Indiana University-Purdue University Indianapolis (IUPUI) / Chromosomal mechanisms of sex determination vary greatly in phylogenetically closely related species, indicative of rapid evolutionary rates. Sex chromosome karyotypes are generally conserved within families; however, many species have derived sex chromosome configurations. Insects display a plethora of sex chromosome systems due to rapid diversification caused by changes in evolutionary processes within and between species. A good example of such a system are insects in the blow fly family Calliphoridae. While cytogenetic studies observe that the karyotype in blow flies is highly conserved (five pairs of autosomal chromosomes and one pair sex chromosome), there is variation in sex determining mechanisms and sex chromosome structure within closely related species in blow flies. The evolutionary history of sex chromosomes in blow fly species have not been fully explored. Therefore, the objective of this research was to characterize the sex chromosome structures in four species of blow flies and investigate the selective forces which have played a role in shaping the diverse sex chromosome system observed in blow flies. The blow fly species used in this study are Phormia regina, Lucilia cuprina, Chrysomya rufifacies and Chrysomya albiceps. Phormia regina,and Lucilia cuprina have a heteromorphic sex chromosome system and are amphogenic (females produce both male and female offspring in equal ratio). In contrast, Chrysomya rufifacies and Chrysomya albiceps, have a homomorphic sex chromosome system, are monogenic (females produce unisexual progeny), have two types of females (arrhenogenic females – male producers and thelygenic females – female producers), and sex of the offspring is determined by the maternal genotype. To accomplish these tasks, a total of nine male and female individual draft genomes for each of the four species (including three individual draft genomes of Chrysomya rufifacies – male, and the two females) were sequenced and assembled providing genomic data to explore sex chromosome evolution in blow flies. Whole genome analysis was utilized to characterize and identify putative sex chromosomal sequences of the four blow fly species. Genomic evidence confirmed the presence of genetically differentiated sex chromosomes in P. regina and L. cuprina; and genetically undifferentiated sex chromosomes in C. rufifacies and C. albiceps. Furthermore, comparative analysis of the ancestral Dipteran sex chromosome (Muller element F in Drosophila) was determined to be X-linked in P. regina and L. cuprina contributing to sex chromosome differentiation but not sex-linked in C. rufifacies and C. albiceps. Evolutionary pressures are often quantified by the ratio of substitution rates at non-synonymous (dN) and synonymous (dS) sites. Substitution rate ratio analysis (dN/dS) of homologous genes indicated a weaker purifying selection may have contributed to the loss of sex-linked genes in Muller element F genes of the undifferentiated sex chromosome as compared to the differentiated sex chromosome system. Overall, the results presented herein greatly expands our knowledge in sex chromosome evolution within blow flies and will reinforce the study of sex chromosome evolution in other species with diverse sex chromosome systems.

Changes in the structure, content and morphology of chromosomes accumulate over evolutionary time and contribute to cell, developmental and organismal biology. The axolotl (Ambystoma mexicanum) is an important model for studying these changes because: 1) it provides important phylogenetic perspective for reconstructing the evolution of vertebrate genomes and amphibian karyotypes, 2) its genome has evolved to a large size (~10X larger than human) but has maintained gene orders, and 3) it possesses potentially young sex chromosomes that have not undergone extensive differentiation in the structure that is typical of many other vertebrate sex chromosomes (e.g. mammalian XY chromosomes and avian ZW chromosomes). Early chromosomal studies were performed through cytogenetics, but more recent methods involving next generation sequencing and comparative genomics can reveal new information. Due to the large size and inherent complexity of the axolotl genome, multiple approaches are needed to cultivate the genomic and molecular resources essential for expanding its utility in modern scientific inquiries. This dissertation describes our efforts to improve the genomic and molecular resources for the axolotl and other salamanders, with the aim of better understanding the events that have driven the evolution of vertebrate (and amphibian) chromosomes. First, I review our current state of knowledge with respect to genome and karyotype evolution in the amphibians, present a case for studying sex chromosome evolution in the axolotl, and discuss solutions for performing analyses of large vertebrate genomes. In the second chapter, I present a study that resulted in the optimization of methods for the capture and sequencing of individual chromosomes and demonstrate the utility of the approach in improving the existing Ambystoma linkage map and generating targeted assemblies of individual chromosomes. In the third chapter, I present a published work that focuses on using this approach to characterize the two smallest chromosomes and provides an initial characterization of the huge axolotl genome. In the fourth chapter, I present another study that details the development of a dense linkage map for a newt, Notophthalmus viridescens, and its use in comparative analyses, including the discovery of a specific chromosomal fusion event in Ambystoma at the site of a major effect quantitative trait locus for metamorphic timing. I then describe the characterization of the relatively undifferentiated axolotl sex chromosomes, identification of a tiny sex-specific (W-linked) region, and a strong candidate for the axolotl sex-determining gene. Finally, I provide a brief discussion that recapitulates the main findings of each study, their utility in current studies, and future research directions. The research in this dissertation has enriched this important model with genomic and molecular resources that enhance its use in modern scientific research. The information provided from evolutionary studies in axolotl chromosomes shed critical light on vertebrate genome and chromosome evolution, specifically among amphibians, an underrepresented vertebrate clade in genomics, and in homomorphic sex chromosomes, which have been largely unstudied in amphibians.

