octor of Philosophy(PhD) / Molecular Evolution is the key to explain the divergence of species and the origin of life on earth. The main task in the study of molecular evolution is the reconstruction of evolutionary trees from sequences data of the current species. This thesis introduces a novel algorithm for inferring evolutionary trees from genetic data using quartet-based approach. The new method recursively merges sub-trees based on a global statistical provided by the global quartet weight matrix. The quarte weights can be computed using several methods. Since the quartet weights computation is the most expensive procedure in this approach, the new method enables the parallel inference of large evolutionary trees. Several techniques developed to deal with quartets inaccuracies. In addition, the new method we developed is flexible in such a way that can combine morphological and molecular phylogenetic analyses to yield more accurate trees. Also, we introduce the concept of critical point where more than one possible merges are possible for the same sub-tree. The critical point concept can provide information about the relationships between species in more details and show how close they are. This enables us to detect other reasonable trees. We evaluated the algorithm on both synthetic and real data sets. Experimental results showed that the new method achieved significantly better accuracy in comparison with existing methods.

<p>Estimating substitution frequencies at sites that influence (Ka) and do not influence (Ks) the amino acid sequence is important for understanding the dynamics of molecular sequence evolution and the selective pressures that have shaped genetic variation. </p><p>The aim of this work was to gain a deeper understanding of the driving forces of substitution frequency variation in human pathogens. <i>Rickettsia prowazekii</i>, the causative agent of epidemic typhus and <i>Helicobacter pylori</i>, which has been implicated in gastric diseases were used as model systems. A specific focus was on the evolution of orphan genes in <i>Rickettsia</i>. Additionally, adaptive sequence evolution and factors influencing protein evolutionary rates in <i>H. pylori</i> were studied.</p><p>The comparative sequence analyses of orphan genes using Typhus Group (TG) and Spotted Fever Group (SFG) <i>Rickettsia</i>, indicate that orphan genes in the SFG correspond to pseudogenes in the TG and that pseudogenes in the SFG correspond to extensively degraded gene remnants in the TG. The analysis also showed that ancestral gene sequences could be reconstructed from extant gene remnants of closely related species. The studies of split genes in <i>R. conorii</i> indicate that many of the small fragmented ORFs are probably pseudogenes. Analysis of the <i>H. pylori </i>carbamoyl phosphate synthetase provided an opportunity to understand natural selection acting on a protein undergoing adaptive evolution. Factors such as network properties, protein-protein interactions, gene essentiality and chromosomal position on protein evolutionary rates in <i>H. pylori</i> were studied, of which antigenicity and gene location were identified as the strongest factors. </p><p>In conclusion, high Ka/Ks ratios in human pathogens may reflect either adaptive sequence evolution or gene deterioration. Distinguishing between the two is an important task in molecular evolution and also of great relevance for medical microbiology and functional genomics research.</p>

Estimating substitution frequencies at sites that influence (Ka) and do not influence (Ks) the amino acid sequence is important for understanding the dynamics of molecular sequence evolution and the selective pressures that have shaped genetic variation. The aim of this work was to gain a deeper understanding of the driving forces of substitution frequency variation in human pathogens. Rickettsia prowazekii, the causative agent of epidemic typhus and Helicobacter pylori, which has been implicated in gastric diseases were used as model systems. A specific focus was on the evolution of orphan genes in Rickettsia. Additionally, adaptive sequence evolution and factors influencing protein evolutionary rates in H. pylori were studied. The comparative sequence analyses of orphan genes using Typhus Group (TG) and Spotted Fever Group (SFG) Rickettsia, indicate that orphan genes in the SFG correspond to pseudogenes in the TG and that pseudogenes in the SFG correspond to extensively degraded gene remnants in the TG. The analysis also showed that ancestral gene sequences could be reconstructed from extant gene remnants of closely related species. The studies of split genes in R. conorii indicate that many of the small fragmented ORFs are probably pseudogenes. Analysis of the H. pylori carbamoyl phosphate synthetase provided an opportunity to understand natural selection acting on a protein undergoing adaptive evolution. Factors such as network properties, protein-protein interactions, gene essentiality and chromosomal position on protein evolutionary rates in H. pylori were studied, of which antigenicity and gene location were identified as the strongest factors. In conclusion, high Ka/Ks ratios in human pathogens may reflect either adaptive sequence evolution or gene deterioration. Distinguishing between the two is an important task in molecular evolution and also of great relevance for medical microbiology and functional genomics research.

