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DNA methylation is an epigenetic modification that plays an important role in development and differentiation. The patterns of DNA methylation are largely established in early embryogenesis and maintained during development. Abnormal DNA methylation patterns have been associated with many human diseases, including cancer. Despite its importance, little is currently known about the mechanisms that determine DNA methylation patterns throughout the genome. To shed light on the molecular mechanisms that regulate DNA methylation, this study investigates whether DNA methylation patterns are established and maintained normally when human DNA is placed into a heterologous murine environment as opposed to its natural, endogenous chromosomal environment. Here, a previously generated transgenic mouse model, containing 117 kb of human DNA bearing the human α-globin cluster and all of its known regulatory elements, was analysed. The pattern of DNA methylation of the endogenous human α-globin cluster was compared with that of the transgenic cluster in the background of mouse embryonic stem cells (ESCs) and tissues. It was found that, although the normal human DNA methylation pattern was largely established and maintained in a mouse background, the region immediately around the human α-globin genes themselves is generally less methylated in mouse compared to human ESCs. It was found that regions adjacent and up to 2kb from the CpG islands (CGIs), so-called CGI shores, were unusually hypomethylated: this seems to be the result of an extension of CGIs in humanised mouse (hm) ESCs compared to human (h) ESCs. Furthermore, this hypomethylation appeared to increase during development in both erythroid and non-erythoid cells. To identify any cis-regulatory sequences responsible for the hypomethylated state of human CGI shores in the mouse, 2-4 kb human test sequences containing the CGI associated with the human α-globin 2 (α2) gene and its adjacent hypomethylated shore were re-integrated into the mouse α-globin locus via recombination-mediated cassette exchange (RMCE). Human CGI shores became hypomethylated in the context of the re-integrated test sequences, indicating that the appearance of hypomethylation is determined by the underlying human DNA sequence in the test fragments. In summary, the data presented here reveal that human CGIs become extended when placed in a mouse background leading to hypomethylation of human CGI shores in the mouse compared to the pattern of methylation at the normal endogenous human locus. These findings suggest that species-specific factors determine DNA methylation near CGIs. The transgenic mouse model provides an excellent system to dissect out species-specific regulation of CGI shore methylation. Furthermore, this study lays the foundation for future experiments addressing the role of DNA methylation in regulating human gene expression in the murine context, and examining the validity of transgenic mouse models for the study of human gene regulation.

Left/right (L/R) asymmetries, differences in morphology between the otherwise mirrored left- and right-hand sides of the body, are found in animals across the Bilateria. For many years it was thought that the mechanisms for establishment of these asymmetries had evolved separately in the three superphyla that constitute the Bilateria, but the discovery in 2009 that the TGF-beta ligand Nodal shares a conserved role in the Deuterostomia and Lophotrochozoa has re-ignited debate and interest in this field. In this thesis, work examining the establishment and maintenance of L/R asymmetries in the lophotrochozoan superphylum is presented, aimed at uncovering the wider conservation of these pathways across the Bilateria. Illumina sequencing and a range of de novo assembly techniques were used to derive genomic and transcriptomic data respectively for two primary model organisms, the limpet Patella vulgata and the serpulid annelid Pomatoceros lamarckii. Additionally, collaborative work lead to the derivation of transcriptomes for two other mollusc species and the genome of the monogont rotifer Brachionus plicatilis. A range of analysis was performed on these novel resources and is detailed here, with particular reference to the transcription factor cassettes contained in these datasets. These sequence resources formed the basis for examination of the breaking of initial symmetry in these model organisms. Known read-outs of correct establishment of L/R asymmetry, the expression of genes Nodal and Pitx on the right of the body, were codified in the course of normal development in P. vulgata. Pharmacological inhibitors of genes implicated in the establishment of L/R asymmetry, particularly ATPase ion channels, were then applied to embryos. After development, markers of normal development were assayed for signs of bilateral inversion. Although radialised phenotypes were observed, it is unclear whether these are specifically the result of L/R asymmetry defects. The localisation of ATPase mRNA and serotonin, often posited as a small molecule potential morphogen, were also assayed, although no conclusions could be drawn as to a role in the establishment of L/R asymmetry for these molecules, counter to some evidence from vertebrates. Once symmetry is broken, the TGF-beta pathway is responsible for the communication, specification and maintenance of tissue identity across the L/R axis. The novel sequence resources described in this thesis provided a comprehensive window into this signalling cassette, and detailed here is a treatment of the TGF-beta pathway within the Lophotrochozoa. Ligand diversity has increased markedly in some clades, while signal transduction and regulatory steps are relatively unchanged. This work has increased our knowledge of lophotrochozoan biology and particularly the mechanisms underpinning the establishment of asymmetry in this under-researched clade, however, much remains to be discovered about the ultimate origin of asymmetry itself.

Not all transcribed DNA encodes protein, and some of these noncoding RNAs (ncRNAs), such as roX1 and roX2, may play important roles in the cell. The functional roles of the majority of these, however, remain largely unknown. In this thesis, I first used EST and mRNA evidence to define 2,788 lincRNA loci within the Drosophila melanogaster genome. I suggest that up to 1,652 of these are functional, as 1,411 show evidence for significant evolutionary constraint while 241 fast-evolving loci are enriched in short RNA species. A distinct set of 1,119 lincRNA loci were defined by RNA-seq, the vast majority of which show clear primary sequence constraint. Their expression profiles and enrichment in particular chromatin domains indicate that these lincRNAs are likely involved in developmental regulation. I also identified 42 potential analogous lincRNAs with shared genomic locations between Drosophila and mouse. Constrained, non-embryonic lincRNAs defined by ESTs are transcribed preferentially in the vicinity of protein-coding genes encoding transcription factors and I demonstrated that one of these, which I name dEvf-2, positively regulates the expression of its genomically adjacent transcription factor, Dll, in cell culture. Finally, I used a reverse genetics approach to search for lincRNA promoter mutations and examined the effect of these on lincRNA expression. My findings suggest that many, previously unknown, functional lincRNAs exist within the Drosophila genome and are worthy of further in-depth experimental investigation.

