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.

With rapid advances in sequencing technologies and precipitous decreases in cost, public sequence databases have increased in size apace. However, experimental characterization of novel genes and their products remains prohibitively expensive and time consuming. For these reasons, bioinformatics approaches have become increasingly necessary to generate hypotheses of biological function. Phylogenomic approaches use phylogenetic methods to place genes, chromosomes, or whole genomes within the context of their evolutionary history and can be used to predict the function of encoded proteins. In this thesis, two new phylogenomic methods and software implementations are presented that address the problems of subcellular localization prediction and functional divergence prediction within protein families respectively. Most of the widely used programs for subcellular localization prediction have been trained on model organisms and ignore phylogenetic information. As a result, their predictions are not always reliable when applied to phylogenetically divergent eukaryotes, such as unicellular protists. To address this problem, PhyloPred-HMM, a novel phylogenomic method was developed to predict sequences that are targeted to mitochondria or mitochondrion-related organelles (hydrogenosomes and mitosomes). This method was compared to existing prediction methods using an existing test dataset of mitochondrion-targeted sequences from well-studied groups, sequences from a variety of protists, and the whole proteomes of two protists: Tetrahymena thermophila and Trichomonas vaginalis. PhyloPred-HMM performed comparably to existing classifiers on mitochondrial sequences from well-studied groups such as animals, plants, and Fungi and better than existing classifiers on diverse protistan lineages. FunDi, a novel approach to the prediction of functional divergence was developed and tested on 11 biological datasets and two large simulated datasets. On the 11 biological datasets, FunDi appeared to perform comparably to existing programs, although performance measures were compromised by a lack of experimental information. On the simulated datasets, FunDi was clearly superior to existing methods. FunDi, and two other prediction programs, was then used to characterize the functional divergence in two groups of plastid-targeted glyceraldehyde-3-phosphate dehydrogenases (GAPDH) adapted to roles in the Calvin cycle. FunDi successfully identified functionally divergent residues supported by experimental data, and identified cases of potential convergent evolution between the two groups of GAPDH sequences.

This thesis investigates species diversity and patterns of molecular evolution in Arachnida through DNA barcoding. The first chapter assesses mite species richness through comprehensive sampling at a subarctic location in Canada. Barcode analysis of 6279 specimens revealed nearly 900 presumptive species with high rates of turnover between major habitat types, demonstrating the utility of DNA barcoding for biodiversity surveys of understudied taxa. The second chapter explores nucleotide composition, indel occurrence, and rates of amino acid evolution in Arachnida. The results suggest a significant shift in nucleotide composition in the arachnid subclasses of Pulmonata (GC = 37.0%) and Apulmonata (GC = 34.2%). Indels were detected in five apulmonate orders, with deletions being much more common than insertions. Finally, rates of amino acid evolution were detected among the orders, and were negatively correlated with generation length, suggesting that generation time is a significant contributor to variation in molecular rates of evolution in arachnids.

In this thesis, I present a model of multidomain evolution with associated algorithms and software for phylogenetic analysis of multidomain families, as well as applications of this novel methodology to case-studies and the human genome. Phylogenetic analysis is of central importance to understanding the origins and evolution of life on earth. In biomedical research, molecular phylogenetics has proved an essential tool for practical applications. Current molecular phylogenetic methods are not equipped, however, to model many of the unique characteristics of multidomain families. Genes that encode this large and important class of proteins are characterized by a mosaic of sequence fragments that encode structural or functional modules, called domains. Multidomain families evolve via domain shuffling, a process that includes insertion, internal duplication, and deletion of domains. This versatile evolutionary mechanism played a transformative role in major evolutionary transitions, including the emergence of multicellular animals and the vertebrate immune system. Multidomain families are ill-suited to current methods for phylogeny reconstruction due to their mosaic composition. Different regions of the same protein may have different evolutionary histories. Moreover, a protein may contain domains that also occur in otherwise unrelated proteins. These attributes pose substantial obstacles for phylogenetic methods that require a multiple sequence alignment as input. In addition, current methods do not incorporate a model of domain shuffling and hence, cannot infer the events that occurred in the history of the family. I address this problem by treating a multidomain family as a set of co-evolving domains, each with its own history. If the family is evolving by vertical descent from a conserved set of ancestral domains, then all constituent domains will have the same phylogenetic history. Disagreement between domain tree topologies is evidence that the family evolved through processes other than speciation and gene duplication. My algorithms exploit this information to reconstruct the history of domain shuffling in the family, as well as the timing of these events and the ancestral domain composition. I have implemented these algorithms in software that outputs the most parsimonious history of events for each domain family. The software also reconstructs a composite family history, including duplications, insertions and losses of all constituent domains and ancestral domain composition. My approach is capable of more detailed and accurate reconstructions than the widely used domain architecture model, which ignores sequence variation between domain instances. In contrast, my approach is based on an explicit model of events and captures sequence variation between domain instances. I demonstrate the utility of this method through case studies of notch-related proteins, protein tyrosine kinases, and membrane-associated guanylate kinases. I further present a largescale analysis of domain shuffling processes through comparison of all pairs of domain families that co-occur in a protein in the human genome. These analyses suggest that (1) a remarkably greater amount of domain shuffling may have occurred than previously thought and (2) that it is not uncommon for the same domain architecture to arise more than once through independent events. This stands in contrast to earlier reports that convergent evolution of domain architecture is rare and suggests that incorporating sequence variation in evolutionary analyses of multidomain families is a crucial requirement for accurate inference.

