The field of experimental evolution is burgeoning under the power of microbial systems. Our ability to manipulate experimental design for use with microbes is only limited by our imagination. This thesis is a study that uses Pseudomonas fluorescens, a soil dwelling bacterium, as an experimental tool for understanding evolutionary processes. The evolution of cooperation has been a thorny issue for many years, because it initially seems to contradict the intrinsically selfish concepts established in Darwin’s theory of evolution by natural selection. Advances in microbiology and the ability to test important evolutionary theories using microbes, provides an exciting opportunity for those working in the field of experimental evolution. This thesis uses P. fluorescens to investigate four aspects of the evolution of cooperative behaviour organised into four results chapters (Chapters 2-5). The first describes the genotypic and phenotypic diversity of 26 independently derived ‘wrinkly spreader’ genotypes in order to analyse the genetic and phenotypic variation among morphotypes. Mutations were identified in 25 of the 26 wrinkly spreaders including a new locus mws and three new genes of known loci wspE, awsR and awsO. This new genetic information provided additional insight into the molecular causes of the wrinkly spreader phenotype. Multivariate analysis of the phenotypic traits revealed that wspF mutants were phenotypically distinct from other morphotypes at a level below the ecological niche. The second chapter extended existing studies on the evolution of wrinkly spreader genotypes within the well-known Haystack model for evolution in group-structured populations, by studying the population dynamics of cooperative genotypes with and without group structure, in a multi-level selection one framework. It was shown that the time spent in a haystack affects the fitness of cooperators, because the longer group-generation treatment conformed to the predictions of the Haystack model, while the shorter group-generation treatment did not. The third chapter was an investigation into how the fitness of the emergent group-level phenotype formed by cooperating wrinkly spreader cells was dependent on the density of wrinkly spreader cells. Contrary to prediction, no density dependence was observed when calculated in a multi-level selection one framework, but rather it was determined that the emergent fitness was dependent on time, implicating a role for a development-like process. The final results chapter of this thesis incorporated the hypothesised role for a development-like process into a novel theoretical model for the evolution of multicellularity in which fitness would be determined in a multi-level selection two framework. Novel apparatus and experimental design were developed to determine if it were possible to observe a response to a selective regime that selected simultaneously at the level of the individual cell and the level of the group of cells. A significant response was shown after only six group-generation cycles. In summary, this thesis exploits P. fluorescens as an experimental tool to gain insight into complex ecological and evolutionary phenomena such as cooperation, biofilm formation and the evolution of multicellularity, and provides insight into the molecular causes of the cooperation among wrinkly spreader genotypes.

Microbes can protect their plant and animal hosts against infection by pathogens, parasites and parasitoids. These ‘defensive microbes' can provide a powerful line of defence beyond the host response and are becoming attractive candidates for disease control. In this thesis, I investigated how defensive microbes can interact with pathogens over evolutionary time by measuring the effects of co-passaging a defensive microbe (Enterococcus faecalis) and a pathogen (Staphylococcus aureus) within the Caenorhabditis elegans model host. In Chapter 1, I found that co-passaging drove the evolution of reduced pathogen virulence as a by-product of adaptation to microbe-mediated defence. Moreover, I show that the mechanism of pathogen resistance to the defensive microbe can determine the direction of virulence evolution. In chapter two, I discovered that the co-passaged defensive microbe and pathogen populations had undergone coevolutionary interactions within host populations via fluctuating selection dynamics. I then showed that these dynamics resulted in patterns of pathogen local adaptation and increased genetic divergence. Finally, in chapter three, I revealed that these coevolutionary interactions significantly affected the costs and benefits of the defensive microbes to their hosts, but that the relationship between these costs and benefits prevented the transition of defensive microbes across the mutualism-parasitism continuum. Together, this thesis uncovers the potential for defensive microbes to shape the evolution of pathogens and demonstrates that defensive microbes can be an evolutionarily dynamic but stable form of host resistance towards infectious disease. As such, the data presented in this thesis have important implications for how we study host-parasite interactions in nature and question our current understanding of virulence evolution, pathogen local adaptation and the origin of defensive microbes.