Sex chromosomes experience distinct evolutionary environments, due to their unusual pattern of inheritance, and studies of sex chromosome evolution can shed light on the fundamental evolutionary forces acting across the genome as a whole. Here, I combine genomic and transcriptomic data across a wide range of avian species to explore the evolutionary processes governing sex chromosome evolution. Birds are female heterogametic and therefore it is possible, via comparisons with male heterogametic species, to identify the fundamental factors driving sex chromosome evolution, versus those associated with sex. In this thesis, I uncover a complex mosaic of recombination suppression between the Z and W chromosomes, characterized by repeated and independent divergence of gametologs, together with ongoing genetic exchange. Additionally, I highlight the role of mating system, and interplay between evolutionary forces, in driving coding and expression evolution on the Z and W chromosomes. My findings indicate that although the Z chromosome is masculinized for male-specific effects, the magnitude of genetic drift acting on Z-linked genes is elevated in promiscuous relative to monogamous mating systems. In contrast, evolution of the female-limited W chromosome is governed predominately by purifying selection. Together, my results suggest that the role of the Z chromosome in encoding sexual dimorphisms may be limited, but that W-linked genes play a significant role in female-specific fitness. In conclusion, my findings reveal the power of mating system in shaping broad patterns of genome evolution.

The first draft sequence of the human genome was published over a decade ago, yet interpreting the functional importance of nucleotides in genomes is still an ongoing challenge. I took a comparative genomic approach to identify functional sequence using signatures of natural selection in DNA sequences. Mutations that are purged or propagated by selection mark sequences of significance for biological fitness. I developed and refined methods for estimating the quantity of sequence constrained with respect to insertions and deletions (indels) between two genome sequences, a quantity I termed αselIndel. This sequence is evolving more slowly than surrounding neutral sequence due to the purging of deleterious indel variants, and thus this sequence is likely to be functional. I estimated αselIndel between diverse mammalian and avian species pairs, and found a strong negative correlation between αselIndel and the divergence between the species’ genome sequences. This implies that functional sequence turns over rapidly as it is lost and gained over time. I quantified the variable levels of sequence constraint, and rates of sequence turnover, for different types of human biochemically annotated element. Furthermore, I found that similar rates of functional turnover have occurred across mammalian and avian evolution. Finally, I identified positively selected amino acid residues that may be important for Darwin’s finch beak development, and found evidence of adaptively evolving reproductive proteins in the ancestral songbird lineage. Collectively these results demonstrate the wide-spread nature of lineage-specific functional sequence with implications for understanding species traits and the use of model organisms to inform human biology.

The development of behavioural resistance in Anopheles mosquitoes is an important threat to the sustainability of current vector interventions, which selectively target mosquitoes that are found indoors. Changes in the following behaviour have been reported in several Anopheles vectors: biting time, host preference, biting and resting behaviour. However, a critical review of the published literature reveals that behavioural resistance has not been demonstrated clearly in Anopheles, highlighting important limitations in using entomological data to investigate the evolution of behaviour. The availability of next generation sequencing and low-cost custom genotyping technologies makes it possible to investigate behavioural resistance using genetic information as an alternative approach. Using such technologies, this thesis aims to investigate whether Anopheles vectors are evolving towards outdoor-biting behaviour ('exophagy') by examining the genetic difference between the indoor- and -outdoor population of the main African malaria vectors, An. coluzzii and An. gambiae from The Gambia and Uganda, respectively. We also investigated the role of olfactory-related genes in driving the potential genetic difference between the two populations. We found limited divergence between the two populations overall, however close inspection identified SNPs potentially associated with outdoor-biting behaviour as well as genomic regions displaying elevated levels of differentiation between the two populations. Most notably, gustatory receptors, Gr11 and Gr12 and the odour receptor, OR1 were identified as well as, genes related to transcription, signal transduction and catalysis. We discuss the potential role of these genes in determining biting behaviour and suggest that further investigation of these genomic regions is required to confirm the early-stage divergence of indoor- and outdoor-biting Anopheles populations.

The Mercurialis annua L. (Euphorbiaceae) species complex comprises a group of closely related lineages that present a wide range of sexual-systems, making it a valuable model for the study of plant sexual-system evolution. Within this polyploid complex, diploid populations are dioecious, and polyploid populations either monoecious or androdioecious (males coexist with functional hermaphrodites). The primary aim of this thesis was to use patterns of genetic diversity to elucidate the evolutionary origin and maintenance of the sexual-system diversity in M. annua. The phylogeny of the M. annua complex was reconstructed using chloroplast and ITS DNA sequence. This, in conjunction with morphometric analysis, showed that both hexaploid M. annua, and a novel species from the Canary Islands (newly described here as Mercurialis canariensis), were allopolyploid in origin. Such an origin for hexaploid M. annua suggests that androdioecy may have been able to arise in this group as a consequence of hybridisation between a monoecious lineage, tetraploid M. annua, and a dioecious lineage, M. huetii. Artificial crosses were used to show that hexaploid M. annua has disomic marker inheritance, and a statistical approach was developed to quantify genetic diversity and differentiation in polyploids with disomic inheritance. Strong gradients in genetic (allozyme) diversity at a pan-European scale were used to infer the existence of separate glacial refugia for dioecious and monoecious races of M. annua, at the eastern and western ends of the Mediterranean basin, respectively. A metapopulation model had previously been proposed to explain the ecological maintenance of androdioecy in M. annua. Here, population-level patterns of genetic diversity were used as an indirect test of this model. The discovery of lower within-population diversity, and of greater genetic differentiation between populations, for monoecious populations than for androdioecious populations was consistent with the metapopulation model, and suggests that androdioecy is maintained by the occurrence of regular local extinction.

