Staphylococcus aureus is a notorious human pathogen associated with severe nosocomial and community-acquired infections. In addition, S. aureus is a major cause of animal diseases including skeletal infections of poultry and bovine and ovine mastitis, which are a large economic burden on the broiler chicken and dairy farming industries. The population structure of S. aureus associated with humans has been well studied. However, despite the prevalence of S. aureus infections in broiler flocks, our understanding of the diversity of poultry S. aureus is very limited. In this study, multilocus sequence typing was performed on 48 strains of S. aureus isolated from broiler chickens on farms in 6 countries on 4 different continents, in addition to 9 isolates from different species of reared game and wild birds in Scotland. This was followed by fine scale population genetic analysis of a subset of strains by single nucleotide polymorphism discovery. These studies reveal that the majority of S. aureus isolates from broiler chickens are the descendants of a single human-to-poultry host jump by a subtype of the worldwide human clonal complex 5 (CC5) clonal lineage unique to Poland. In contrast to human subtypes of the CC5 radiation, which demonstrate strong geographic clustering, the poultry CC5 clade was distributed in different continents, consistent with wide dissemination via the global poultry industry distribution network. In order to establish the molecular basis for avian specificity in the CC5 poultry clade, whole genome sequences were determined for a sequence type 5 (ST5) poultry isolate from Ireland and a basal human associated ST5 MRSA strain from Poland. Sequence analysis revealed that the poultry CC5 clade has undergone genetic diversification from its human progenitor strain by acquisition of novel mobile genetic elements from an avian-specific accessory gene pool, and by the inactivation of several proteins important for human disease pathogenesis. In order to examine the importance of positive selection in the adaptation of S. aureus to poultry and for S. aureus evolution, in general, genome-wide analysis of the ratio of synonymous to non-synonymous substitutions was performed on 30 strains from 3 humans and other animals, from diverse lineages. Positive selection has affected proteins from the majority of functional categories, resulting in diversification of the proteome, metabolome and replication capacity, which may be associated with adaptation of S. aureus to diverse environments. For several proteins, an elevated rate of non-synonymous substitutions unique to animal-associated lineages is consistent with a role for these proteins in host adaptation. Taken together, the results of this study have determined the evolutionary history of a major new animal pathogen that has undergone rapid avian host adaptation and intercontinental dissemination. The data highlight the importance of gene acquisition and loss and positive selection in the adaptive evolution of S. aureus.

Transposable elements (TEs), also known as jumping genes, are DNA sequences capable of mobilizing and replicating within the genome. In mammals, it is not uncommon for 50% of the genome to be derived from TEs, yet they remain an underutilized tool for tracking evolutionary change. With the increasing number of publicly funded genome projects and affordable access to next-generation sequencing platforms, it is important to demonstrate the role TEs may play in helping us understand evolutionary patterns. The research presented herein utilizes TEs to investigate such patterns at the genomic, specific, and generic levels in three distinct ways. First at the genomic level, an analysis of the historical TE activity within the thirteen-lined ground squirrel (Spermophilus tridecemlineatus) shows that non-LTR retrotransposon activity has been declining for the past ~26 million years and appears to have ceased ~5 million years ago. Since most mammals, and all other rodents studied to date, have active TEs the extinction event in S. tridecemlineatus makes it a valuable model for understanding the factors driving TE activity and extinction. Second, we examined TEs as factors impacting genomic and species diversity. We found that DNA transposon insertions in Eptesicus fuscus, appear to have been exapted as miRNAs. When placed within a phylogenetic context a burst of transposon-driven, miRNA origination and the vespertilionid species radiation occurred simultaneously ~30 million years ago. This observation implies that lineage specific TEs could generate lineage specific regulatory pathways, and consequently lineage specific phenotypic differences. Finally, we utilized TEs to investigate their phylogenetic potential at the level of genus. In particular a method was developed that identified, over 670 thousand Ves SINE insertions in seven species of Myotis for use in future phylogenetic studies. Our method was able to accurately identify insertions in taxa for which no reference genome was available and was confirmed using traditional PCR and Sanger sequencing methods. By identifying polymorphic Ves insertions, it may be possible to resolve the phylogeny of one of the largest species radiations in mammals.

