Sexual selection is pervasive in nature and plays an important role in the evolution of biological diversity both within and among sexual species. However, while we have a good understanding of how competition for reproductive opportunities and mate choice can drive the evolution of exaggerated secondary sexual traits, much less is known about how sexual selection interacts with other forms of natural selection and the consequences such interactions may have for adaptation to novel environments, the purging of deleterious mutations, and population divergence/speciation. In my thesis, I carried out a series of experiments with the fruit fly Drosophila melanogaster to test hypotheses regarding the operation of sexual selection and to broaden our understanding of how sexual selection may influence adaptation and diversification. Theory suggests that natural and sexual selection may align to promote adaptation and the purging of deleterious mutations, although the harm imposed by sexual conflict may counter this. In two separate experiments, I find no evidence that sexual selection promotes adaptation to a novel environment and, rather than aligning with natural selection, I find that the effects of sexual conflict may cause sexual selection to hamper the purging of deleterious mutations. With respect to diversification, sexual conflict has been suggested to be an important, non-ecological driver of population divergence. However, the traits involved in sexual conflict may also affect nonsexual fitness and natural selection may therefore act to constrain diversification. Using an evolution experiment, I demonstrate ecologically-dependent parallel evolution of traits involved in sexual conflict, providing evidence for ecology’s importance in divergence via sexual conflict. Overall, my work has shed light on the interaction of natural and sexual selection and the consequences this may have beyond the evolution of exaggerated sexual displays and armaments.

Ageing reduces fitness, but how ageing evolves is still unclear. Evolutionary theory of ageing hinges on the fundamental principal that the force of natural selection declines with age. This principle has yielded two important predictions: 1) the evolution of faster ageing in populations under high rate of extrinsic mortality; and 2) the evolution of faster ageing in a sex that experiences higher rates of extrinsic mortality. However, an emerging new theory argues that when the extrinsic mortality is not random but instead selects on traits showing positive genetic correlation with lifespan, increased mortality should lead to the evolution of increased lifespan. Such condition-dependent mortality is also expected to increase the robustness in the population, resulting in increased deceleration of mortality in late-life. Similarly, high sex-specific mortality can result in increased sex-specific selection on traits that have positive pleiotropic effects on lifespan in the affected sex. This thesis is based on two experimental evolution studies in Caenorhabditis remanei. The first experiment was designed to disentangle the effects of the rate (high or low) and the source (random or condition-dependent) of mortality on the evolution of lifespan and ageing. Reduced lifespan evolved under high rate of random mortality, whereas high condition-dependent mortality, imposed by heat-shock, led to the evolution of increased lifespan (Paper I). However, while female reproduction increased under condition-dependent mortality, male reproduction suffered, suggesting a role for sexual antagonism in maintaining genetic variation for fitness (Paper II). Besides, long lifespan and high fecundity evolved at a cost of slow juvenile growth rate in females (Paper III). Moreover, high condition-dependent mortality led to the evolution of lower rate of intrinsic mortality in late-life (Paper IV). The second experiment showed that evolution of sexual dimorphism in lifespan is driven by the factors that cause sex-specific mortality and cannot be predicted from differences in mortality rate alone. Specifically, high condition-dependent mortality renders males less prone to ageing than females despite higher rates of male mortality (Paper V). The strength of this thesis is the reconfirmation of the earlier findings combined with support for the new theory. Rather than further complicating the matter, the inclusion of the new ideas should help explain some empirical results that are inconsistent with the classic theory, as well as provide a more comprehensive picture of ageing evolution.

In this thesis, I investigated the role of parental care in evolution. Parents provide the environment in which offspring develop and therefore have a large influence on their offspring's phenotypes, and so are in prime position to influence evolutionary processes. I used an experimental approach, and focused on the burying beetle, Nicrophorus vespilloides. The burying beetle is a perfect system for this question: they exhibit elaborate biparental care which is correlated with rapid speciation in the Nicrophorus genus. I started with a thorough exploration of burying beetle ecology and how the guild structure and interspecific competition in local populations can shape phenotypic evolution of my focal species, N. vespilloides. Interspecific competition shapes how the carrion niche is partitioned, which feeds back onto the evolution of body size within Nicrophorus reducing competition. The evolution of parental care in this genus likely facilitated its adaptive radiation, as parental care is linked with body size, both within and across species. But to what extent does the ecology shape the production and maintenance of phenotypic and genetic variation? I then use a quantitative genetic approach to show that body size and development time of N. vespilloides shows no additive genetic variation. Evolution of these fitness related traits can only occur through maternal effects or sibling effects. I tested this prediction by mimicking the radiation of the burying beetles by imposing my own selection on body size when parents could care for their offspring and when they could not. The presence of post-hatching parental care dramatically changed how populations responded to selection, through a combination of cooperation between parents and offspring, and cooperation between offspring. As well as shaping the evolutionary potential of populations, an experimental change in parental care can induce new selective forces, favouring adaptive novelties for the new social environment. Larvae evolving without parental care evolved disproportionately larger mandibles when small to better adapt them to a life without care. Much is known about the evolution of parental care across the animal kingdom, but what happens next: are the burying beetles a "one-off"? I compiled data across the arthropods comparing clades that exhibit post-hatching parental care with their sister clades and show that clades with care are more species rich. While the mechanism may not be the same as with Nicrophorus, I discussed other potential mechanisms that may be at play in the role of parents in evolution.