This thesis addresses gaps that exist in the theory and knowledge of ancient DNA (aDNA). Much of the underlying basis of the field has been neglected in the excitement that followed the first aDNA studies. Therefore the results of many studies have been based on untested assumptions about the nature of post mortem DNA damage, sample preservation, contamination, and the efficacy of sample decontamination techniques. The validity of such results is questionable if the assumptions prove false. Hydrolytic post mortem DNA damage may modify recovered aDNA sequences. This thesis reports new insights into the biochemical basis of, predisposition of certain sequences and nucleotide positions towards, and subsequent effects of, such damage. Parallels of post mortem damage with in vivo mutation also enable insights into DNA sequence evolution. The long-term survival of DNA, and contamination of samples with exogenous DNA are two related problems characteristic to aDNA. The survival of endogenous DNA within bone, teeth and hair samples, the susceptibility of such samples to contamination, and the efficacy of decontamination techniques used to remedy such problems are investigated. The results highlight serious flaws in using bone and teeth as a DNA source. In contrast, the results demonstrate that hair may present a valuable DNA source for future studies. Numerous studies have reported the retrieval of ancient pathogen DNA from human samples. Analyses of the DNA content within teeth extracted from putative victims of the 2nd plague argue that such studies are at great risk from DNA degradation, and contamination arising due to environmental microorganisms. An extrapolation of these results using basic physical and chemical theory is used to evaluate the potential survival of aDNA in ancient Egyptian remains. This suggests that positive results from such samples are unlikely.

Studies of the evolution of the major histocompatibility complex (MHC) have been central to the understanding sexual selection and pathogen-mediated selection. The European badger Meles meles is well suited for exploring such questions because of its life history characteristics, reproductive biology and mating system. In this thesis, I examined both MHC class I and class II genes. Seven putatively functional sequences were found for class I genes and four for class II DRB genes. Evidence of past balancing selection of both genes was demonstrated by the dNdS ratio, by positive selection at the antigen-binding site (ABS) and by trans-species polymorphism of alleles within other mustelids and carnivores. MHC class I genes also showed evidence of concerted evolution, but domains showed different evolutionary histories. MHC genes may influence microbiota and odour of an individual and influence mating preferences. I examined the bacterial community of the subcaudal gland secretion and demonstrated a high number of bacterial species (56 operational taxonomic units), which cubs exhibited a higher diversity than adults. The microbiota may lead to an individual-specific odour as a cue signaling the MHC genotype of potential mating partners. I report the first evidence for a MHC- based mating preference in carnivores. Female badgers showed a MHC-assortative mate choice towards breeding with males that had functionally similar MHC genes, for MHC class II DRB genes. This applied to neighbouring-group matings. I also found considerable annual fluctuation in the occurrence of MHC-based mate choice. Based on genome-wide background in the same mating randomizations I found no evidence of inbreeding, which indicated that MHC similarity was apparently the actual target of mate choice. In line with MHC-assortative mate choice, MHC heterozygosity had no influence on the co-infection status. Individual MHC alleles did, however, associate with resistance and susceptibility to specific pathogens, suggesting that MHC diversity may be driven and maintained by pathogen-mediated selection through rare-allele advantages and/or fluctuating selection. My study of genetic characteristics, mate choice and pathogen pressures in a wild population revealed past and contemporary evolutionary process of the MHC genes. This increases knowledge of how the MHC may affect mating behaviour and sexual selection, ultimately influencing population processes.

Human populations in Africa have high levels of genetic, cultural and linguistic diversity. Despite this, only a small proportion of African populations have been studied from a genetics perspective. There is a particular dearth of information for Southern Africa, even though this region is one of the few places where hunter-gatherer, pastoralist and farmer populations remain, and where interaction between these groups can be studied. This thesis analyses novel populations from Lesotho, Namibia and South Africa, for both maternal and paternal markers, in order to provide a more accurate understanding of the history of this region. Analysis of biodemographic data for these populations highlights signatures of specific cultural traits. Interestingly, contrary to expectations, results indicate that in the patrilocal Basotho there are no differences in within and between area mtDNA and NRY variation, despite observations of a higher female than male migration rate. Females move preferentially at shorter distances than males, minimising the impact of the higher female migration rate. Further analysis of the Southern African samples indicates that the genetic composition of these populations is different to previously studied populations from Sub-Saharan Africa. Notably, there is a significantly higher maternal hunter-gatherer component, potentially as a result of an archeologically defined static frontier which existed along the Maloti/Drakensberg escarpment. Analysis of Namibian samples provides additional information about the history of populations with different lifestyles in the region, with support for a link between Southern Africa pastoralists and East Africa, while other Namibian populations appear genetically different from previously studied populations. Overall, this work demonstrates the diversity of populations in Southern Africa, improves understanding of the history of this region, and also emphasises the value of having access to geographically and ethnically well-defined samples.