The substantial difference in ionic concentration and osmotic pressure between marine and freshwater environments creates a barrier to dispersal that relatively few metazoan lineages have been able to cross during the evolution of life on earth. Only about half of animal phyla have representatives in both marine and freshwater environments. Even within the phyla that contain freshwater species there are often large clades that continue to be exclusively marine. Interestingly, though, among some of the clades with freshwater species, this transition has occurred repeated. In order to begin to better understand the mechanisms that have allowed some marine lineages to colonize freshwater environments, I investigated the role of gene duplications in this process. First, using published annelid genomes I compared the gene copy number of the Na+/K+-ATPase alpha subunit gene family, the plasma membrane Ca2+ ATPase (PMCA) gene family, and the sarcoplasmic reticulum Ca2+ (SERCA) gene family between marine and freshwater species. I also used gene tree/species tree reconciliation to infer the time of those duplication events. There was a burst of duplications of the Na+/K+-ATPase alpha subunit gene that coincides with the colonization of freshwater habitats by annelids. The evidence of such a burst of duplications for the PMCA or SERCA gene families is inconclusive. Next, in order to increase the sample size and look for more gene families that were involved in the transition to freshwater habitats I downloaded 11 genomes from spiralian animals. I looked for specific gene families that showed a significant increase in size in freshwater species compared to marine species and identified the Na+/K+-ATPase alpha subunit gene family among others. I also used GO enrichment analysis to determine which GO terms were overrepresented in gene families that expanded along freshwater lineages and found terms related to ion transfer to be most common. Finally, I examined available mollusk genomes to compare size of the gene families of interest from the spiralian analyses between marine and freshwater mollusk species. I again found the Na+/K+-ATPase alpha subunit gene family to show a significant increase in size in the freshwater species. How marine animals were able to colonize freshwater habitats is one of the great questions in metazoan evolution and this work represents an important early step in understand this process.

Background: Long terminal repeats (LTRs, consisting of U3-R-U5 portions) are important elements of retroviruses and related retrotransposons. They are difficult to analyse due to their variability. The aim was to obtain a more comprehensive view of structure, diversity and phylogeny of LTRs than hitherto possible. Results: Hidden Markov models (HMM) were created for 11 clades of LTRs belonging to Retroviridae (class III retroviruses), animal Metaviridae (Gypsy/Ty3) elements and plant Pseudoviridae (Copia/Ty1) elements, complementing our work with Orthoretrovirus HMMs. The great variation in LTR length of plant Metaviridae and the few divergent animal Pseudoviridae prevented building HMMs from both of these groups. Animal Metaviridae LTRs had the same conserved motifs as retroviral LTRs, confirming that the two groups are closely related. The conserved motifs were the short inverted repeats (SIRs), integrase recognition signals (5' TGTTRNR ... YNYAACA 3'); the polyadenylation signal or AATAAA motif; a GT-rich stretch downstream of the polyadenylation signal; and a less conserved AT-rich stretch corresponding to the core promoter element, the TATA box. Plant Pseudoviridae LTRs differed slightly in having a conserved TATA-box, TATATA, but no conserved polyadenylation signal, plus a much shorter R region. The sensitivity of the HMMs for detection in genomic sequences was around 50% for most models, at a relatively high specificity, suitable for genome screening. The HMMs yielded consensus sequences, which were aligned by creating an HMM model (a 'Superviterbi' alignment). This yielded a phylogenetic tree that was compared with a Pol-based tree. Both LTR and Pol trees supported monophyly of retroviruses. In both, Pseudoviridae was ancestral to all other LTR retrotransposons. However, the LTR trees showed the chromovirus portion of Metaviridae clustering together with Pseudoviridae, dividing Metaviridae into two portions with distinct phylogeny. Conclusion: The HMMs clearly demonstrated a unitary conserved structure of LTRs, supporting that they arose once during evolution. We attempted to follow the evolution of LTRs by tracing their functional foundations, that is, acquisition of RNAse H, a combined promoter/polyadenylation site, integrase, hairpin priming and the primer binding site (PBS). Available information did not support a simple evolutionary chain of events.