Antibiotic resistance is a major threat to public health. Understanding how it evolves, and the genes that underlie resistance, is the main goal of my Ph.D. research. After a resistance mutation arises, it’s fate within a pathogen population will be etermined in part by its fitness: mutations that suffer little or no fitness cost are more likely to persist in the absence of antibiotic treatment. My research centers on understanding this process better by gaining knowledge about the spectrum of fitness effects associated with antibiotic resistance mutations. Using a meta-analysis framework I find that, across a range of antibiotics and pathogens, on average single resistance mutations exhibit fitness costs in the absence of drug, however, there are instances of cost-free mutations. To evaluate the conditions leading to the persistence of resistance in the absence of antibiotic, I use experimental evolution of the opportunistic pathogen Pseudomonas aeruginosa and the antibiotic ciprofloxacin to investigate the phenotypic and genetic differences associated with constant and fluctuating drug treatment. I find that fluctuating drug treatment leads to the evolution of cost-free resistance. At the genetic level, cost-free resistance is the result of second-site mutations that compensate for the fitness cost associated with ciprofloxacin-resistance mutations. Further examination of the resistance mutations shows a lack of epistatic interactions between co-occurring mutations that confer resistance within a single isolate. To investigate the repeatability of the genetic causes of resistance, I execute a second evolution experiment using multiple clinical strains of P. aeruginosa adapting to a constant ciprofloxacin selective pressure. I find a remarkable lack of parallel evolution at the genomic level both within and between different P. aeruginosa strains. I have shown that antibiotic resistance is costly, and that these costs can be ameliorated by second-site mutations that readily arise over short time scales. Additionally, different strains of the same bacteria can gain resistance through a diverse set of genetic mutations. On an applied level these results are not positive; combating resistance evolution will be difficult because pathogens can easily compensate fitness costs of resistance, and resistance itself can be gained via a large number of genetic targets.

Sexual selection is pervasive in nature and plays an important role in the evolution of biological diversity both within and among sexual species. However, while we have a good understanding of how competition for reproductive opportunities and mate choice can drive the evolution of exaggerated secondary sexual traits, much less is known about how sexual selection interacts with other forms of natural selection and the consequences such interactions may have for adaptation to novel environments, the purging of deleterious mutations, and population divergence/speciation. In my thesis, I carried out a series of experiments with the fruit fly Drosophila melanogaster to test hypotheses regarding the operation of sexual selection and to broaden our understanding of how sexual selection may influence adaptation and diversification. Theory suggests that natural and sexual selection may align to promote adaptation and the purging of deleterious mutations, although the harm imposed by sexual conflict may counter this. In two separate experiments, I find no evidence that sexual selection promotes adaptation to a novel environment and, rather than aligning with natural selection, I find that the effects of sexual conflict may cause sexual selection to hamper the purging of deleterious mutations. With respect to diversification, sexual conflict has been suggested to be an important, non-ecological driver of population divergence. However, the traits involved in sexual conflict may also affect nonsexual fitness and natural selection may therefore act to constrain diversification. Using an evolution experiment, I demonstrate ecologically-dependent parallel evolution of traits involved in sexual conflict, providing evidence for ecology’s importance in divergence via sexual conflict. Overall, my work has shed light on the interaction of natural and sexual selection and the consequences this may have beyond the evolution of exaggerated sexual displays and armaments.