The Neutral Indel Model is able to predict accurately the distribution of indel events in alignments of neutrally evolving genomic sequence. Here, I apply this model to a diverse range of metazoan species pairs, to a number of ends. First, I apply the Neutral Indel Model to alignments of genome sequences for species within the mammalian clade in order to estimate the quantities of functional DNA shared between species pairs. I demonstrate that as the evolutionary divergence between species pairs increases, estimates of functional sequence drop off dramatically. This pattern is not replicated in extensive simulations of genome sequence alignments, suggesting that functional (and mostly non-coding) sequence is turning over at a rapid rate. I also estimate that between 200 and 300 Mb (6.5-10%) of the human genome is under evolutionary constraint, a considerably higher quantity of sequence than has been estimated by previous whole genome analyses. Second, extending my analyses to consider more diverse metazoan species, I provide estimates for functional bases within organisms’ genomes that appear to mirror our conceptions of organismal complexity. Thirdly, I develop the Neutral Indel Model as a method for assessing genome sequence quality, by quantifying indel errors within alignments of closely related (ds < 0.1) species pairs. Applying this method to six primate genome sequence assemblies, I demonstrate that the frequency of indel error events per base varies up to six-fold. Further to this, I show that second generation sequencing technologies can be used to create high quality genome sequence assemblies and to ameliorate errors in pre-existing assemblies. Finally, I analyse patterns of indel mutations in primate transposable elements and show that indels are not randomly distributed within these sequences due to regularly spaced homo-nucleotide motifs.

During human immunodeficiency virus 1 (HIV-1) infection, the viral population is in constant battle with the host immune system. The cytotoxic T-lymphocyte (CTL) response, a branch of the adaptive immune response, is implicated in viral control and can drive viral evolution in the infected host population. Endogenous viral peptides, or ‘epitopes’, are presented to CTLs by human leukocyte antigen (HLA) class I molecules on the surface of infected cells where they may be identified as non-self. Mutations in or proximal to a viral epitope can result in ‘escape’ from CTLs targeting that epitope. The repertoire of epitopes which may be presented is dependent upon host class I HLA types. As such, reversion may occur after transmission due to changes in viral fitness and selection in the context of a new HLA background. Thus, parameters describing the dynamics of CTL escape and reversion are key to understanding how CTL responses within individuals relate to HIV-1 sequence evolution in the infected host population. Escape and reversion can be studied directly using biological assays and longitudinal viral sequence data, or indirectly by considering viral sequences across multiple hosts. Indirect approaches include tree based methods which detect associations between host HLA and viral sequence but do not estimate rates of escape and reversion, and ordinary differential equation (ODE) models which estimate these rates but do not consider the dependency structure inherent in viral sequence data. We introduce two models which estimate escape and reversion rates whilst accounting for the shared ancestry of viral sequence data. For our first model, we lay out an integrated Bayesian approach which combines genealogical inference and an existing epidemiological model to inform escape and reversion rate estimates. Using this model, we find evidence for correlation between escape rate estimates across widely separated geographical regions. We also observe a non-linear negative correlation between in vitro replicative capacity and escape rate. Both findings suggest that epistasis does not play a strong role in the escape process. Although our first model worked well, it had some key limitations which we address in our second method. Notably, by making a series of approximations, we are able account for recombination and analyse very large datasets which would be computationally infeasible under the first model. We verify our second approach through extensive simulations, and use the method to estimate both drug and HLA associated selection along portions of the HIV-1 genome. We test the results of the model using existing knowledge, and determine a collection of putative selected sites which warrant further investigation. Finally, we find evidence to support the notion that the CTL response played a role in HIV-1 subtype diversification.

Following their dispersal out of Africa, humans colonised all continents of the world save one, Antarctica. Whilst Europe was initially peopled soon after this exodus, paleoclimatic, archaeological, and historical evidence suggest that successive waves and migrations of people have contributed to the population resident in Europe today. I therefore examined the impact of past events on the European population through the analysis of DNA sampled both from contemporary Europeans, and from worldwide populations pertinent to its history. I genotyped and analysed data from the Y chromosomes of over 2,000 haplogroup R-M269 European men from over 30 different populations and, in combination with comparable datasets gathered from the literature, show that there it is not possible to assign a date to the origin of this lineage in Europe, and thus that any conclusion as to the ancient or recent spread of this lineage in Europe is unfounded. I also show that commonly used Y chromosome lineage dating techniques based on STR variation are biased by the markers used and conclusions based on such dates should be viewed with a large amount of caution. I next use genome-wide SNP data from 1,550 individuals from 95 worldwide populations to explore the population structure of Europe and present an analysis of the detailed structure of Europe in a novel analytical framework using ChromoPainter and fineSTRUCTURE. Admixture analysis based this data reveals distinct genomic inputs to peripheral European populations, from North Africa, Sub-Saharan Africa, the Middle East, and East Asia, and provides dates for this admixture within the last 1,000 years that correspond to the emergence and decline of empires and kingdoms in these regions of Europe. This novel analysis highlights the importance of recent historical events on European population structure, but also suggests a degree of ancient structure across European populations. Taken together, these analyses demonstrate the substantial effects of both ancient and recent migrations and mixture on the contemporary genetic structure of Europe.

Characterising and understanding factors that affect the rate of molecular evolution in proteins has played a major part in the development of evolutionary theory. The early analyses of amino acid substitutions stimulated the development of the neutral theory of molecular evolution, which later evolved into the nearly neutral theory. More recent work has lead to a better understanding of the role selection plays at the molecular level, but there is still limited understanding of how higher levels of protein organisation affect the way natural selection acts. The investigation of this question is the central aim of this thesis, which is addressed via the analysis of selective pressures in secondary protein structures in insects, mammals and fungi. The analyses for the first two groups were conducted using publically available datasets. To conduct the analyses in fungi, genome sequence data from the fungal genus Microbotryum (sequenced in our laboratory) was assembled and annotated, resulting in the development of a number of bioinformatics tools which are described here. The fungal, insect and mammalian datasets were interrogated with regard to a number of structural features, such as protein secondary structure, position of a site with regard to adaptively evolving sites, hydropathy and solvent-accessibility. These features were correlated with the signals of positive and purifying selection detected using phylogenetic maximum likelihood and Bayesian approaches. I conclude that all of the factors examined can have an effect on the rate of molecular evolution. In particular, disordered and hydrophilic regions of the protein are found to experience fewer physiochemical constraints and contain a higher proportion of adaptively evolving sites. It is also revealed that positively selected residues are ‘clustered’ together spatially, and these trends persist in the three taxa. Finally, I show that this variation in adaptive evolution is a result of both selective events and physiochemical constraint.