<p>Recombination is the process in which genetic material is exchanged between one's homologous chromosome pairs during egg or sperm development (meiosis). Recombination is necessary for proper segregation of chromosomes during meiosis, and also plays a role in purging deleterious mutations, accelerating adaptation, and influencing the distribution of genomic features over evolutionary time. While recombination is clearly an important process, recombination rate is known to vary within and between individuals, populations, and species. Furthermore, what causes this variation remains relatively unknown. Using empirical and sequenced based estimates of recombination rate for the closely related species <italic>Drosophila pseudoobscura</italic> and <italic>Drosophila miranda</italic>, I seek to understand where recombination happens across the genome, to what extent recombination changes between species, and what genomic features are responsible for these changes. These data will deepen our understanding of mechanisms determining the recombination landscape, and shed light on generalized patterns and exceptions of recombination rate variation across the tree of life.</p> / Dissertation

Cytochromes c are electron transfer proteins in which haem is covalently attached to the polypeptide chain via thioether bonds formed from thiol groups of the two cysteines and the two vinyl groups of haem. This attachment is a post translational process and in many species of bacteria as many as approximately twelve gene products, the functions of which are largely unknown, are required. In Gram-negative bacteria the assembly of the c-type cytochromes occurs in the periplasm. Cytochrome c₅₅₂ from the thermophilic organism Hydrogenobacter thermophilus is known to be expressed in the cytoplasm of Escherichia coli. This unique example of cytoplasmic assembly of a c-type cytochrome has previously been postulated to result from insertion of haem into the folded apoform of the cytochrome followed by non-catalysed attachment of the haem. This postulate is supported by the present work which has shown that the cytoplasmic assembly of this cytochrome c₅₅₂ continues in the absence of the E. coli ccm genes which are needed for 'normal' c-type cytochrome assembly in that organism. Attempts to test the postulate of spontaneous assembly of the cytochrome c₅₅₂ with in vitro experiments require large amounts of cytochrome c₅₅₂ and its apo protein. A number of procedures for preparing these proteins were investigated. Although a T7-based expression produced lower amounts than was expected, its use led to detection of the apo form of cytochrome c₅₅₂ in E. coli. It was shown that this apoform has some secondary structure, whereas mitochondrial apocytochrome c has a random coil conformation. This observation is consistent with, but does not prove, the postulate for cytochrome c₅₅₂ assembly. It was unexpectedly found that a strain of E. coli that produces abnormally large amounts of its endogenous c-type cytochromes also made large amounts of cytoplasmic cytochrome c₅₅₂. NMR studies on this material are consistent with a single and 'normal' attachment of the haem to the polypeptide. Thus the unusual cytoplasmic assembly was not different from the usual periplasmic assembly that occurs in the H. thermophilus itself. In E. coli there is a periplasmic cytochrome b₅₆₂ that is presumed to acquire its haem in the periplasm. Some of the ccm genes, required for c-type cytochrome assembly, are postulated to code for a system that transports haem to the periplasm. Cytochrome b- ₅₆₂ synthesis was not blocked by the absence of these genes. This implies that haem provision for cytochrome b₅₆₂ synthesis occurs independently of the ccm system. Apocytochrome b<sub>562 could be detected in E. coli with the ratio apo:holo being higher in a strain that produces c-type cytochromes to relatively low levels. It is suggested that the synthesis of both cytochrome b₅₆₂ and c-type cytochromes is at least partly a reflection of the rate of production of haem by the cells.