The plastid genomes of land plants are generally highly conserved in gene content and order, genome organization, and rates of sequence evolution; however, a few groups have experienced genomic change. The previously published sequence of Pelargonium X hortorum (Geraniaceae) reveals the largest, most rearranged plastid genome among land plants, and rate heterogeneity and genomic change have been documented in the monocot family Poaceae. Three initiatives were taken to characterize plastid genome evolution better in these groups. First, I estimate and compare genome-wide rates of sequence evolution in Geraniaceae genes relative to other angiosperms. An analysis of nucleotide substitutions for 72 plastid genes from 47 angiosperms, including nine Geraniaceae, shows that values of dN are accelerated in ribosomal protein and RNA polymerase genes. dN/dS, an indicator of selection, is significantly elevated in the same two classes of genes and ATPase genes. Second, I sequenced three additional Geraniaceae plastid genomes (Erodium texanum, Geranium palmatum, and Monsonia speciosa) and compare these sequences to each other, P. X hortorum, and other rosids. Geraniaceae plastid genomes are highly variable in size, gene content and order, and base composition. The genome of M. speciosa is among the smallest land plant plastid genomes, and one copy of the IR region in E. texanum has been lost. Gene/intron loss and gene duplication are rampant in Geraniaceae plastid genomes, and a number of losses are phylogenetically inconsistent. To explain the unusual rates and patterns of genome evolution in Geraniaceae, I propose a model of aberrant DNA repair coupled with altered gene expression. Lastly, I characterize genome evolution in the family Poaceae and order Poales. There has been a recent surge in the availability of Poaceae sequences, but a comprehensive analysis of genome evolution had not been performed that included any non-grass Poales taxa. I present the sequence of Typha latifolia (Typhaceae), the first non-grass Poales sequenced to date, and I show that Poaceae plastid genomes exhibit increased genomic rearrangements and nucleotide substitutions. These analyses show the extent of lineage-specific rate acceleration on the branch leading to Poaceae and deceleration during the diversification of the family. / text

It has long been evident that genome size is not an accurate measure of organismal complexity. This paradox was “solved” with the discovery of nonfunctional and selfish DNA in the 1970s. However, emerging from this explanation was an enigma of complexity. Neither neutral nor adaptive models can account for all genome size variation across the tree of life. An organism with intraspecific variation is needed to investigate the functional role of genome size differences. Here I use different populations of the seed beetle, Callosobruchus maculatus, with a known intraspecific genome size variation of ~4%. It has previously been shown that a larger genome is associated with higher scores in fitness-related traits for this species. In this study, genome size is regressed with phenotypic plasticity along three different environmental gradients. Genome size did not correlate with plasticity in mass and development time along environmental gradients of temperature and host types. However, the results show that larger genomes are consistent with higher canalization of fitness under different food regimes. This further supports the idea that natural selection acts on genome size variation in this species.

Scleractinian corals represent the foundation species of one of the most diverse and productive ecosystem on earth, coral reefs. Corals not only constitute the trophic basis of these ecosystems, but also provide essential habitats and shelter for a wide variety of marine species, many of which are commercially relevant. They also provide other important ecosystem services such as food provision, shoreline protection and opportunities for ecotourism. Despite the ecological importance of corals, very little is known about how their soft-bodied ancestor evolved the ability to form a calcified skeleton and became the ecosystem builders they are today. Corallimorpharia are closely related to reef-building corals but lack the ability to form calcified skeletons. Here we assembled and annotated two draft genomes of the corallimorpharians, Amplexidiscus fenestrafer and Discosoma sp., and further provided an online interface to facilitate the use of these resources. The two genomes can not only inform on the current evolutionary gap in genomic resources for the subclass of Hexacorallia but also provide important resources for comparative genomic studies aiming at understanding the evolution of coral specific traits. Our broad phylogenomic approach using whole genome data, including phylogenetic analyses of nuclear encoding genes as well as genome-wide presence/absence information and synteny conservation from six hexacorallian species, provides robust evidence that corallimorpharians are a monophyletic sister group of scleractinians, therefore rejecting the “naked coral” hypothesis. Being the closest non-calcifying relative of scleractinian corals, corallimorpharians appear to be the best candidates to understand the evolutionary origin of coral calcification. Molecular divergence analysis of scleractinian coral and Corallimorpharia genes suggests that the soft-bodied ancestor of corals evolved the ability to calcify within approximately 80 million years after the divergence of these two orders. To uncover the molecular basis of coral skeletal formation and growth, we integrate genomic and transcriptomic data as well as skeletal proteomic data, and show that gene and domain duplications have been the main evolutionary mechanisms underlying the evolution of calcification in scleractinian corals.

