Canary grasses (Phalaris, Poaceae) include 21 species widely distributed throughout temperate and subtropical regions of the world with centers of diversity in the Mediterranean Basin and western North America. The genus contains annual/perennial, endemic/cosmopolitan, wild, and invasive species with basic numbers of x=6 (diploid) and x=7 (diploid/tetraploid/hexaploid). The latter display vastly greater speciation and geographic distribution. These attributes make Phalaris an ideal platform to study species diversification, dispersal, historic hybridization, polyploidy events, and chromosome evolution in the grasses. This body of research presents the first molecular phylogenetic and phylogeographic reconstruction of the genus based on the nuclear ITS and plastid trnT-F DNA regions allowing species relationships and the importance of polyploidy in speciation to be assessed. Divergence dates for the genus were determined using Bayesian methods (BEAST, version 1.6.2) and historic patterns of dispersal were analyzed with RASP (version 2.1b). Self-incompatibility and the feasibility of hybridization between major groups within the genus were studied with a series of greenhouse experiments. Acetocarmine and fluorescent staining techniques were used to study the morphology of the chromosomes in a phylogenetic context and the nuclear DNA content (C values) was quantified using flow cytometry. Four major clades were revealed in the genus with cytological and geographic affinities leading to the establishment of two subgenera and four sections in the first comprehensive infrageneric treatment of Phalaris. Divergence dating revealed a Miocene emergence (20.6-8.4 MYA) for the genus which is concurrent with studies of other genera in the Aveneae tribe. The hypothesis stating that Phalaris originated in the Mediterranean Basin and dispersed to the New World via a western route leading to a secondary center of diversification in western North America was supported by phylogeographic and cytological analyses. An empirical study comparing the weight, length, and width of the florets by morphological type and cytotype revealed significant differences that support a dispersal advantage among the New World and Arundinacea species. The x=6 species displayed greater intraspecific C value variation, higher DNA content per haploid chromosome set, and a distinct karyotype compared with the x=7 species indicating a complex history of chromosome evolution. / Ph. D.

No description available.

<p>Given the emerging epidemic of renal disease in HIV+ patients and the fact that HIV DNA and RNA persist in the kidneys of HIV+ patients despite therapy, it is necessary to understand the role of direct HIV-1 infection of the kidney. HIV-associated kidney disease pathogenesis is attributed in large part to viral proteins. Expression of Vpr in renal tubule epithelial cells (RTECs) induces G2 arrest, apoptosis and polyploidy. The ability of a subset of cells to overcome the G2/M block and progress to polyploidy is not well understood. Polyploidy frequently associates with a bypass of cell death and disease pathogenesis. Given the ability of the kidney to serve as a unique compartment for HIV-1 infection, and the observed occurrence of polyploid cells in HIV+ renal cells, it is critical to understand the mechanisms and consequences of Vpr-induced polyploidy. </p><p>Here I determined effects of HIV-1 Vpr expression in renal cells using highly efficient transduction with VSV.G pseudotyped lentiviral vectors expressing Vpr in the HK2 human tubule epithelial cell line. Using FACS, fluorescence microscopy, and live cell imaging I show that G2 escape immediately precedes a critical junction between two distinct outcomes in Vpr+ RTECs: mitotic cell death and polyploidy. Vpr+ cells that evade aberrant mitosis and become polyploid have a substantially higher survival rate than those that undergo complete mitosis, and this survival correlates with enrichment for polyploidy in cell culture over time. Further, I identify a novel role for ATM kinase in promoting G2 arrest escape and polyploidy in this context. In summary, my work identifies ATM-dependent override of Vpr-mediated G2/M arrest as a critical determinant of cell fate Vpr+ RTECs. Further, our work highlights how a poorly understood HIV mechanism, ploidy increase, may offer insight into key processes of reservoir establishment and disease pathogenesis in HIV+ kidneys.</p> / Dissertation