Membrane proteins are found in all kingdoms of life and are essential for cellular interactions with the environment. Although a large research effort have been put into this group many membrane proteins remains uncharacterized, both in terms of function and evolutionary history. We have estimated the component of α-helical membrane proteins within the human proteome; the membrane proteome. We found that the human membrane proteome make up 27% of all protein, which we could classify the majority of into 234 families and further into three major functional groups: receptors, transporters or enzymes. We extended this analysis by determining the membrane proteome of 24 organisms that covers all major groups of eukaryotes. This comprehensive membrane protein catalog of over 100,000 proteins was utilized to determine the evolutionary history of all membrane protein families throughout eukaryotes.  We also investigated the evolutionary history across eukaryotes of the antiviral Interferon induced transmembrane proteins (IFITM) and the G protein-coupled receptor (GPCR) superfamily in detail.  We identified ten novel human homologs to the IFITM proteins, which together with the known IFITMs forms a family that we call the Dispanins. Using phylogenetic analysis we show that the Dispanins first emerged in eukaryotes in a common ancestor of choanoflagellates and animals, and that the family later expanded in vertebrates into four subfamilies. The GPCR superfamily was mined across eukaryotic species and we present evidence for a common origin for four of the five main human GPCR families; Rhodopsin, Frizzled, Adhesion and Secretin in the cAMP receptor family that was found in non-metazoans and invertebrates, but has been lost in vertebrates. Here we present the first accurate estimation of the human proteome together with comprehensive functional and evolutionary classification and extend it to organisms that represents all major eukaryotic groups. Moreover, we identify a novel protein family, the Dispanins, which has an evolutionary history that has been formed by horizontal gene transfer from bacteria followed by expansions in the animal lineage. We also study the evolution of the GPCR superfamily throughout eukaryotic evolution and provide a comprehensive model of the evolution and relationship of these receptors.