No description available.

The allocation of resources to male or female progeny, or to male or female reproductive function more generally, is one of the most important life history decisions a sexually reproducing individual must ever make. Sex determination is thus a fundamental process, yet the mechanisms which control it are surprisingly diverse. In this thesis, I examine sex determination in the plant species Mercurialis annua L. (Euphorbiaceae). I assess the mechanism of sex determination operating in dioecious and androdioecious populations of M. annua and also investigate the conservation and evolution of sex-determining mechanisms across the annual mercury clade, the lineages of which display exceptional variation in sexual system. First, using crosses, I establish that sex in dioecious M. annua is controlled by a single-locus genetic mechanism, consistent with recent work that identified a single male-linked DNA marker in the species. My search for new sex-linked genes revealed none, however, suggesting that M. annua possesses at most a small non-recombining region around sex-determining loci. Why many dioecious plants lack heteromorphic sex chromosomes is still poorly understood and I consider explanations for this. I extend my investigation by comparing genetic diversity between loci that differ in their linkage to the sex-determining locus. I find a single male-linked marker to possess significantly lower diversity than autosomal loci, but no difference in the diversity of partially sex-linked and non-sex-linked genes. I also assess the conservation of a sex-linked marker among annual mercury lineages and conduct crosses between lineages to examine the conservation of sex determination. My findings indicate a conserved mechanism of single-locus genetic sex determination and I consider the role polyploidisation and hybridisation have played in sexual system evolution and the modification of sex-determining mechanisms in the clade. Finally, I assess the presence of environmental sex determination in androdioecious M. annua, concluding that although male frequency is not influenced by growing density, a degree of sexual lability exists in the lineage.

<div>Chromosomal mechanisms of sex determination vary greatly in phylogenetically closely related species, indicative of rapid evolutionary rates. Sex chromosome karyotypes are generally conserved within families; however, many species have derived sex chromosome configurations. Insects display a plethora of sex chromosome systems due to rapid diversification caused by changes in evolutionary processes within and between species. A good example of such a system are insects in the blow fly family Calliphoridae. While cytogenetic studies observe that the karyotype in blow flies is highly conserved (five pairs of autosomal chromosomes and one pair sex chromosome), there is variation in sex determining mechanisms and sex chromosome structure within closely related species in blow flies. The evolutionary history of sex chromosomes in blow fly species have not been fully explored. Therefore, the objective of this research was to characterize the sex chromosome structures in four species of blow flies and investigate the selective forces which have played a role in shaping the diverse sex chromosome system observed in blow flies. The blow fly species used in this study are Phormia regina, Lucilia cuprina, Chrysomya rufifacies and Chrysomya albiceps. Phormia regina,and Lucilia cuprina have a heteromorphic sex chromosome system and are amphogenic (females produce both male and female offspring in equal ratio). In contrast, Chrysomya rufifacies and Chrysomya albiceps, have a homomorphic sex chromosome system, are monogenic (females produce unisexual progeny), have two types of females (arrhenogenic females – male producers and thelygenic females – female producers), and sex of the offspring is determined by the maternal genotype. </div><div>To accomplish these tasks, a total of nine male and female individual draft genomes for each of the four species (including three individual draft genomes of Chrysomya rufifacies – male, and the two females) were sequenced and assembled providing genomic data to explore sex chromosome evolution in blow flies. Whole genome analysis was utilized to characterize and identify putative sex chromosomal sequences of the four blow fly species. Genomic evidence confirmed the presence of genetically differentiated sex chromosomes in P. regina and L. cuprina; and genetically undifferentiated sex chromosomes in C. rufifacies and C. albiceps. Furthermore, comparative analysis of the ancestral Dipteran sex chromosome (Muller element F in Drosophila) was determined to be X-linked in P. regina and L. cuprina contributing to sex chromosome differentiation but not sex-linked in C. rufifacies and C. albiceps. Evolutionary pressures are often quantified by the ratio of substitution rates at non-synonymous (dN) and synonymous (dS) sites. Substitution rate ratio analysis (dN/dS) of homologous genes indicated a weaker purifying selection may have contributed to the loss of sex-linked genes in Muller element F genes of the undifferentiated sex chromosome as compared to the differentiated sex chromosome system. Overall, the results presented herein greatly expands our knowledge in sex chromosome evolution within blow flies and will reinforce the study of sex chromosome evolution in other species with diverse sex chromosome systems.</div><div><br></div>