This research focuses on non-Long Terminal Repeat (non-LTR) retrotransposons in the African malaria mosquito, Anopheles gambiae and other mosquito species. An unprecedented diversity of non-LTRs was discovered by genome analysis of the An. gambiae genome assembly. One hundred and four families were found by a reiterative and comprehensive search using the conserved reverse transcriptase domains of known non-LTRs from a number of organisms as the starting queries. These families range in copy number from a few to approximately 2000 and occupy at least 3% of the genome. An. gambiae non-LTRs represent 8 of the 15 previously defined clades, plus two novel clades, Loner and Outcast, raising the total number of known clades to 17. The first invertebrate L1 clade representatives were also found. All clades except one have families with sequence characteristics suggesting recent activity. Juan, a non-LTR of the Jockey clade originally discovered in the mosquito Culex pipiens quinquefasciatus (Mouches et al. 1991), has been implicated in horizontal transfer in three non-sibling species of the Aedes genus (Mouches, Bensaadi, and Salvado 1992). PCR was used to obtain sequences from 18 mosquito species of six genera. Phylogenetic analysis demonstrates predominant vertical inheritance of Juan elements among these species. There is strong evidence from sequence analysis supporting the recent activity of Juan in several divergent species. We hypothesize that the sustained activity (versus quick inactivation) of non-LTRs in mosquitoes may contribute to the diversity we observe in the An. gambiae genome today. Promoter and transcriptional analyses were performed for several families previously identified as potentially active elements based on sequence analysis. RT-PCR results indicate that transcripts are present in An. gambiae cell lines that contain sequences corresponding to 13 of 15 tested non-LTR families. The 5' UTRs of An. gambiae non-LTRs from the I, Jockey, and L1 clades support basal transcription in divergent mosquito cell lines from 3 species. The Jen-1 5'UTR did not support transcription in Ae. aegypti and had low activity in Ae. albopictus. In summary, this research shows that Non-LTRs have been highly successful genomic elements that have flourished in many divergent mosquito species. / Ph. D.