Une grande partie des organismes hermaphrodites, qu’il s’agisse de plantes ou d’animaux, est capable de se reproduire par autofécondation, comme alternative à la fécondation croisée. Or les modèles théoriques prédisent un ensemble de conséquences évolutives importantes liées à l’autofécondation. La première prédiction est qu'une population pratiquant l'autofécondation est moins sensible à la dépression de consanguinité qu'une population à reproduction croisée, car une partie de la dépression a été « purgée », c’est-à-dire que les allèles délétères récessifs sont éliminés par la sélection naturelle plus facilement en autofécondation. Cette purge entraine en retour une sélection positive sur l’autofécondation. On attend aussi chez ces populations l’évolution de traits facilitant l’autofécondation (par ex., des fleurs fermées), ainsi qu’une réallocation de ressources de la fonction mâle vers la fonction femelle, en raison d’une sélection sexuelle réduite sur la fonction mâle. Une reproduction par autofécondation va aussi considérablement affecter la variabilité disponible en raison d’une taille efficace de population divisée par deux, augmentant les effets de dérive. Par ailleurs, la moindre efficacité de la recombinaison va augmenter la sensibilité aux interférences sélectives (sélection d’arrière-plan, balayage sélectif) et diminuer la probabilité de fixer plusieurs mutations avantageuses dans le même génome. En d’autres termes, l’autofécondation conduit à un fardeau génétique plus lourd, et diminue les capacités d'adaptation et l’efficacité de la sélection naturelle. On prédit donc que les espèces autofécondantes ont une probabilité d’extinction plus grande que les espèces allofécondantes – elles constituent un cul-de-sac évolutif. Ces prédictions ont pour l’essentiel été évaluées chez des plantes, voire ne l’ont pas été du tout. L’objectif de cette thèse est d’apporter des éléments permettant de les tester chez des animaux, les escargots hermaphrodites d’eau douce. Pour ce faire, nous avons opté pour une approche d’évolution expérimentale permettant de contrôler régime de reproduction, conditions environnementales et pressions de sélection. Notre modèle d’étude est Physa acuta, une espèce allofécondante qui est capable de se reproduire par autofécondation et nous avons des lignées expérimentales se reproduisant soit en allofécondation stricte soit alternant avec une génération d’autofécondation depuis 20 à 30 générations au laboratoire. La première expérience montre que non seulement la dépression de consanguinité est largement purgée en une dizaine de génération d’autofécondation, mais aussi que le temps d’attente (un trait positivement corrélé au taux d’allofécondation) a fortement diminué. Nous n’observons en revanche aucune réallocation sur la fonction femelle. La deuxième expérience dans laquelle nous avons comparé la réponse à la sélection sur un trait morphologique en autofécondation et en allofécondation montre qu’une population en autofécondation répond d’abord mieux car les allèles sont progressivement placés à l’état homozygote mais cet avantage s’épuise rapidement probablement à cause des interférences sélectives car en trois générations elles commencent à répondre plus lentement que la même population en allofécondation (le trait considéré était la forme de la coquille). Ces travaux apportent des éléments nouveaux quant à notre compréhension de l’évolution de l’autofécondation, et proposent des éléments expérimentaux novateurs quant à la moindre adaptabilité des espèces autofécondantes. / Many hermaphroditic organisms, either plants or animals, are able to reproduce by self-fertilization, at least alternatively with cross fertilization. Theoretical models predict several important consequences linked to this mating system. The first prediction is that a selfing population is less sensitive to inbreeding depression than an outcrossing one, because part of the depression can be « purged » meaning that the recessive deleterious alleles are easier to eliminate by natural selection under selfing. This purge creates a positive feedback to favour self fertilization. In these circumstances, we also expect the evolution of traits facilitating self fertilization (for example closed flowers) and a reallocation of resources from the male to the female function, because sexual selection is reduced in the male function. Self-fertilization also affects standing variation, as the effective population size is divided by two, enhancing the effects of drift. In addition, recombination becomes inefficient, increasing the extent of selective interference among loci (background selection, selective sweep) and decreasing the probability to fix