La méiose est un processus fondamental qui conditionne la formation de gamètes et assure la stabilité des génomes tout en générant de la diversité par brassage génétique. La régularité méiotique nécessite la formation de crossing-overs (CO) exclusivement entre chromosomes homologues. Cette condition est plus difficile à remplir chez les espèces allopolyploïdes qui présentent plusieurs jeux de chromosomes toujours susceptibles de recombiner ensemble. Bien que la polyploïdie soit omniprésente chez les plantes, on connait peu de choses sur le déroulement de la méiose chez ces espèces. Au cours de ma thèse, je suis intéressée à l’effet de la polyploïdie sur la formation et sur la fréquence de CO en utilisant le colza (Brassica napus, AACC, 2n=38) comme modèle d’étude. J’ai notamment cherché à comprendre : (1) quel est l’effet du niveau de ploïdie sur la fréquence de crossovers, et (2) l’origine des variations de COs observées chez les plantes allohaploïdes (AC) produites à partir de différentes variétés de colza, en utilisant une palette d’approches cytologiques et cytogénétiques. Mes travaux ont permis de montrer que le niveau de ploïdie induit une augmentation de la fréquence de crossovers, et que cette augmentation est plus importante dans un contexte triploïde que tétraploïde. J’ai ainsi montré que la fréquence de CO augmente progressivement du diploïde (1,6 CO/bivalent) vers le tétraploïde (2 CO/bivalents) et quelle est maximale chez le triploïde (2,8 CO/bivalent). En ce qui concerne la deuxième question, j’ai montré que la différence entre les allohaploïdes de colzas apparaît tardivement au cours de la méiose. Elle semble être liée à une capacité différente à former des CO en fonction de la variété utilisée pour produire ces allohaploïdes et non pas à une différence dans la reconnaissance de l’homologie. Un de mes résultats original est que la protéine HEI10, impliquée dans la voie de formation des CO interférents, présente une dynamique différente entre les deux variétés, que ce soit à l’état euploïde (AACC) qu’allohaploïde (AC).Mes résultats conduisent à s’interroger sur la relation entre (i) la régulation du nombre de CO formés entre chromosomes homologues et (ii) la suppression des CO entre chromosomes non homologues chez les espèces allopolyploïdes. / Meiosis is a fundamental process required to produce gametes, ensure genome stability and generate diversity within species by creating new chromosome/allele combinations. For all these outcomes the exclusive formation of crossovers (CO) between homologous chromosomes is required. This condition is more difficult to fulfil in allopolyploid species that have more than two sets of chromosomes still able to recombine together. Although polyploidy has been particularly prevalent in plants, little is known about meiosis in polyploids. During my thesis I have analyzed the effect of polyploidy on CO formation and frequency, using oilseed rape (Brassica napus, AACC, 2n=38) as model. My work aimed to investigate (i) the effect of ploidy level on the rate of meiotic COs and (ii) the causes for the observed difference in CO rate between allohaploid plants (AC) produced from different B. napus varieties. To address these questions, I have combined a series of cytological, immunocytological and cytogenetical analyses.My work first indicates that polyploidization leads to increase CO frequency. I showed that the number of COs progressively increases from the diploid (1,6 CO/bivalent) to the tetraploid (2 CO/bivalent) and is maximal in the triploid (2,8 CO/bivalent). In the second part, I have shown that the difference of meiotic behaviors between B. napus allohaploids appears at a late stage of meiosis. This difference seems to be due to a difference in the propensity to form CO between the two varieties rather than a difference in the stringency of homology recognition. This difference could be related to the difference in the pattern and/or chronology of HEI10 (a key protein involved in the interfering CO pathway) signals along chromosomes during prophase I in both euploids (AACC) and allohaploids (AC).My results thus puts under the spotlight the link that may exist between (i) the regulation of CO rate between homologous chromosomes and (ii) the suppression of COs between non-homologous chromosomes in polyploid species.