Methylation of CpG islands located at the promoter regions is a mechanism which controls gene silencing and expression. Hyper or hypo methylation of these sites on promoter sequences have been associated with many diseases, like cancer. Even though promoter CpG islands and their methylation profiles are important regulators of gene expression, the exact mechanism of gene silencing through methylation is not known. Here, we have investigated the status of promoter CpG methylation under various evolutionary pressures by calculating the differences in promoter CpG content and methylation profiles at different pass points. In order to determine the list of genes under each category we have analyzed and compared the orthologs among 58 genomes available through ENSEMBL. The total number of CpG dinucleotides at the promoter regions of all groups of genes have been calculated and compared. Additionally, we have compared the experimentally determined methylation profiles of these CpG&#039 / s between human blood cells and fibroblast cells. While the promoter CpG content changed through common to newer genes, the number of the CpG units methylated found to be consistent. Here, we present the functional level analysis of common gene lists at different pass points and report the differences of the promoter CpG content and the methylation profiles among these groups with distinct evolutionary conservation status. We have also observed the conservation status of individual methylated CpG units on the low and high methylated genes. Our analysis revealed that the surrounding methylation content had a positive effect on the conservation of individual CpG&rsquo / s.

Many bacteria live in close association with other organisms such as plants and animals, with important implications for both health and disease. This thesis investigates bacteria that are well adapted to live inside an animal host, and describes the molecular evolutionary processes underlying host-adaptation, based on bacterial genome comparisons. Insect-transmitted bacteria of the genus Bartonella infect the red blood cells of mammals, and we investigate host adaptation and genome evolution in this genus. In Bartonella, many host-interaction systems are encoded in a highly variable chromosomal segment previously shown to be amplified and packaged into bacteriophage particles. Among all genes imported into the Bartonella ancestor, we identify the short gene cluster encoding these phage particles as the most evolutionary conserved, indicating a strong selective advantage and a role in niche adaptation. We also provide an overview of the remarkable evolutionary dynamics of type IV and type V secretion systems, including a detailed analysis of the type IV secretion system trw. Our results highlight the importance of recombination and gene conversion in the evolution of host-adaptation systems, and reveal how these mutational mechanisms result in strikingly different outcomes depending on the selective constraints. In the insect endosymbionts Buchnera and Blochmannia, we show that genes frameshifted at poly(A) tracts can remain functional due to transcriptional slippage. Selection against poly(A) tracts is very inefficient in these genomes compared to other bacteria, and we discuss why this can lead to increased rates of gene loss. Using the human pathogen Helicobacter pylori as a model, we provide a deeper understanding of why highly expressed genes evolve slowly. This thesis emphasizes the power of using complete genome sequences to study evolutionary processes. In particular, we argue that knowledge about the complex evolution of duplicated gene segments is crucial to understand host adaptation in bacteria.