In this thesis I apply new sequencing technologies and analytical methods derived from genomics and computer science to the neuroanatomy of gene expression. The first project explores characteristics of gene expression across adult neocortical layers in a representative mammal – the mouse. Amongst the thousands of genes and transcripts differentially expressed across layers, I found common functional characteristics of genes that define certain layers, candidate cases of isoform switching, and over a thousand apparent long intergenic non-coding RNA transcripts. The second project compares patterns of gene expression in the structurally diverged adult derivatives of the pallium in mice and chickens. Overall, gene expression levels were moderately correlated between the two species. While expression patterns of ‘marker’ genes were only poorly conserved in these regions, there nevertheless was significant conservation of cross-species marker genes for homologous structures, cell types and functionally analogous regions. Many aspects of these data from both projects can now be easily browsed and searched from custom-built web interfaces. In addition to generating unprecedented genome-wide resources for the neuroscience community to explore the functional and structural dimensions of gene expression amongst different pallial regions in mammals and birds, this work also provides new insights into the widespread evolutionary shuffling of adult marker gene expression.

The study of sex allocation is one of the most successful areas in evolutionary biology: its theoretical predictions have been supported by experimental, observational and comparative approaches. Here, I develop sex allocation theory as follows: (1) I use fertility insurance theory to predict the sex ratio strategy of the malaria parasite, in response to human medical interventions that increase mortality and decrease fertility of the parasite’s various sexual stages; (2) Haplodiploidy has been suggested as a driver of the evolution of eusociality, as under this genetic system a female may be more related to her sister than to her own offspring. I examine a model considering queen versus worker control over the sex ratio of the colony and show that haplodiploidy alone does not explain the evolution of helping; (3) I follow up this study of the haplodiploidy hypothesis by examining the idea that split-sex ratios may favour the evolution of eusociality in haplodiploid species. I study the two mechanisms of split sex ratios, that are found in natural populations and may have been important in the transition to eusociality: queen virginity and queen replacement. I focus on the impact of worker reproduction by considering the effect of woker producing a fraction of the colony offspring and by considering variation in the workers’ offspring sex ratio. My analysis shows that worker reproduction does not promote the evolution of helping in haplodiploid species; (4) I examine the evolution and function of a sterile soldier caste in parasitoid wasps from the genus Encyrtidae. Two main functions have been hypothesized for the emergence of soldiers: spiteful mediation of a sex ratio conflict in mixed-sex broods, and altruistic protection and 7 facilitation of the development of relatives. I develop a model considering variation in the oviposition behaviour of females, that may produce single-sex or mixed-sex broods. I show that, in accordance with previous theory, females are expected to produce more soldiers than males, under the sex ratio conflict hypothesis. I also show that one of the consequences of this costly conflict is that females are favoured to produce single-sex broods over mixed-sex broods.

This work aimed to investigate the evolutionary origin of the involvement of the HIF Prolyl Hydroxylases (PHDs) in oxygen sensing. The α/β-heterodimer HIF (Hypoxia Inducible Factor) is a master regulator of oxygen homoeostasis in metazoans. In the nucleus, HIF binds to the Hypoxia Responsive Elements and forms a transcriptional complex that activates the transcription of a multitude of downstream genes. Under normoxic conditions, the Fe(II)- and oxygen-dependent PHDs catalyse 4R-prolyl-hydroxylation of the HIF α-subunit, which subsequently leads to its degradation. It had previously been proposed that the evolution of the HIF-pathway, shared by all metazoans but not found in other organisms, is linked to the rapid diversification of multicellular life during the Cambrian Explosion. This work investigates the structural and biochemical properties of a PHD of the basal metazoan Trichoplax adhaerens (taPHD), and a PHD-like enzyme of the protist Monosiga brevicollis (mbP4H). Two crystal structures of taPHD were obtained (1.2-1.3 Å), one containing a Trichoplax adhaerens HIFα subunit peptide (taODD). Comparison with crystal structures of human PHD2 showed a high degree of conservation of structural features and enzyme-substrate interactions. The prolyl-residue of taODD, shown to be hydroxylated by taPHD, is occupying the C4-endo conformation in the crystal structure, supporting the previously proposed mechanism of HIFα hydroxylation by PHD2 in humans. A conservation of biochemical properties with human PHD2, such as the formation of a stable enzyme-Fe(II)-2OG complex, was observed and could therefore be key to oxygen sensing by the PHDs. mbP4H was shown to catalyse 4R-prolyl-hydroxylation of taODD. It was proposed that the native substrate of mbP4H is a protein containing a prolyl-hydroxylation site similar to taODD, possibly with a YXXLAP motif. The study of biochemical properties and substrate selectivity of mbP4H suggests that the precursor of PHDs may have had similar properties to mbP4H. Further work on mbP4H could therefore yield clues about the evolutionary origin of HIF-prolyl hydroxylases in oxygen sensing and probe the previously proposed connection between metazoan life and HIF–mediated oxygen sensing.