A poliploidia induzida é uma ferramenta valiosa para o melhoramento genético de plantas, resultando em genótipos com características superiores aos diploides correspondentes. Considerando a importância da cultura de eucalipto para o setor florestal e para a economia brasileira, o presente trabalho teve como objetivo estabelecer um protocolo de poliploidização in vitro para eucalipto, utilizando a espécie Eucalyptus urophylla ST Blake. Segmentos nodais contendo gemas laterais de plântulas desenvolvidas in vitro foram utilizados como explantes para os testes de indução. Os tratamentos avaliados consistiram na imersão e agitação dos explantes em soluções de colchicina nas concentrações: 0; 0,125% e 0,250% em tempos de exposição de 12 e 24 horas e posterior inoculação in vitro em meio de cultura para multiplicação. Após 60 dias, as brotações emitidas foram analisadas por citometria de fluxo e constatou-se indivíduos diploides, tetraploides e mixoploides. Estas foram micropropagadas para a obtenção de mudas clonais ao final do processo. As mudas obtidas foram avaliadas por meio de análises morfológicas organográficas e anatômicas para verificar possíveis alterações resultantes da poliploidização in vitro. Os tratamentos promoveram a indução de 16 poliploides, sendo 11 mixoploides, quatro tetraploides e um octaplóide. A eficência máxima de indução obtida foi de 28% de poliploides no tratamento com 0,125% de colchicina por 12 horas sob agitação, com a obtenção tanto de mixoploides como de tetraploides puros. O protocolo geral de micropropagação utilizado, apesar de algumas perdas durante as etapas, garantiu a obtenção de altos percentuais de mudas clonais inteiramente tetraploides, comprovando a eficiência da técnica no isolamento de setores poliploides. A análise morfo-anatômica das mudas mostrou diferenças significativas em relação às características estomáticas, apontando este tipo de análise como indicador de poliploidização na separação preliminar dos indivíduos. A estrutura anatômica das folhas, no entanto, não sofreu modificações significativas, com exceção da espessura e área foliar da epiderme adaxial. A análise de componentes principais realizada para algumas variáveis morfo-anatômicas demonstrou que, apesar da análise estatística não ter apontado diferenças significativas, existe uma tendência em agrupar os genótipos diploides e tetraploides, diferenciando-os principalmente em relação às características estomáticas, espessura foliar, área do mesofilo, parênquimas paliçádico e lacunoso e espaço intercelular. Os resultados obtidos neste trabalho instigam a continuidade do estudo em diversas possibilidades de abordagens, como o estudo do desenvolvimento das mudas em condições de campos, anatomia da madeira e avaliação do comportamento meiótico. / The induced polyploidy is a valuable tool for genetic improvement of plants, resulting in genotypes with superior characteristics to their corresponding diploid. Considering the Eucalyptus culture importance to the forest sector and Brazilian economy, the aim of this study was establish an in vitro polyploidy protocol for eucalyptus, using the species Eucalyptus urophylla ST Blake. Nodal segments containing lateral buds developed from in vitro seedlings were used as explants for induction tests. The treatments tested consisted of soak and shake the explants in colchicine solutions at the concentrations: 0; 0.125% and 0.250% in soaking by periods of 12 or 24 hours and subsequent in vitro inoculation in culture medium for multiplication. After 60 days, the emitted shoots were classified by flow cytometry in levels of ploidy (diploid, tetraploid and mixoploids) and were subsequently micropropagated to obtain clonal seedlings at the end of the process. The clonal seedlings were evaluated using morphological and anatomical analysis to check the possible changes resulting from the in vitro polyploidy. The treatments promoted the induction of sixteen polyploids, being eleven mixoploids, four tetraploid and one octaploid. The maximum induction efficiency was obtained in 28% of polyploidy in the treatment with 0.125% of colchicine by soaking period of 12 hours, having obtained both mixoploids and pure tetraploids. The general protocol of micropropagation used, although presenting some losses during the steps, guaranted the inducing of high percentages of enterelly tetraploid, proving the efficiency of micropropagation in the isolation of polyploid sectors. The morpho-anatomical analysis of the poliploidy seedlings showed significant differences in relation to stomatal characteristics, indicating this type of analysis as polyploidy indicator in the preliminary separation of individuals. The anatomical structure of the leaves, however, did not change significantly, except for the thickness and leaf area of the adaxial epidermis. The principal component analysis (PCA) performed for some morphological and anatomical variables demonstrated that despite the statistical analysis did not have pointed out significant differences, there is a tendency to group the diploid and tetraploid genotypes, differentiating them mainly in relation to stomatal characteristics, leaf thickness, mesophyll area, palisade and spongy parenchyma areas and intercellular space. The results of this work instigate to continue the study in several possibilities of approaches, such as the study of the development of seedlings in fields conditions, wood anatomy and study of meiotic behavior.

