Antibiotic resistance is generally associated with a cost in terms of reduced competitive fitness in the absence of antibiotics. Despite this 'cost of resistance', the cessation of antibiotic treatment does not result in significant reductions in the prevalence of resistance. The maintenance of resistance, in spite of the costs, has been attributed to the rarity of reversion mutations, relative to compensatory mutations at other loci in the genome. However, the large size of bacteria populations, and the potential for migration, suggest that reversion mutations should occasionally be introduced to resistant populations. In this thesis, I show that additional mechanisms can prevent fixation of reversion mutations even if they do occur. Using an experimental evolution approach, with rifampicin resistance in Pseudomonas aeruginosa as a model system, I measured the costs of resistance in several environments and followed the adaptive dynamics of resistant populations where a sensitive lineage had invaded by migration. The results suggest that several additional mechanisms contribute to the maintenance of antibiotic resistance. Most rifampicin resistance mutations are not unconditionally costly in all environments, suggesting that migration between environments could maintain a resistant reservoir population. In environments where resistance is initially costly, the fixation of a revertant is not guaranteed, even if introduced through migration. Revertant fixation was impeded or prevented by clonal interference from adaptation in the resistant strain. Revertants that did successfully replace the resistant strain were forced to adapt to do so. Contrary to assumptions in the existing literature, fitness in the resistant strains was not recovered by general compensatory mutations, but instead by adaptive mutations specific to the environment. The data challenge several assumptions about the maintenance of antibiotic resistance: that resistance mutations are always costly, that the rarity of back mutations prevents the reversion of resistance, and that resistant strains recover fitness by compensatory mutations.

Sperm play a pivotal role in determining the reproductive success of individuals whose sperm must compete directly with that of others. I used sperm precedence assays and experimental evolution to examine the role of sperm traits in the reproductive success of hermaphrodites and males in the androdioecious nematode Caenorhabditis elegans. First, sperm size and the rate of reproduction were analyzed, in the context of male-male sperm competition, for evidence of natural heritable variation. Sperm size proved to be a strong indicator of second-male sperm precedence in the genotypes examined. Second, I tested the theoretically predicted effect of larval development time on the number of self-sperm produced by hermaphrodites. I demonstrated that a short larval development period favored the evolution of fewer sperm, inline with theoretical predictions. These results provide important insights into C. elegans reproductive biology and more generally to our understanding of the evolution of reproductive systems.

Sperm play a pivotal role in determining the reproductive success of individuals whose sperm must compete directly with that of others. I used sperm precedence assays and experimental evolution to examine the role of sperm traits in the reproductive success of hermaphrodites and males in the androdioecious nematode Caenorhabditis elegans. First, sperm size and the rate of reproduction were analyzed, in the context of male-male sperm competition, for evidence of natural heritable variation. Sperm size proved to be a strong indicator of second-male sperm precedence in the genotypes examined. Second, I tested the theoretically predicted effect of larval development time on the number of self-sperm produced by hermaphrodites. I demonstrated that a short larval development period favored the evolution of fewer sperm, inline with theoretical predictions. These results provide important insights into C. elegans reproductive biology and more generally to our understanding of the evolution of reproductive systems.

The interplay between physiology and evolution in microorganisms is extremely relevant from the stand-point of human health, the environment, and biotechnology; yet microbial physiology and microbial evolution largely continue to grow as disjoint fields of research. The goal of this dissertation was to use experimental evolution to study methylamine metabolism in Methylobacterium extorquens species. Methylotrophs like the M. extorquens species grow on reduced single carbon compounds and are the largest biological sink for methane. M. extorquens AM1, the model system for the study of aerobic methylotrophy, has an unstable genome and severe growth defects as a result of laboratory domestication. First, I describe the genomic, genetic, and phenotypic characterization of a new model system for the study of aerobic methylotrophy: M. extorquens PA1. This strain has a stable genome, was recently isolated from a known ecological niche, and is closely related to AM1. Whereas PA1 grew 10-50% faster than AM1on most substrates, it was five-fold slower on methylamine. The PA1 genome encodes a poorly characterized but ecologically relevant N-methylglutamate pathway whereas AM1 also encodes the well-characterized methylamine dehydrogenase for methylamine oxidation. I characterized the genetics of the N-methylglutamate pathway in PA1 to resolve a linear topology that requires the formation of two, unique amino acid intermediates during methylamine oxidation. I also showed that methylamine metabolism via the N-methylglutamate pathway routes carbon flux in a manner completely different from previous instances of methylotrophy. Next, I evolved replicate populations of PA1 on methylamine for 150 generations. Based on the empirical heuristic that the initial fitness is negatively correlated to the rate of adaptation, it was expected that the fitness gain would be rapid. However, methylamine fitness did not improve at all; adaptive constraints led to evolutionary recalcitrance despite low initial fitness. These adaptive constraints were alleviated by the horizontal gene transfer of an alternate, functionally degenerate metabolic module. Finally, I uncovered ecologically distinct roles for two functionally degenerate routes for methylamine oxidation pathways in the AM1 genome; the highly expressed, efficient route is primarily used for growth and the tightly regulated, energetically expensive route is used for assimilating nitrogen in methylamine-limiting environments.