Parmi plus de 20000 espèces de trématodes hermaphrodites, les Schistosomatidae ont un statut particulier car ils sont gonochoriques (i.e. deux sexes séparés). Le gonochorisme chez ces espèces, et leur dimorphisme sexuel, seraient en fait une stratégie d’adaptation à leur habitat : le système veineux des vertébrés à sang chaud, dont l’Homme. Malgré un mode chromosomique de déterminisme du sexe (i.e. hétérogamétie femelle ZW), les individus mâles et femelles demeurent phénotypiquement identiques durant tous les stades larvaires de leur cycle de vie hétéroxène. La différenciation sexuelle n’a lieu qu’après l’infestation de leur hôte définitif. Dans ce travail, nous nous sommes intéressés aux facteurs moléculaires déclenchant cette différenciation chez Schistosoma mansoni. Nous avons établi le profil d’expression sexe-dépendant de gènes conservés de la cascade de détermination/différenciation chez les animaux : les DMRT (Double-sex and Male-abnormal-3 Related Transcription Factors). Nous avons par ailleurs généré un transcriptome comparatif mâle/femelle (RNA-seq) sur 5 stades de développement in vivo, dont 3 stades « schistosomules » inédits. Cela nous a permis d’identifier de potentiels gènes « clés » de la différenciation sexuelle et de souligner l’importance de l’interaction hôte-parasite. Enfin, par la combinaison de cette approche transcriptomique et d’une analyse épigénomique (ChIP-seq), nous avons montré une dynamique de la compensation de dose génique au cours du cycle de vie chez les femelles ainsi que la mise en place d’une stratégie transcriptionnelle particulière chez les mâles, optimisant leur développement dans l’hôte et ainsi, leur succès reproducteur. / Parasitic flatworms include more than 20.000 species that are mainly hermaphrodites. Among them, the hundred species of Schistosomatidae are intriguing because they are gonochoric. The acquisition of gonochorism in these species is supposed to provide genetic and functional advantages to adapt to their hosts: warm-blooded animals. Sex of schistosomes is genetically determined at the time of fertilization (i.e. ZW female heterogametic system). However, there is no phenotypic dimorphism through all the larval stages of its complex lifecycle: sexual dimorphism appears only in the definitive host. The molecular mechanisms triggering this late sexual differentiation remain unclear, and this is precisely the topic of our present work. We performed transcriptomic (RNA-Sequencing and quantitative-PCRs) and structural (ChIP-Sequencing) analyses at different stages of Schistosoma mansoni development. Here, we present data suggesting that the sexual differentiation relies on a combination of genetic and epigenetic factors. In a genetic point of view, we show a sex-associated expression of the DMRT genes (Double-sex and Mab-3 Related Transcription Factors) that are known to be involved in sex determination/differentiation through all the animal kingdom. In addition, we propose new potential sex-determining key genes and a pivotal role of host-pathogen interaction at the time of development. In a structural point of view, we highlight a dynamic status of dosage compensation in females and chromatin modifications in males. This intense remodeling reveals a specific transcriptomic strategy which optimizes male development and beyond that, schistosomes reproductive success.