The evolutionary options of a population are strongly influenced by the avail- ability of adaptive mutants. In this thesis, I use the concept of neutral networks to show that neutral drift can actually increase the accessibility of adaptive mu- tants, and therefore facilitate adaptive evolutionary change. Neutral networks are groups of unique genotypes which all code for the same phenotype, and are connected by simple point mutations. I calculate the size and shape of the networks in a small but exhaustively enumerated space of RNA genotypes by mapping the sequences to RNA secondary structure phenotypes. The qual- itative results are similar to those seen in many other genotype–phenotype map models, despite some significant methodological differences. I show that the boundary of each network has single point–mutation connections to many more phenotypes than the average individual genotype within that network. This means that paths involving a series of neutral point–mutation steps across a network can allow evolution to adaptive phenotypes which would otherwise be extremely unlikely to arise spontaneously. This can be likened to walking along a flat ridge in an adaptive landscape, rather than traversing or jumping across a lower fitness valley. Within this model, when a genotype is made up of just 10 bases, the mean neutral path length is 1.88 point mutations. Furthermore, the map includes some networks that are so convoluted that the path through the network is longer than the direct route between two sequences. A minimum length adaptive walk across the genotype space usually takes as many neutral steps as adaptive ones on its way to the optimum phenotype. Finally I show that the shape of a network can have a very important affect on the number of generations it takes a population to drift across it, and that the more routes between two sequences, the fewer generations required for a population to find an advantageous sequence. My conclusion is that, within the RNA map at least, the size, shape and connectivity of neutral networks all have a profound effect on the way that sequences change and populations evolve, and by not considering them, we risk missing an important evolutionary mechanism.

Fundamental to the complexity of the nervous system is the precise regulation in space and time of the production, maturation, and migration of neurons in the developing embryo. This is eloquently seen in the forming cranial sensory ganglia (CSG) of the peripheral nervous system. Placodes, which are transient pseudostratified neuroepithelia in the surface ectoderm of the embryo, are responsible for generating most of the neurons of the CSG. Placodal progenitors commit to the neuronal fate and delaminate from the epithelium as immature, multipolar neuroblasts. These neuroblasts reside in a staging area immediately outside the placode. Differentiation of the neuroblasts is intimately coupled to their adoption of a bipolar morphology and migration away from the staging area to the future site of the CSG. Thus the forming CSG is a highly tractable model to anatomically separate the three phases of a neuroblast’s lifetime: from neuroepithelial progenitor (in the placode), to immature neuroblast (in the staging area), to mature neuron (in the migratory stream). In this thesis, I used the forming CSG as a model to investigate the role of Regulator of G protein Signalling 3 (RGS3) in neuroblast commitment and differentiation. Promoters within introns of the RGS3 locus generate isoforms in which N-terminal sequences are sequentially truncated, but C-terminal sequences are preserved. Intriguingly, I found that expression of these isoforms in the forming CSG is temporally co-linear with their genomic orientation: longer isoforms are exclusively expressed in the progenitor placode; a medium isoform is expressed exclusively in the neuroblast staging area; and the shortest isoforms are expressed in the neuronal migratory stream. Furthermore, through loss- and gain-of-function experiments, I demonstrated that each of these isoforms plays a specific role in the differentiation state in which it is expressed: placode-expressed isoforms negatively regulate neurogenesis; the neuroblast-expressed isoform negatively regulates differentiation; and the neuron-expressed isoforms negatively regulate neuronal migration. The negative regulatory role which all isoforms play in different cell-biological contexts is intriguing in light of the fact that they all share a C-terminal RGS domain, which canonically negatively regulates G protein signalling. Through domain mutation and deletion, I showed that the RGS and N-terminal domains are important for the function of each isoform. Thus temporally co-linear expression within the RGS3 locus generates later-expressed isoforms which lack the regulatory N-terminal domains of the earlier-expressed isoforms, giving them new license to perform different biochemical functions. Lastly, I investigated the conservation and evolution of RGS3 and its isoforms. RGS3 was found to be present in all extant metazoans, and results from this thesis implicate it as the founding member of the R4 subfamily of RGS proteins. Furthermore, in the early vertebrate lineage, a critical domain was lost. This is intriguing in light of the fact that placodes in their stereotypic forms also emerged early in the vertebrate lineage. Ectopic overexpression of the full-length invertebrate RGS3 protein prevented pseudostratification of the vertebrate placode, suggesting that the domain loss in the early vertebrate lineage was important for the evolution of pseudostratified placodes and the expansion of the vertebrate nervous system. In summary, the work in this thesis has uncovered a previously unseen model of transcriptional regulation of a single locus: intragenic temporal co-linearity. Furthermore, the demonstrated functions of this regulation have profound implications on the generation and differentiation of vertebrate neurons, as well as the evolution of the vertebrate nervous system.

Transcription factors of the helix-loop-helix (HLH) gene family are widespread in the animal kingdom. Among them, members of HLH subfamilies such as ASCL, Neurogenin, NeuroD, COE, Atonal, Oligo, NSCL, Hairy/E(spl) and Hey (here referred to as neural HLH genes) have been shown to be fundamental for the development of the nervous system. They are expressed at different time periods of neuronal differentiation, from the specification of ectoderm towards a neural lineage, to the ultimate differentiation of neurons. Few HLH genes have been identified in the lamprey; however, considering the wide diversity of HLH gene subfamilies in metazoans, including vertebrates, it is very likely that lampreys possess a large repertoire of HLH genes in their genome. In the present study, the identification of several HLH genes in the lamprey genome, as well as the isolation and expression of different lamprey neural HLH genes is reported. As expected, a wide repertoire of HLH genes was identified in the sea lamprey (Petromyzon marinus) genome. On the other hand, the identification and expression analysis of different neural HLH genes of the ASCL, Neurogenin, COE and Hairy/E(spl) in the brook lamprey Lampetra planeri showed an overall conservation with other vertebrates, both at the sequence and expression pattern levels. In addition, novel features of the lamprey nervous system are revealed, such as the identification of possible new sensory cranial placodes in pharyngeal arches. Furthermore, these genes can serve as molecular markers for different cranial placodes and dorsal root ganglia (DRG), and their expression also highlights the presence of a ventricular zone in the brain and spinal cord, along with a complementary marginal zone. Finally, with the use of a Notch pathway inhibitor in developing L. planeri embryos, the regulation of expression of the isolated genes by the Notch signaling pathway was shown to be generally conserved between lampreys and gnathostomes in the spinal cord. This functional study also revealed that the lamprey spinal cord likely presents an independent developmental programme from the brain. All together, the present study shows that the analysis of neural HLH genes represents an excellent tool to understand the lamprey nervous system.