Antimicrobial resistance is a serious and imminent threat to human health, though its rise may be controlled with improved stewardship strategies that limit the emergence and spread of resistant strains. Motivated by theoretical models from population genetics and ecology, my M.Sc. experimentally evaluates how varying drug availability in either time or space impacts the prevalence of resistance in a population. By experimentally evolving Pseudomonas aeruginosa under different antibiotic selection regimes in vitro, I show that spatial, but not temporal, drug free refuges delay the fixation of resistance by promoting the coexistence of sensitive and resistant genotypes. Second, I establish that this polymorphism is underlain by a trade-off between resistance and growth rate in the absence of antibiotic that underpins the maintenance of diversity through negative frequency dependent selection. Third, I demonstrate that spatially varied drug selection cannot prevent the fixation of resistance because continued selection leads to the evolution of resistant types that pay smaller costs of resistance and gradually displace sensitive strains. These results provide insight into the fate of diversity under long-term selection and highlight the value of incorporating the principles of evolutionary ecology into antimicrobial resistance stewardship.

Life on Earth is incredibly diverse. The process of diversification that gives rise to this diversity is not the same for all lineages. Diversification is often driven by ecological opportunity. Pseudomonas aeruginosa is an opportunistic pathogen present in a variety of environments that causes chronic lung infections in cystic fibrosis (CF) patients. It diversifies rapidly within the CF lung and CF lung-like environments. Here we aim to assess both ecological and genetic factors in diversification of several strains of P. aeruginosa. We evolved 12 replicate populations of 8 different strains of P. aeruginosa in a nutritionally complex (LB) and simple environment (MIN) for 750 generations. We then measured diversity over time by observing the number of colony morphologies in each population every 250 generations. We also measured competitive fitness relative to the ancestor for endpoint populations. To provide a more complete analysis, phylogeny was factored into our statistical models. First, we found no significant differences in diversification between populations evolved in LB versus MIN media. Ancestor population size had no significant effect on diversification. We found that in both selection environments, CF strains diversified less than environmental strains, but this difference was marginally significant and only present when comparing these two niches directly and excluding acute strains. Finally, we found no correlation between gains in fitness and endpoint diversity. Our results suggest that diversification is limited by niche specialization (domestication) of P. aeruginosa to the CF lung.

Darwin first proposed sexual selection as a process to explain the evolution of extravagant morphological traits in males. Despite being potentially detrimental to individual survival, such traits evolve because they increase a male's reproductive success, and provide a net benefit to their fitness. Mate competition is the source of sexual selection, and healthier, more vigorous males are likely to be superior competitors. Because most genes are likely to impact an individual’s health/vigour, sexual selection should act across much of the genome to favour the same alleles as natural selection, thereby promoting adaptation. On the other side of the coin, adaptation to an environment should enhance male sexual fitness, since it is likely to increase the overall health/vigour of individuals within a population, though tests of this prediction are rare and results are mixed. Taking advantage of a long-term evolution experiment involving replicate populations of Drosophila melanogaster, I performed a reciprocal transplant in which the sexual fitness of males was compared when raised in an environment to which they are well adapted and in one to which they are not. I improved on past tests via a comprehensive measure of male sexual fitness that included pre- and post-copulatory reproductive success in a competitive assay under conditions that closely mirrored those to which the populations have been evolving. I found that sexual fitness was higher in locally-adapted males from these experimental populations, a result that was consistent across environments that also manipulated the context in which mate competition occurred.