Salmonella enterica serovar Typhimurium is a common cause of salmonellosis among humans and animals worldwide. In Australia, Typhimurium is responsible for over half of the salmonellosis cases. The Anderson phage-typing scheme is the primary means of long-term surveillance of Typhimurium outbreak isolates, and has played an important role in epidemiology. However, there exist quite a number of strains of Typhimurium that cannot be defined by the phage-typing scheme. Furthermore, the knowledge of evolutionary relationships among isolates of different phage types is still very limited and the genetic basis of phage type variation remains largely unknown. To address these issues, this study focused on molecular typing and evolution of Typhimurium. Fluorescent amplified-fragment length polymorphism (AFLP) was applied to 46 Typhimurium isolates comprising nine phage types in Australia using the restriction enzymes MseI and EcoRI and MseI +1 / EcoRI +1 primer pair combinations. The selected phage types, DT9, DT135, DT64, DT44, DT126, DT12a, DT1, DT141 and DT108, have been dominant or frequent phage types in animal and human infections in Australia in recent years. AFLP in the present study showed a very good discrimination power with Simpson index of diversity of 0.98, 35 different AFLP patterns were observed in the 46 isolates studied. The tree based on AFLP patterns showed good correlation with phage type, grouped most Typhimurium isolates by phage type, and differentiated all nine phage types. Furthermore, 84 phage-type specific polymorphic AFLP fragments, for which presence or absence correlated with phage type (including 25 with one exception to phage-type specificity) were observed in the 46 strains studied. Eighteen phage-type specific AFLP fragments were cloned and sequenced. Sixteen are of known genes or have a homologue in the databases. It was found a predominance of phage and plasmid genes rather than mutational changes in the AFLP fragments studied. Of the 18 cloned and sequenced AFLP fragments, only four relate to mutational changes in the S. enterica chromosome, the other 14 comprise DNA of mobile elements: nine are phage related, three are plasmid related and two are gain of DNA from unknown origin. Twelve of the 18 sequenced phage-type specific AFLP markers are polymorphic because the DNA is present or absent as indicated by Southern hybridization. Two of these markers were successfully used in preliminary PCR-based typing of 30 DT9 and 29 DT135 isolates from worldwide collections. 27 of the 30 DT9 isolates and all DT135 isolates tested were correctly categorized. The results implied a good potential to use the sequence of these fragments as the basis for a multiplex PCR or a microarray based molecular �phage� typing method for Typhimurium. This thesis also studied the molecular evolutionary relationships among the same set of 46 Typhimurium isolates using mutational changes detected by AFLP, or analysis of intergenic regions and their flanking genes in genome sequences. The complete genome sequence of Typhimurium LT2 was analysed by computer modelled AFLP. The polymorphic AFLP fragments, which matched with the modelled LT2 AFLP fragments, were amplified and sequenced by LT2 genome based primers to determine the changes. Forty-nine intergenic regions with higher pairwise differences between LT2 and Typhi CT18 were amplified and sequenced using LT2 genome based primers for one isolate of each phage type. 51 polymorphic sites were detected consisting of 18 in AFLP fragments and 33 in intergenic regions or their flanking genes. PCR-RFLP (restriction fragment length polymorphism) and SNaPshot were used to further investigate the distribution of the single nucleotide polymorphisms (SNPs) detected in intergenic regions in all isolates studied. Of the 18 mutational changes detected in AFLP fragments, eight were indels (insertions / deletions) and ten single base substitutions. Of the eight indels, four were in genes, three in intergenic regions, and one covered adjacent intergenic and coding regions. The four indels in genes all caused frameshift mutations, including three single base indels and one 19 bp deletion. Of the ten substitutions, one was in an intergenic region and nine in genes comprising three synonymous and six non-synonymous substitutions. Of the 33 polymorphic sites detected from sequences of 23 intergenic regions and their flanking genes, one was IS200 insertion and 32 single nucleotide polymorphisms (SNPs), of which 30 were single base substitutions and two were single base indels. Nine of the 33 variations were found in the flanking genes, which were all single base substitutions comprising four synonymous, four non-synonymous substitutions and one non-sense mutation. More non-synonymous than synonymous substitutions were found for those in coding regions within Typhimurium, indicating that slightly deleterious intraspecies mutations can be fixed within clones, such as various lineages of Typhimurium. The 51 polymorphic sites, which were inferred from sequences of both mutation related AFLP fragments, and intergenic regions and their flanking genes, gave a single phylogenetic tree of the 46 Typhimurium isolates studied. All sequences involved were compared with the homologous sequences in the available S. enterica genome sequences for serovars Typhi, Paratyphi A, Gallinarum, Enteritidis and Pullorum and this enabled the determination of the direction of the mutational changes in the isolates studied and the root of the phylogenetic tree. There were only two events inferred to have occurred twice, the remaining 49 polymorphisms can be explained by a single event. The data indicated that Typhimurium has a very strong clonal structure with a very low level of recombination over the time for diversification of Typhimurium as majority of clonal variations are from point mutations rather than recombination. The phylogenetic tree based on mutational changes showed that most Typhimurium isolates of a given phage type are in the same evolutionary group, but that some phage types appear to have arisen more than once. Comparison of the phylogenetic tree with AFLP data gave examples of unrelated isolates of a given phage type having common AFLP fragments comprising plasmid or phage genes, supporting the view that phage type can be determined by presence of specific phages or plasmids. The mutation-based tree showed that six of the nine phage types studied appeared to have a single origin, at least for the isolates studied. It also found that DT1 and DT44 had two independent origins even for the limited set of strains used. The distribution of DT12a isolates into two groups could be explained that the group of three DT12a isolates were derived from the other group of four DT12a isolates, where the root of the tree might be. The data also confirmed that DT64 arose from DT9. The phylogenetic tree that was generated based on essentially mutational changes provides clear relationships of the closely related Typhimurium isolates with high level of consistency and reasonable confidence. This study provided one of the few analyses of relationships of isolates within a clone. Matching actual AFLP with computer modeled AFLP and sequencing intergenic regions provide very good new strategies to identify mutational polymorphisms and to study the molecular evolutionary relationships in the closely related isolates.

The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on April 1, 2010). Vita. Thesis advisor: R. Michaels Roberts. "May 2008" Includes bibliographical references