The development of behavioural resistance in Anopheles mosquitoes is an important threat to the sustainability of current vector interventions, which selectively target mosquitoes that are found indoors. Changes in the following behaviour have been reported in several Anopheles vectors: biting time, host preference, biting and resting behaviour. However, a critical review of the published literature reveals that behavioural resistance has not been demonstrated clearly in Anopheles, highlighting important limitations in using entomological data to investigate the evolution of behaviour. The availability of next generation sequencing and low-cost custom genotyping technologies makes it possible to investigate behavioural resistance using genetic information as an alternative approach. Using such technologies, this thesis aims to investigate whether Anopheles vectors are evolving towards outdoor-biting behaviour ('exophagy') by examining the genetic difference between the indoor- and -outdoor population of the main African malaria vectors, An. coluzzii and An. gambiae from The Gambia and Uganda, respectively. We also investigated the role of olfactory-related genes in driving the potential genetic difference between the two populations. We found limited divergence between the two populations overall, however close inspection identified SNPs potentially associated with outdoor-biting behaviour as well as genomic regions displaying elevated levels of differentiation between the two populations. Most notably, gustatory receptors, Gr11 and Gr12 and the odour receptor, OR1 were identified as well as, genes related to transcription, signal transduction and catalysis. We discuss the potential role of these genes in determining biting behaviour and suggest that further investigation of these genomic regions is required to confirm the early-stage divergence of indoor- and outdoor-biting Anopheles populations.

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Studies of the evolution of the major histocompatibility complex (MHC) have been central to the understanding sexual selection and pathogen-mediated selection. The European badger Meles meles is well suited for exploring such questions because of its life history characteristics, reproductive biology and mating system. In this thesis, I examined both MHC class I and class II genes. Seven putatively functional sequences were found for class I genes and four for class II DRB genes. Evidence of past balancing selection of both genes was demonstrated by the dNdS ratio, by positive selection at the antigen-binding site (ABS) and by trans-species polymorphism of alleles within other mustelids and carnivores. MHC class I genes also showed evidence of concerted evolution, but domains showed different evolutionary histories. MHC genes may influence microbiota and odour of an individual and influence mating preferences. I examined the bacterial community of the subcaudal gland secretion and demonstrated a high number of bacterial species (56 operational taxonomic units), which cubs exhibited a higher diversity than adults. The microbiota may lead to an individual-specific odour as a cue signaling the MHC genotype of potential mating partners. I report the first evidence for a MHC- based mating preference in carnivores. Female badgers showed a MHC-assortative mate choice towards breeding with males that had functionally similar MHC genes, for MHC class II DRB genes. This applied to neighbouring-group matings. I also found considerable annual fluctuation in the occurrence of MHC-based mate choice. Based on genome-wide background in the same mating randomizations I found no evidence of inbreeding, which indicated that MHC similarity was apparently the actual target of mate choice. In line with MHC-assortative mate choice, MHC heterozygosity had no influence on the co-infection status. Individual MHC alleles did, however, associate with resistance and susceptibility to specific pathogens, suggesting that MHC diversity may be driven and maintained by pathogen-mediated selection through rare-allele advantages and/or fluctuating selection. My study of genetic characteristics, mate choice and pathogen pressures in a wild population revealed past and contemporary evolutionary process of the MHC genes. This increases knowledge of how the MHC may affect mating behaviour and sexual selection, ultimately influencing population processes.

During human immunodeficiency virus 1 (HIV-1) infection, the viral population is in constant battle with the host immune system. The cytotoxic T-lymphocyte (CTL) response, a branch of the adaptive immune response, is implicated in viral control and can drive viral evolution in the infected host population. Endogenous viral peptides, or ‘epitopes’, are presented to CTLs by human leukocyte antigen (HLA) class I molecules on the surface of infected cells where they may be identified as non-self. Mutations in or proximal to a viral epitope can result in ‘escape’ from CTLs targeting that epitope. The repertoire of epitopes which may be presented is dependent upon host class I HLA types. As such, reversion may occur after transmission due to changes in viral fitness and selection in the context of a new HLA background. Thus, parameters describing the dynamics of CTL escape and reversion are key to understanding how CTL responses within individuals relate to HIV-1 sequence evolution in the infected host population. Escape and reversion can be studied directly using biological assays and longitudinal viral sequence data, or indirectly by considering viral sequences across multiple hosts. Indirect approaches include tree based methods which detect associations between host HLA and viral sequence but do not estimate rates of escape and reversion, and ordinary differential equation (ODE) models which estimate these rates but do not consider the dependency structure inherent in viral sequence data. We introduce two models which estimate escape and reversion rates whilst accounting for the shared ancestry of viral sequence data. For our first model, we lay out an integrated Bayesian approach which combines genealogical inference and an existing epidemiological model to inform escape and reversion rate estimates. Using this model, we find evidence for correlation between escape rate estimates across widely separated geographical regions. We also observe a non-linear negative correlation between in vitro replicative capacity and escape rate. Both findings suggest that epistasis does not play a strong role in the escape process. Although our first model worked well, it had some key limitations which we address in our second method. Notably, by making a series of approximations, we are able account for recombination and analyse very large datasets which would be computationally infeasible under the first model. We verify our second approach through extensive simulations, and use the method to estimate both drug and HLA associated selection along portions of the HIV-1 genome. We test the results of the model using existing knowledge, and determine a collection of putative selected sites which warrant further investigation. Finally, we find evidence to support the notion that the CTL response played a role in HIV-1 subtype diversification.