Ethanol tolerance in Saccharomyces cerevisiae is a complex polygenic trait. As a toxin, ethanol damages multiple cell constituents as well as being both a substrate and product of the metabolism of S.cerevisiae. This complexity has made ethanol tolerance difficult to study. Deletion screens have identified hundreds of genes that impair ethanol tolerance by their absence and hence might help survival in high ethanol environments. Similarly, expression studies have revealed genes that respond to ethanol shock, but it is unclear whether those genes are likely targets for improvement of ethanol tolerance in strains growing normally. In addition, those yeasts that are currently commercially exploited for their high ethanol tolerance in the brewing and bioethanol industries are commonly aneuploid or polyploid which makes it difficult to correlate particular features of their genotype with the ethanol tolerant phenotype. Experimental evolution can reveal genetic changes that change competitive fitness. It is practical to run numerous competitions in parallel between isogenic S.cerevisiae strains for hundreds of generations under ethanol stress, after which whole genome sequencing can identify the genetic changes. Fluorescent tagging of those strains can reveal small changes in population dynamics. We propagated 144 populations in batch culture for between 100 and 200 generations under 4 ethanol regimes (0%, 4.5%, 6.5% and ramped 0-10%). We monitored the progress of evolution by using mixtures of two fluorescently tagged, but otherwise isogenic, haploid, hoΔ (site-specific endonuclease deletant) founder strains (DeLuna et al 2008). Population dynamics measured using fluorescently labelled strains indicated that changes had occurred in competitive fitness due to adaptive evolution. Cell-size measurement and flow cytometry showed that evolved populations were diploid or triploid and the transition to higher ploidy occurred more rapidly with increasing ethanol stress. During the experimental evolution three strains evolved the capacity to grow on organic acids. We have sequenced the complete genomes of eight evolved strains. These strains are confirmed as being diploid, but not aneuploid. Sequencing of evolved strains revealed mutations that have not been previously characterised in deletion or expression studies of ethanol or organic acid tolerance in S.cerevisiae. Both increasing ploidy, to produce triploids as well as diploids, and the acquisition of organic acid tolerance under ethanol stress are unexpected outcomes that have implications for future work.

Naturally competent bacterial species, which self-induce the recombination mechanism of transformation, are wide spread across the bacterial tree-of-life. However, it remains unclear why competence has evolved in these bacteria. Although it is likely that exact explanations will be different for each species, a common selective factor cannot be excluded. Currently, three dominant hypotheses, which focus on the transformation function, try to explain the benefits of competence. Firstly, competence is thought to increase the rate of adaptation by combining beneficial alleles in single genotypes. Secondly, competence can repair DNA-damage by replacing the damaged DNA fragments with undamaged ones. Thirdly, the DNA uptake during competence is used to recycle environmental DNA fragments for nutrients. One of the naturally competent species is the Gram-positive Streptococcus pneumoniae, which is an opportunistic pathogen generally inhabiting the naso-pharyngeal area of young children. Competence in S. pneumoniae is regulated via density dependent extracellular signaling peptide. Here I use a combination of experiments designed around knockout mutants of the signaling mechanism and next-generation sequencing methods to test the first two hypotheses in S. pneumoniae. First, I extend on the DNA-for-repair hypothesis by showing that competent populations of S. pneumoniae are better protected not only against a DNA-damaging agent, but also against protein synthesis inhibitors. However, the mechanisms underlying this protection differ between types of stress. DNA-damage requires the full process of transformation, while protection against protein synthesis inhibitors only requires the activation of the competent cell state. This shows that benefits of competence cannot be totally explained by the benefits of transformation. Second, I use a long-term evolution experiment, where competent and non-competent strains are kept in the presence and absence of periodic stress, to determine the importance of competence for the generation of genetic variation. I find that competence does not increase the rate of adaptation in S. pneumoniae. The fitness of evolved competent populations was significantly lower than those of non-competent populations evolved over the same period of time. However, the intrinsic costs of competence are mitigated by the addition of short periods of stress exposure. These results confirm the prediction of the fitness associated recombination (FAR) hypothesis that competence is favoured in low-fitness situations. Thirdly, whole genome re-sequencing of the evolved populations allowed me to explore genomic evolution next to fitness changes. The genomic data revealed that competence reduces the mutational load of deleterious mutations rather than generating combinations of beneficial alleles. In addition I show several case of parallel genomic evolution within each treatment and across treatments. This shows that parallel evolution is not restricted by genotypic background (competence) or environment (periodic stress). Finally, these results show that competence has evolved in populations of S. pneumoniae as a mechanism to deal with various forms of stress.