Genomic organization of the major histocompatibility complex (MHC) can profoundly influence gene function and multigene family evolution. Situated at the interface of individual genetic variation and the adaptive immune response, MHC class I and II loci are intensively studied for disease associations and used as markers of adaptive genetic variation in evolutionary ecology research. Genomic sequence of MHC-containing cosmid clones from the red-billed gull (Larus scopulinus, Charadriiformes: shorebirds, gulls, and allies) was obtained for comparative analysis of avian MHC evolution. MHCI polymorphism was further investigated using cDNA library screening and locus-specific genotyping protocols. This first information regarding MHC organization and MHCI variation in charadriiforms suggests a complex evolutionary history to MHC architecture in birds. Duplication of MHCIIα loci in tandem MHCIIα/β pairs and their proximity to MHC-region gene COL11A2 are similar to arrangements in nonavian vertebrates, and contrast with the “minimal essential” MHC of the chicken (Gallus gallus, Galliformes: gamebirds). MHCI–TAP2 organization is shared with Galloanserae (gamebirds + waterfowl), as is a proposed major classical function for this MHCI gene. In contrast, the placement of MHCI genes adjacent to sequence from chromosomes 3, 5, and 22 of the chicken and zebra finch (Taeniopygia guttata, Passeriformes: perching birds) indicates interchromosomal rearrangements in birds and the possible genomic dispersal of nonclassical MHCI genes in the red-billed gull. Screening for avian malaria, genetic parentage tests, and field data from red-billed gulls at Kaikoura Peninsula, New Zealand were combined with MHCI genotypes to investigate relationships with disease and reproduction. Plasmodium infection was confirmed in red-billed gulls, and breeding condition was negatively associated with malarial infection and positively related to variation at the putative major MHCI locus. A low rate of extrapair paternity was identified across thirteen breeding seasons. Partners without extrapair young (EPY) had greater MHCI dissimilarity than was expected by chance, whereas lower individual MHCI variation and elevated hatching failure existed for pairs with EPY. In addition to contributing to studies of MHC evolution, sexual selection, and disease dynamics in the New Zealand avifauna, this research will facilitate studies of MHC genes in related charadriiforms, many of which are of conservation concern.

Genomic organization of the major histocompatibility complex (MHC) can profoundly influence gene function and multigene family evolution. Situated at the interface of individual genetic variation and the adaptive immune response, MHC class I and II loci are intensively studied for disease associations and used as markers of adaptive genetic variation in evolutionary ecology research. Genomic sequence of MHC-containing cosmid clones from the red-billed gull (Larus scopulinus, Charadriiformes: shorebirds, gulls, and allies) was obtained for comparative analysis of avian MHC evolution. MHCI polymorphism was further investigated using cDNA library screening and locus-specific genotyping protocols. This first information regarding MHC organization and MHCI variation in charadriiforms suggests a complex evolutionary history to MHC architecture in birds. Duplication of MHCIIα loci in tandem MHCIIα/β pairs and their proximity to MHC-region gene COL11A2 are similar to arrangements in nonavian vertebrates, and contrast with the “minimal essential” MHC of the chicken (Gallus gallus, Galliformes: gamebirds). MHCI–TAP2 organization is shared with Galloanserae (gamebirds + waterfowl), as is a proposed major classical function for this MHCI gene. In contrast, the placement of MHCI genes adjacent to sequence from chromosomes 3, 5, and 22 of the chicken and zebra finch (Taeniopygia guttata, Passeriformes: perching birds) indicates interchromosomal rearrangements in birds and the possible genomic dispersal of nonclassical MHCI genes in the red-billed gull. Screening for avian malaria, genetic parentage tests, and field data from red-billed gulls at Kaikoura Peninsula, New Zealand were combined with MHCI genotypes to investigate relationships with disease and reproduction. Plasmodium infection was confirmed in red-billed gulls, and breeding condition was negatively associated with malarial infection and positively related to variation at the putative major MHCI locus. A low rate of extrapair paternity was identified across thirteen breeding seasons. Partners without extrapair young (EPY) had greater MHCI dissimilarity than was expected by chance, whereas lower individual MHCI variation and elevated hatching failure existed for pairs with EPY. In addition to contributing to studies of MHC evolution, sexual selection, and disease dynamics in the New Zealand avifauna, this research will facilitate studies of MHC genes in related charadriiforms, many of which are of conservation concern.