Laboratory bacterial populations provide ideal opportunities to experimentally test theories in ecology and evolutionary biology. I used a model laboratory microbial system, Pseudomonas fluorescens SBW25, to address an array of questions on the origin, maintenance, and functional role of biodiversity, and the evolution of biotic interactions. My thesis reports experiments with the following conclusions. (1) The extent of diversification in P. fluorescens populations is not affected by the presence of an interspecific competitor P. putida, although the early stage of the diversification in one environment (spatially homogeneous environment) could be speeded up by the competitor. (2) Niche and neutral mechanisms simultaneously contribute to the maintenance of phenotypic diversity in P. fluorescens populations; but the operation of niche processes does not lead to a positive effect of biodiversity on ecosystem functioning. (3) The competitive interactions among bacterial phenotypes are generally transitive, and competitive hierarchies inferred from pair-wise competition are fairly consistent to those from multi-species competition. (4) The niche complementarity and selection effects evaluated by random assembly biodiversity experiments can be used to predict the functional consequences of particular non-random species extinction scenarios. (5) P. fluorescens does not show an evolutionary trade-off in using several carbon substrates (glucose, galactose and trehalose), and evolution in environments containing these resources results in imperfect generalists; migration among populations may speed up fitness evolution of some generalists. (6) Biofilm formation at the air-broth interface by wrinkly spreader phenotypes in P. fluorescens is a cooperative behaviour which is costly to individuals but benefits the group; this behaviour could be exploited by smooth morph phenotypes. The cooperators and cheats in this system show reciprocal antagonistic coevolution in resistance and cheating performance.

During the evolution of sex, different sexual conflicts arise. Sexual conflicts reduce fitness of the opposite sex. That is why several mechanisms have evolved to resolve them, which leads to rapid and unpredictable co-evolution of male and female traits involved in reproduction. This rapid co-evolution of male and female reproductive traits driven by sexual conflict can further lead to reproductive isolation resulting in speciation.             I used the hermaphroditic fungus Neurospora crassa, which has two mating types, as a model organism. Mating types are proxy to sex, because both are needed for sexual reproduction, but they are not limited to either sex role. However by using male pheromone knock-out lines, I created an evolutionary setup, where either mating type is forced to adapt to its restricted sex role. After 21 sexual generations of adaptive co-evolution, I tested if mating types had adapted to the assigned sex by measuring fitness (production of sexual spores called ascospores). I used three evolutionary setups (lines): Δccg4 lines, where mat A is female and mat a is adapted to the male role, Δmfa1 lines, where conversely mat A is adapted to the male role and mat a is female, and wild-type lines used as controls, where both mating types have maintained and adapted to both sex roles. And discovered one Δccg4 line, which indeed adapted to the newly assigned sex roles. At generation 15 and 21 I obtained mixed results for the presence of sexual conflict by correlating male and female fitness in hermaphroditic partner mat a in this line, however I found a sexual conflict also in the asexual growth, where male role is associated with increased, but female role with decreased mycelium growth rate. This work will further allow to study genomic mechanisms underlying this adaptation.

Sexual interactions play an important role in generating sexual selection and antagonistic co-evolution. These forces can shape differences between the sexes, but also have the potential to generate population divergence and contribute to speciation. The aim in this thesis was to provide new insights into the genes involved in different stages of female sexual interactions, using Drosophila as a model system. In chapter 2 I tested whether a candidate gene (period) that influences species-specific rhythmic characteristics in male courtship song in D. melanogaster also has a pleiotropic effect on female song preference. Using mutant and transgenic strains I found support for this. In chapter 3 I examined further how females respond to the song at the level of gene expression, using microarrays. Expression profiles revealed modest changes in transcripts abundance overall, which were dominated by antennal olfactory genes, neuropeptide encoding genes and immunity genes. Many of these have previously been found to respond to mating. In chapter 4 I therefore studied further two of these genes, TurandotM and TurandotC and their role in female post-mating fitness. Using RNA interference I found that knocking down these genes influenced immediate fecundity. In chapter 5 I focused on analysing post-mating gene expression patterns in relation to sexual selection in D. pseudoobscura using microarrays. I explored the consequences of experimental variation in female promiscuity on gene expression divergence as a whole, and in response to mating. I found large-scale expression divergence between monandrous and polyandrous females after 100 generations of experimental evolution. Experimental polyandry increased the expression of genes that show female-biased expression in wild-type individuals and decreased male- biased gene expression. Females experiencing no sexual selection showed the opposite pattern. Out of the genes affected by mating, the majority showed increased expression in polyandrous compared to monandrous females, with enrichment e.g. in oogenesis-related genes.