Dans ce travail de thèse j'aborde d'un point de vue expérimental le rôle de l'hétérogénéité environnementale (spatiale et temporelle) comme moteur de diversification biologique. Il comporte une synthèse générale sur les principales thématiques abordées pendant ma thèse suivie d'une présentation des publications dérivées de mes propres recherches et de collaborations. Dans la synthèse je commence par présenter la biodiversité, je souligne l'importance d'étudier les conditions qui favorisent son émergence et son maintien pour mieux comprendre son organisation. Je présente le type de diversité biologique sur lequel se concentre mon travail ainsi que l'échelle d'organisation que j'ai considérée: la metacommunauté. J'évoque aussi l'intérêt croissant des écologistes pour prendre en considération les échelles évolutives dans les approches modernes des communautés. Plus loin, je présente l'évolution expérimentale comme l'outil qui m'a permis d'explorer les aspects écologiques et évolutifs du maintien et de l'émergence de la diversité en milieu hétérogène avant d'explorer certains de ces aspects théoriques. Pour finir, j'explore la relation diversité-fonctionnement comme une approche pour étudier les conséquences de l'actuel déclin de la biodiversité. Les trois premiers articles explorent différents aspects de la diversification de la bactérie Pseudomonas fluorescens SBW25 dans un environnement spatialement hétérogène (métacommunauté) après ~500 générations d'évolution. Les résultats des trois articles sont issus d'une même expérience d'évolution. Dans un premier article, j'ai exploré l'effet du niveau de dispersion sur la diversité régionale. Cette dernière s'est révélée maximale pour des niveaux de dispersion intermédiaires. Ce gradient de diversité à l'échelle régionale était lié avec une plus forte productivité résultant d'une meilleure complémentarité écologique. Dans le deuxième, j'ai mesuré la diversité à l'échelle locale (communautés) et j'ai montré une augmentation de la diversité pour des niveaux de dispersion intermédiaires (mass-effect) mais qui n'aboutit pas à une plus forte productivité. Cet article souligne l'importance de considérer l'échelle ainsi que les mécanismes de coexistence pour comprendre la relation entre diversité et fonctionnement des écosystèmes. Dans le troisième (en préparation) j'explore l'effet de la dispersion sur le patron d'adaptation locale. Les résultats préliminaires montrent qu'un patron d'adaptation locale n'est pas un résultat obligé de l'évolution en milieu spatialement hétérogène. Le niveau de dispersion semble modifier considérablement le patron d'adaptation avec une plus forte adaptation locale moyenne pour des niveaux de dispersion intermédiaire. Le quatrième article présente une nouvelle expérience d'évolution avec laquelle j'ai exploré le rôle du grain environnemental dans l'émergence et le maintien de la diversité dans un environnement temporellement fluctuant. Après ~300 générations d'évolution j'ai constaté que les traitements de grain intermédiaire présentaient la plus haute diversité. En temps que collaborateur j'ai également participé à la rédaction d'un article de revue sur le concept de spécialisation en écologie. Cet article inclut une synthèse des différentes définitions de ce concept ainsi que des méthodes pour la mesurer. Pour finir j'ai participé à la réalisation d'une expérience avec des bactéries marines Méditerranéennes visant à explorer la dynamique évolutive de la relation biodiversité-fonction dans des conditions favorisant soit la spécialisation écologique soit l'émergence de généralistes. Après 64 jours d'évolution, le résultat général est une relation positive entre productivité et richesse spécifique des assemblages. L'évolution de généralistes aboutit à une productivité moyenne plus forte que chez les spécialistes alors que la pente de la relation diversité –fonction est plus forte pour des spécialistes. / In this work I propose an experimental approach to the study of the impact of spatial and temporal environmental heterogeneity on biological diversification. It is composed of two main parts: first, a general review of the principal subjects I have explored during my thesis and then a presentation of the manuscripts from my own research and from different collaboration projects. In the synthesis I first present what is biological diversity (Biodiversity) and I underline the importance of considering the conditions to its emergence and maintenance in the understanding of its organization. I present the kind of diversity I'm interested in and the scale of organization I have considered: metacommunities. I also present the interest of current ecology research in including evolutionary processes on the modern concept of communities. Then, I present experimental evolution as a tool allowing the exploration of the ecological and evolutionary aspects of the maintenance and emergence of diversity in heterogeneous environments and I also present some of its theoretical aspects. Finally, I explore the biodiversity-ecosystem functioning relationship as a way to explore the consequences of diversity erosion. The first three manuscripts explore different aspects of a ~500 generation experiment of diversification of the bacteria Pseudomonnnas fluorescens SBW25 in a spatially heterogeneous environment (metacommunities). On the first manuscript, I have explored the effect of dispersal on regional diversity. The results show that diversity was maximal for intermediate dispersal levels. This gradient of diversity at the regional scale was positively related to a higher productivity as a result of higher ecological complementarity among genotypes. In a second article, I have measured the local diversity. The result was a higher increase in diversity at intermediate dispersal levels (mass-effect) but with no impact on productivity. This article underlines the importance of considering the mechanisms of coexistence when studying the diversity-ecosystem functioning relationship. Then, I present the preliminary results on the impact of dispersal on the pattern of local adaptation. The results show that a pattern of local adaptation is not an obligate outcome of evolution in spatially heterogeneous environments. Intermediate dispersal levels enhanced the emergence of local adaptation patterns. On a fourth article, I present the results of an experimental test of effect of the environmental grain on the emergence and maintenance of diversity in a temporally fluctuating environment by using Pseudomonas fluorescens SBW25. After ~300 generations of evolution, I have found that diversification was maximal at an intermediate environmental grain. I have also collaborated in the writing of a review about ecological specialization concept. This synthesis includes the different available definitions of ecological diversification and the different methods used to measure it. Finally, I have participated in an experiment using Mediterranean Sea bacteria exploring the dynamics of the diversity-ecosystem functioning relationship under too different selective scenarios favoring either specialization or the evolution of generalists. After 64 days of evolution, the result was a positive relationship between species richness and productivity for both specialists and generalists. Generalists performed better than specialists for any species richness level while the steepness of the relationship was higher for specialists.