<p>Asexual reproduction and polyploidy are relatively rare in animals with chromosomal sex determination and always represent a derived condition. To accomplish asexual reproduction several changes in gene expression are required in the mechanism of oogenesis. Polyploidy increases the cell volume and also gives rise to alterations in general physiology. Nevertheless, there are asexual animals that not only survive but seem to be doing better than their sexual progenitors. This is expressed in the distribution pattern called geographical parthenogenesis. Using molecular phylogeny, I here examine the evolution of Otiorynchid weevils, mainly <i>Otiorhynchus scaber</i> and <i>sulcatus</i> in an attempt to trace the evolutionary history and find out what causes the variation in success of different parthenogens. I also evaluate the contribution of asexuality, hybridity and polyploidy as explanations behind geographical parthenogenesis in insects. I conclude that what is called <i>O. scaber</i> is, in fact, a set of geographical polyploids as polyploidy and not asexuality explains the difference in clonal success. I also argue that <i>O. sulcatus</i> is a recently formed clonal species of non-hybrid origin that may well be a good example of a true general purpose genotype. I find little support for asexuality or a hybrid origin as explanations behind geographical parthenogenesis in insects. Finally, I argue that polyploidy in all eukaryotes should be seen as an opportunity for the species evolution, not as a limitation that ensures the demise of the taxa.</p>

Asexual reproduction and polyploidy are relatively rare in animals with chromosomal sex determination and always represent a derived condition. To accomplish asexual reproduction several changes in gene expression are required in the mechanism of oogenesis. Polyploidy increases the cell volume and also gives rise to alterations in general physiology. Nevertheless, there are asexual animals that not only survive but seem to be doing better than their sexual progenitors. This is expressed in the distribution pattern called geographical parthenogenesis. Using molecular phylogeny, I here examine the evolution of Otiorynchid weevils, mainly Otiorhynchus scaber and sulcatus in an attempt to trace the evolutionary history and find out what causes the variation in success of different parthenogens. I also evaluate the contribution of asexuality, hybridity and polyploidy as explanations behind geographical parthenogenesis in insects. I conclude that what is called O. scaber is, in fact, a set of geographical polyploids as polyploidy and not asexuality explains the difference in clonal success. I also argue that O. sulcatus is a recently formed clonal species of non-hybrid origin that may well be a good example of a true general purpose genotype. I find little support for asexuality or a hybrid origin as explanations behind geographical parthenogenesis in insects. Finally, I argue that polyploidy in all eukaryotes should be seen as an opportunity for the species evolution, not as a limitation that ensures the demise of the taxa.