The rapid accumulation of numerous sequenced genomes has provided a golden opportunity for ancestral state reconstruction studies, especially in the whole genome reconstruction area. However, most ancestral genome reconstruction methods developed so far only focus on gene or replicon sequences instead of whole genomes. They rely largely on either detailed modeling of evolutionary events or edit distance computation, both of which can be computationally prohibitive for large data sets. Hence, most of these methods can only be applied to a small number of features and species. In this dissertation, we describe the design, implementation, and evaluation of an ancestral genome reconstruction system (REGEN) for bacteria. It is the first bacterial genome reconstruction tool that focuses on ancestral state reconstruction at the genome scale instead of the gene scale. It not only reconstructs ancestral gene content and contiguous gene runs using either a maximum parsimony or a maximum likelihood criterion but also replicon structures of each ancestor. Based on the reconstructed genomes, it can infer all major events at both the gene scale, such as insertion, deletion, and translocation, and the replicon scale, such as replicon gain, loss, and merge. REGEN finishes by producing a visual representation of the entire evolutionary history of all genomes in the study. With a model-free reconstruction method at its core, the computational requirement for ancestral genome reconstruction is reduced sufficiently for the tool to be applied to large data sets with dozens of genomes and thousands of features. To achieve as accurate a reconstruction as possible, we also develop a homologous gene family prediction tool for preprocessing. Furthermore, we build our in-house Prokaryote Genome Evolution simulator (PEGsim) for evaluation purposes. The homologous gene family prediction refinement module can refine homologous gene family predictions generated by third party de novo prediction programs by combining phylogeny and local gene synteny. We show that such refinement can be accomplished for up to 80% of homologous gene family predictions with ambiguity (mixed families). The genome evolution simulator, PEGsim, is the first random events based high level bacteria genome evolution simulator with models for all common evolutionary events at the gene, replicon, and genome scales. The concepts of conserved gene runs and horizontal gene transfer (HGT) are also built in. We show the validation of PEGsim itself and the evaluation of the last reconstruction component with simulated data produced by it. REGEN, REconstruction of GENomes, is an ancestral genome reconstruction tool based on the concept of neighboring gene pairs (NGPs). Although it does not cover the reconstruction of actual nucleotide sequences, it is capable of reconstructing gene content, contiguous genes runs, and replicon structure of each ancestor using either a maximum parsimony or a maximum likelihood criterion. Based on the reconstructed genomes, it can infer all major events at both the gene scale, such as insertion, deletion, and translocation, and the replicon scale, such as replicon gain, loss, and merge. REGEN finishes by producing a visual representation of the entire evolutionary history of all genomes in the study. / Ph. D.

Microsatellites are strings of short DNA motifs (≤6 bp) repeated in tandem across genomes of both prokaryotes and eukaryotes. In 20 years, they became popular genetic markers, successfully employed in the field of genetic mapping and gene hunting, as well as to address various biological questions at the individual, family, population and species level. However, evolutionary and demographic inferences from microsatellite polymorphism are hampered by controversy and ambiguity in the mutational processes of microsatellite sequences. Drawing on new data from genome projects, I review in Chapter 1 the concept of a microsatellite life cycle, which hypothesizes that microsatellites follow a life cycle from birth, through expansion, contraction, death and potentially resurrection. To document and understand this integrative concept of evolution, which could help improve current models of microsatellite evolution, there is an implicit need to study the evolution of microsatellites above the species level. A prerequisite of such comparative studies is therefore to find microsatellite loci that are conserved between different species. The near or full completion of many vertebrate genomes and their alignment against one another offer the ultimate approach to find genomic elements conserved over a large evolutionary scale. In Chapter 2, I present a new comprehensive method to find conserved microsatellites in whole genomes. Using the multiple-alignment of the human genome against those of 11 mammalian and five non-mammalian vertebrates, I examine the genomewide conservation of microsatellites, and challenge the general assumption that microsatellites are too labile to be maintained in distant species. In Chapter 3, I present similar results using the alignment of the newly sequenced platypus genome against those of three mammals, the chicken and the lizard, and incorporate these data into the framework created by the 17-genome analysis. This enlarged dataset was ground for attempting to reconstruct a vertebrate phylogeny from the presence/absence of microsatellites in the different genomes. Maximum parsimony analyses resulted in a tree much similar to that of the current view of the vertebrate phylogeny, while Bayesian analyses showed some discrepancies. This work opens a way for novel theoretical developments regarding the inference of ancestral states of microsatellites. In Chapter 4, I show how knowledge on conserved microsatellite sites can help for the development of a set of comparative primers useful across the Mammalia; implementing a similar protocol, nine conserved dinucleotide repeats were genotyped in 20 unrelated individuals of 18 species (nine sister species) encompassing the mammalian phylogeny, including marsupials and monotremes, and four microsatellites were sequenced in 4 individuals per species. My results emphasize conserved microsatellites as a new resource for genetic mapping and population studies. Finally, in Chapter 5, I recount the unexpected extent of structural change among mammalian orthologous microsatellites, including change of complexity, motif replacement and overall length variability. Altogether, these findings provide a comprehensive framework that may help in many areas of research, including molecular ecology, genome mapping, population genetics, and genome and microsatellite evolution.