The first draft sequence of the human genome was published over a decade ago, yet interpreting the functional importance of nucleotides in genomes is still an ongoing challenge. I took a comparative genomic approach to identify functional sequence using signatures of natural selection in DNA sequences. Mutations that are purged or propagated by selection mark sequences of significance for biological fitness. I developed and refined methods for estimating the quantity of sequence constrained with respect to insertions and deletions (indels) between two genome sequences, a quantity I termed αselIndel. This sequence is evolving more slowly than surrounding neutral sequence due to the purging of deleterious indel variants, and thus this sequence is likely to be functional. I estimated αselIndel between diverse mammalian and avian species pairs, and found a strong negative correlation between αselIndel and the divergence between the species’ genome sequences. This implies that functional sequence turns over rapidly as it is lost and gained over time. I quantified the variable levels of sequence constraint, and rates of sequence turnover, for different types of human biochemically annotated element. Furthermore, I found that similar rates of functional turnover have occurred across mammalian and avian evolution. Finally, I identified positively selected amino acid residues that may be important for Darwin’s finch beak development, and found evidence of adaptively evolving reproductive proteins in the ancestral songbird lineage. Collectively these results demonstrate the wide-spread nature of lineage-specific functional sequence with implications for understanding species traits and the use of model organisms to inform human biology.

Left/right (L/R) asymmetries, differences in morphology between the otherwise mirrored left- and right-hand sides of the body, are found in animals across the Bilateria. For many years it was thought that the mechanisms for establishment of these asymmetries had evolved separately in the three superphyla that constitute the Bilateria, but the discovery in 2009 that the TGF-beta ligand Nodal shares a conserved role in the Deuterostomia and Lophotrochozoa has re-ignited debate and interest in this field. In this thesis, work examining the establishment and maintenance of L/R asymmetries in the lophotrochozoan superphylum is presented, aimed at uncovering the wider conservation of these pathways across the Bilateria. Illumina sequencing and a range of de novo assembly techniques were used to derive genomic and transcriptomic data respectively for two primary model organisms, the limpet Patella vulgata and the serpulid annelid Pomatoceros lamarckii. Additionally, collaborative work lead to the derivation of transcriptomes for two other mollusc species and the genome of the monogont rotifer Brachionus plicatilis. A range of analysis was performed on these novel resources and is detailed here, with particular reference to the transcription factor cassettes contained in these datasets. These sequence resources formed the basis for examination of the breaking of initial symmetry in these model organisms. Known read-outs of correct establishment of L/R asymmetry, the expression of genes Nodal and Pitx on the right of the body, were codified in the course of normal development in P. vulgata. Pharmacological inhibitors of genes implicated in the establishment of L/R asymmetry, particularly ATPase ion channels, were then applied to embryos. After development, markers of normal development were assayed for signs of bilateral inversion. Although radialised phenotypes were observed, it is unclear whether these are specifically the result of L/R asymmetry defects. The localisation of ATPase mRNA and serotonin, often posited as a small molecule potential morphogen, were also assayed, although no conclusions could be drawn as to a role in the establishment of L/R asymmetry for these molecules, counter to some evidence from vertebrates. Once symmetry is broken, the TGF-beta pathway is responsible for the communication, specification and maintenance of tissue identity across the L/R axis. The novel sequence resources described in this thesis provided a comprehensive window into this signalling cassette, and detailed here is a treatment of the TGF-beta pathway within the Lophotrochozoa. Ligand diversity has increased markedly in some clades, while signal transduction and regulatory steps are relatively unchanged. This work has increased our knowledge of lophotrochozoan biology and particularly the mechanisms underpinning the establishment of asymmetry in this under-researched clade, however, much remains to be discovered about the ultimate origin of asymmetry itself.

Sex chromosomes experience distinct evolutionary environments, due to their unusual pattern of inheritance, and studies of sex chromosome evolution can shed light on the fundamental evolutionary forces acting across the genome as a whole. Here, I combine genomic and transcriptomic data across a wide range of avian species to explore the evolutionary processes governing sex chromosome evolution. Birds are female heterogametic and therefore it is possible, via comparisons with male heterogametic species, to identify the fundamental factors driving sex chromosome evolution, versus those associated with sex. In this thesis, I uncover a complex mosaic of recombination suppression between the Z and W chromosomes, characterized by repeated and independent divergence of gametologs, together with ongoing genetic exchange. Additionally, I highlight the role of mating system, and interplay between evolutionary forces, in driving coding and expression evolution on the Z and W chromosomes. My findings indicate that although the Z chromosome is masculinized for male-specific effects, the magnitude of genetic drift acting on Z-linked genes is elevated in promiscuous relative to monogamous mating systems. In contrast, evolution of the female-limited W chromosome is governed predominately by purifying selection. Together, my results suggest that the role of the Z chromosome in encoding sexual dimorphisms may be limited, but that W-linked genes play a significant role in female-specific fitness. In conclusion, my findings reveal the power of mating system in shaping broad patterns of genome evolution.