<p>In an ever-changing world, evolution is an essential process that may allow organisms to adapt to their environment through natural selection. All evolutionary processes act through a single fundamental medium: genetic variation. Polyploidy, or whole genome duplication, is a major mechanism for evolutionary change because it is both widespread across taxa and results in a proliferation of genetic material that evolution can act upon. The key questions addressed here are: (1) How does chromosome pairing during meiosis in allopolyploids affect the magnitude of genetic variation?, (2) How does the genome of polyploids evolve following formation, and what genetic mechanisms govern this evolution?, and (3) How does genetic and genomic evolution in polyploids affect phenotypic evolution? I use the shy monkeyflower, <italic>Mimulus sookensis</italic>, a tetraploid of hybrid origin between <italic>Mimulus guttatus</italic> and <italic>Mimulus nasutus</italic>, to address these focal questions. In order to develop a foundation to aid in interpretation of my findings, I first investigate the evolutionary history of <italic>M. sookensis</italic>. Chromosome counts establish that <italic>M. sookensis</italic> is indeed an allotetraploid, and a review of taxonomic literature reveals that this species is heretofore undescribed. By analysing the patterns of genetic variation at chloroplast and nuclear loci in <italic>M. guttatus</italic>, <italic>M. nasutus</italic>, and <italic>M. sookensis</italic>, I show that <italic>M. sookensis</italic> has recurrently formed from <italic>M. guttatus</italic> and <italic>M. nasutus</italic>. Crossing experiments within <italic>M. sookensis</italic> indicate that recurrent origins can contribute to genetic diversity without contributing to reproductive isolation among independently arisen polyploid lineages.</p><p>To address my focal questions, I take advantage of an intriguing and striking difference in flower size among <italic>M. sookensis</italic>, <italic>M. guttatus</italic>, and <italic>M. nasutus</italic>. The flowers of <italic>M. sookensis</italic> and <italic>M. nasutus</italic> are small and remarkably similar to one another, while the flowers of <italic>M. guttatus</italic> and diploid and tetraploid F1 hybrids between <italic>M. guttatus</italic> and <italic>M. nasutus</italic> are large and showy. This phenotypic divergence in flower size between <italic>M. sookensis</italic> and <italic>M. guttatus</italic>-like hybrids indicates that small flower size has evolved in <italic>M. sookensis</italic>. Using genetic marker data and phenotypic measurements from synthetic neoallotetraploid <italic>Mimulus</italic>, I demonstrate that there are low levels of fragment loss and phenotypic variation in neoallotetraploids; this suggests that homeologous pairing and recombination following polyploidization is not a major source of genetic variation or phenotypic evolution in <italic>M. sookensis</italic>. Analysis of the whole genome sequence of two <italic>M. sookensis</italic> lines reveals that <italic>M. sookensis</italic> is a fixed heterozygote throughout its entire genome, in that it has retained both a <italic>M. guttatus</italic>-like and <italic>M. nasutus</italic>-like subgenome, neither of which have been removed through homeologous recombination. These subgenomes have been homogenized by widespread gene conversion, and do not appear to have been differentially affected by deletions or deleterious mutations. Finally, to directly characterize the genetic architecture of flower size in <italic>M. sookensis</italic>, I cross a large-flowered synthetic neoallotetraploid <italic>Mimulus</italic> to small-flowered <italic>M. sookensis</italic>. I then employ a novel genotyping-by-sequencing approach to identify quantitative trait loci (QTL) associated with flower size. I find that there is one locus that accounts for a large proportion of phenotypic variation, and four other loci also contribute to flower size variation between the parental lines. Some of these loci co-localize with previously identified loci for flower size in diploid <italic>Mimulus</italic>, while others do not. Altogether, genetic marker data, phenotypic analysis of neoallotetraploids, whole genome sequence data, and QTL mapping data suggest that the genetic variation necessary for flower size evolution was likely caused by both gene conversion and new mutations, but not homeologous recombination. These results suggest that trait evolution in polyploids may be affected by the unique attributes of polyploids, but that new mutations are always an important source of genetic variation, regardless of ploidy level.</p> / Dissertation

