est dominé par les infections respiratoires qui constituent la cause majeure de morbidité. Diverses espèces fongiques sont décrites dans ces infections, notamment les Scedosporium et le complexe Rasamsonia argillacea pour lesquels les connaissances restent limitées. Dans ce travail, nous avons évalué l’intérêt de l’amplification des séquences répétitives de l’ADN pour la différenciation spécifique et infraspécifique de ces champignons. La rep-PCR a d’abord été appliquée à l’analyse d'isolats du genre Scedosporium. La rep-PCR a produit des résultats concordants avec ceux obtenus par séquençage du gène de la bêta-tubuline, amplification aléatoire de fragments d’ADN polymorphes et séquençage multi-loci. Elle permet également l’identification précise des espèces au sein du complexe R. argillacea. L’analyse des résultats obtenus sur 116 isolats a montré la capacité de ces champignons à coloniser de manière chronique les voies respiratoires, un même profil électrophorétique étant observé généralement chez un même patient. Enfin, nous avons produit de nouvelles données sur l’écologie des Scedosporium en étudiant différents biotopes au Maroc. Comme précédemment rapporté,ces champignons préfèrent les environnements impactés parles activités humaines. On les rencontre dans des sols de pH neutre, riches en matières organiques et pauvres en sels. Enfin, si S. apiospermum était l’espèce la plus abondante, S. boydii, S. aurantiacum et S. dehoogii ont été rencontrés avec des fréquences comparables. En conclusion, les données générées renforcent nos connaissances sur l’épidémiologie des infections causées par ces pathogènes émergents. / In cystic fibrosis (CF), respiratory infections are the leading cause of morbidity and mortality. Bacteria are the most common causative agents of these infections, but an increasing number of fungal species may also be found, including Scedosporium species and species of the Rasamsonia argillacea complex. In order to improve our knowledge on the epidemiology of these infections, here we evaluated the interest of PCR amplification of repetitive DNA sequences (rep-PCR) for species identification and strain delineation. rep-PCR was applied to the retrospective analysis of a panel of 63 isolates belonging to the Scedosporiumgenus. Results were consistent with those obtained by beta-tubulingene sequencing, random amplification of polymorphic DNA and Multilocus Sequence Typing. rep-PCR also permitted precise species identification in the R. argillacea complex. Analysis of the data obtained on 116 isolates revealed the capacity of these fungi to chronically colonize the airways of patients with CF, a unique electrophoretic profile being observed usually for each patient. Finally, we produced in this thesis new data about the ecology of Scedosporium species by studying different biotopes in Morocco. As previously reported, these fungi are mainly found in human-impacted areas. Also they prefer soils with a neutral pH, a high organic matter content and a very low mineral salts content. In addition, if S. apiospermum was by far the predominant species, S. boydii, S. aurantiacum and S. dehoogii were encountered with similar frequencies. In conclusion, data provided here reinforce our knowledge on the epidemiology of the infections caused by these emerging pathogens.