Bacteria have small, streamlined genomes and evolve rapidly. Their large population sizes allow selection to be the main driver of evolution. With advances in sequencing technologies and precise methods for genetic engineering, many bacteria are excellent models for studying elementary questions in evolutionary biology. The work in this thesis has broadly been devoted to adaptive evolution and fitness effects of different types of mutations. In Paper I we experimentally tested the fitness constrains of horizontal gene transfer (HGT), which could be used to predict how the fixation of HGT events are affected by selection and fitness effects. We found that the majority of the examined HGT inserts were indistinguishable from neutral, implying that extra DNA transferred by HGT, even though it does not confer an immediate selective advantage, could be maintained at transfer-selection balance and serve as a reservoir for the evolution of novel beneficial functions. Paper II examined why four synonymous mutations in rpsT (encoding ribosomal protein S20) reduced fitness, and how this cost could be genetically compensated. We found that the cause for the fitness reduction was low S20 levels and that this lead to a defective subpopulation of 30S subunits lacking S20. In an adaptive evolution experiment, these impairments were compensated by up-regulation of S20 though various types of mutations. In Paper III we continued the studies of how the deleterious rpsT mutations could be compensated. The mutations either down-regulated the global regulator Fis or altered a subunit of the RNA polymerase (rpoA). We found that the decreased S20 levels in the cells causes an assembly defect of the 30S particles and that the fis and rpoA mutations restored the skewed S20:ribosome ratio by both increasing S20 levels and decreasing other ribosomal components. Paper IV examined adaptation of two bacterial species to different growth media. A total of 142 different adaptive mutations were identified and 112 mutants were characterized in terms of fitness. We found that the experimental variation in fitness measurements could be reduced 10-fold by introducing some adaptive mutations prior to the experiment, allowing measurements of fitness differences as small as 0.04%.

Understanding dispersal is a central aim of evolutionary ecology. Theoretical analyses of dispersal have been crucial in identifying key variables which contribute to its evolution and maintenance, but the supporting empirical data remains elusive. Microbes offer a powerful model system on which ecological and evolutionary theory can be experimentally tested with controlled and replicated experiments, and with the convenient malleability of selective pressures and bacterial genomics. Pseudomonas aeruginosa is an ubiquitous, opportunistic pathogen that is able to induce acute or chronic infections in a broad array of hosts. As well as in vivo environments, P. aeruginosa can be found in a range of ecological habitats, from solid to aqueous, and as such requires a variety of dispersal mechanisms (including swimming, gliding on a surfactant and ‘crawling’) for effective colonisation and infectivity. In this thesis, I present a collection of papers which outline empirical ecological and evolutionary experiments to identify the abiotic and biotic forces that shape the evolution of these different dispersal mechanisms, with particular focus on the theoretically important role of kin competition and the structure of the abiotic environment.