As many as 70% of angiosperm species are known to contain polyploids, but many aspects of polyploid evolution are unclear in woody plants. Crataegus is a woody genus of Rosaceae comprising 140-200 species that are widely distributed in the Northern Hemisphere. Several species, particularly those in North America, are shown to contain polyploids. The overall goal of the thesis is to provide a better understanding of polyploid evolution by resolving problems from intergeneric to intraspecific levels in Crataegus using phylogenetic and population genetic approaches. Three major aspects were investigated: (1) Phylogeography of the Old and New World Crataegus; (2) Reproductive system and distribution of cytotypes of the black-fruited series Douglasianae in Pacific Northwest and; (3) Origins, population structure, and genetic diversity of diploid and polyploid species. Phylogenetic analyses of molecular data provide evidences of historical events such as trans-Beringian migrations and North Atlantic vicariance that contributed to modern distribution of Crataegus. Poor resolution and short internal branches in eastern North American species suggest genetic bottlenecks and/or rapid divergence following glaciations. In the Pacific Northwest, polyploids of series Douglasianae show a wider distribution and ecological amplitude than diploids. Parsimony tree and network analyses indicate that autotriploids and allotriploids occur in C. suksdorfii, while tetraploid C. suksdorfii are formed via the triploid bridge followed by introgression of sympatric C. douglasii. At the regional level, microsatellite data indicate a separation of the Pacific coastal diploids and triploids from the Columbia Plateau and Rocky Mountain triploids and tetraploids. High genetic differentiation among C. suksdorfii populations suggests that gene flow is limited by ploidy level differences as well as geographical distance. Within-population multilocus genotypic variation is greatest in sexual diploids, and least in apomictic triploids. Frequent gene flow via seed dispersal contributes to an appreciable level of intrapopulation diversity in apomictic tetraploids, and counterbalances the effects of apomixis and/or self-fertilization, which diminish genetic variation within and between seed families. These findings collectively clarify taxonomy and historical biogeography, provide an explicit reticulation model for polyploid formation, and shed light on evolution of natural populations in woody plants that show heterogeneous ploidy levels and reproductive systems.

As many as 70% of angiosperm species are known to contain polyploids, but many aspects of polyploid evolution are unclear in woody plants. Crataegus is a woody genus of Rosaceae comprising 140-200 species that are widely distributed in the Northern Hemisphere. Several species, particularly those in North America, are shown to contain polyploids. The overall goal of the thesis is to provide a better understanding of polyploid evolution by resolving problems from intergeneric to intraspecific levels in Crataegus using phylogenetic and population genetic approaches. Three major aspects were investigated: (1) Phylogeography of the Old and New World Crataegus; (2) Reproductive system and distribution of cytotypes of the black-fruited series Douglasianae in Pacific Northwest and; (3) Origins, population structure, and genetic diversity of diploid and polyploid species. Phylogenetic analyses of molecular data provide evidences of historical events such as trans-Beringian migrations and North Atlantic vicariance that contributed to modern distribution of Crataegus. Poor resolution and short internal branches in eastern North American species suggest genetic bottlenecks and/or rapid divergence following glaciations. In the Pacific Northwest, polyploids of series Douglasianae show a wider distribution and ecological amplitude than diploids. Parsimony tree and network analyses indicate that autotriploids and allotriploids occur in C. suksdorfii, while tetraploid C. suksdorfii are formed via the triploid bridge followed by introgression of sympatric C. douglasii. At the regional level, microsatellite data indicate a separation of the Pacific coastal diploids and triploids from the Columbia Plateau and Rocky Mountain triploids and tetraploids. High genetic differentiation among C. suksdorfii populations suggests that gene flow is limited by ploidy level differences as well as geographical distance. Within-population multilocus genotypic variation is greatest in sexual diploids, and least in apomictic triploids. Frequent gene flow via seed dispersal contributes to an appreciable level of intrapopulation diversity in apomictic tetraploids, and counterbalances the effects of apomixis and/or self-fertilization, which diminish genetic variation within and between seed families. These findings collectively clarify taxonomy and historical biogeography, provide an explicit reticulation model for polyploid formation, and shed light on evolution of natural populations in woody plants that show heterogeneous ploidy levels and reproductive systems.