<p>Determining the genetic basis of adaptation has become a central focus of evolutionary biology, and the incorporation of increasingly sophisticated analytical tools from molecular biology has made identifying causal genes a practical reality. The work presented herein addresses the effects of pleiotropic constraint on evolutionary change at the level of individual genes and genetic networks. In the first chapter, I combine molecular phylogenetic analyses and direct assays of enzymatic function to determine the evolutionary processes following a gene duplication in the anthocyanin pathway. My results show that, prior to duplication, the DFR gene was constrained from functional improvement by its multiple enzymatic roles. Following duplication, this constraint was released and adaptive evolution proceeded along both paralog lineages. In the second chapter, I determine the molecular genetic basis of a flower color transition that is associated with change in pollinator attraction in morning glories. A regulatory change in a branching gene in the flavonoid biosynthetic pathway restricted flux down the cyanidin-producing branch, conferring nearly exclusive production of red pelargonidin pigment in flowers. I further demonstrate that this regulatory change was restricted to floral tissue, and that ancestral pathway flux predominates in vegetative tissues. I propose that deleterious pleiotropic effects prevented evolutionary change via enzymatic changes in the pathway due to the numerous essential products downstream of this branching point. Together, these two results show that evolutionary change may be constrained by the molecular genetic context in which prospective adaptive mutations occur.</p> / Dissertation

Genes present in multiple copies and genes that contain regions of repetitive sequences can undergo concerted evolution, which results in homogenization of the nucleotide sequence of the genes or repetitive regions. In regions of tandem repeats, this occurs through misalignment of repeat units followed by unequal crossover, which generates two products with differing numbers of repeat units. Gene conversion is thought to lead to one of these products becoming fixed in a species. The homogenous sequence of previously studied genes that have been thought to undergo this process has made it difficult to determine the exact models involved. Here I examine concerted evolution in SM50, a sea urchin gene that encodes a protein involved in biomineralization. The repetitive region in the SM50 gene varies in length between species, and there is variability in each repeat unit as well. I examine the codon usage in SM50 in a variety of species, and discuss how purifying selection, substitutions, concerted evolution, and selection at the level of DNA sequence have played a role in the evolution of this gene. I also examine the structure and sequence of the repeat units, and purpose models that have led to the evolution of the repeat pattern seen in the different species examined. Finally, I have found variation in the number of repeat units within several species. This has allowed us to deduce the specific models of unequal crossover that led to this variation. The unique variation in the repetitive region of SM50 has enabled us to describe a model of how substitutions affect the model of misalignment and unequal crossover.

Plastid genomes of flowering plants are largely identical in gene order and content, but a few lineages have been identified with many gene and intron losses, genomic rearrangements, and accelerated rates of nucleotide substitutions. These aberrant lineages present an opportunity to understand the modes of selection acting on these genomes as well as their long-term stability. My research has focused on two areas within plastid genome evolution in Geraniaceae: first, an investigation of the diversity of unusual plastid genomes in a single genus, Erodium (Geraniaceae) for chapters one and three. Chapter two focuses on the evolution of subunits of the plastid-encoded RNA polymerase (PEP). The first chapter described the loss of plastid-encoded NADPH dehydrogenase (ndh) genes from a clade of 13 Erodium species. Divergence time estimates indicate this clade is less than 5 million years old. This recent loss of ndh genes in Erodium presents an opportunity to investigate changes in photosynthetic function through comparative biochemistry between Erodium species with and without plastid-encoded ndh genes. Second, I examined the evolution of the gene encoding the alpha subunit (rpoA) of PEP in three disparate angiosperm lineages—Pelargonium (Geraniaceae), Passiflora (Passifloraceae), and Annonaceae—in which this gene has diverged so greatly that it is barely recognizable. PEP is conserved in the plastid genomes of all photosynthetic angiosperms. I found multiple lines of evidence indicating that the genes remain functional despite retaining only ~30% sequence identity with rpoA genes from outgroups. The genomes containing these divergent rpoA genes have undergone significant rearrangement due to illegitimate recombination and gene conversion, and I hypothesized that these phenomena have also driven the divergence of rpoA. Third, I conducted a survey of plastid genome evolution in Erodium with the completion of 15 additional whole genomes. Except for Erodium and some legumes, all angiosperm plastid genomes share a quadripartite structure with large and small single copy regions (LSC, SSC) and two inverted repeats (IR). I discovered a species of Erodium that has re-formed a large inverted repeat. Demonstrating a precedent for loss and regain of the IR also impacts models of evolution for other highly rearranged plastid genomes. / text