several advantageous mutations in the same genome. In other words, self-fertilization decreases the adaptive potential and the efficiency of natural selection. We then predict that autogamous species have a higher probability of extinction, this is called the “dead end hypothesis”. Some of these predictions have been tested mainly in plants or not at all. The aim of this thesis is to test them in animals, using freshwater snails as model systems. To this end, we followed an experimental evolution approach using laboratory populations of Physa acuta a preferentially outcrossing snail able to reproduce by self-fertilization. These populations were maintained for 20 to 30 generations either under pure outcrossing or under alternating generations of outcrossing and selfing. In a first experiment we show that inbreeding depression is largely purged after only ten generations of selfing, but also that the waiting time, (a trait positively correlated to the outcrossing rate) decreased largely. We did not observe however any reallocation in favour of the female function. In a second experiment we compared the response to artificial selection on a morphological trait under selfing and outcrossing. We observed that when an outbred population switches to self-fertilization the response to selection is initially enhanced as alleles are progressively made homozygous. However this advantage is quickly offset by selective interference and after no more than three generations selfing populations start to respond to selection more slowly than outcrossing onesThis work brings new elements for the understanding of the evolution of mating systems, and provides empirical support for the lower adaptability of selfing species.

Une plante peut être totalement protégée d'un agent pathogène grâce à un gène majeur de résistance, mais ce dernier peut être rapidement contourné suite à l'apparition et à la propagation de variants pathogènes adaptés. Cette thèse s'intéresse aux mécanismes évolutifs permettant le ralentissement de ce contournement chez les virus de plantes en agissant sur deux forces évolutives majeures, la dérive génétique et la sélection, depuis le niveau de l'hôte jusqu'à celui de la parcelle. D'abord, un modèle épidémiologique stochastique de type SI au niveau d'une parcelle agricole a montré que la dérive génétique pouvait être particulièrement bénéfique au rendement agricole lorsque l'adaptation du virus au gène majeur induit un coût de fitness intermédiaire dans les plantes sensibles. Ensuite, la conception et la validation d'un modèle basé sur des équations déterministes de Lotka-Volterra et des processus stochastiques Dirichlet-multinomiaux a permis de distinguer les effets de la dérive génétique et ceux de la sélection sur des données temporelles de compétition intra-plante entre variants viraux, et de mettre en évidence le contrôle génétique de ces effets par les plantes. Enfin, une analyse de la corrélation entre ces estimations des intensités de dérive génétique et de sélection et une estimation expérimentale de la durabilité d'un gène majeur a montré qu'une forte dérive génétique lors des stades précoces de l'infection augmentait la durabilité du gène majeur. Ces résultats ouvrent des perspectives pour une gestion plus durable de la résistance des plantes, par la sélection de variétés de plantes induisant une forte dérive génétique sur les populations d'agents pathogènes / Plants can be fully protected from their pathogens when they carry major resistance genes, but the efficiency of these genes is limited by the emergence and spread of adapted, resistance-breaking pathogen variants. This thesis studies how evolutionary forces imposed by the plants on pathogen populations may increase the durability of major resistance genes. Using plant viruses as a biological model, this thesis investigates the effect of genetic drift and selection, from the within-host to the host population level. Firstly, a stochastic epidemiological SI model at the field level showed that genetic drift could be particularly beneficial for crop yield when the fitness cost associated with virus adaptation to resistance was intermediate in susceptible plants. Then, the design and validation of a mechanistic-statistical model based on deterministic Lotka-Volterra equations and stochastic Dirichlet-multinomial processes allowed to disentangle the effects of genetic drift from those of selection on temporal data of within-host competition between virus variants. The intensities of genetic drift and selection acting on virus populations were shown to be controlled genetically by the hosts. Finally, a correlation analysis between these estimations of genetic drift and selection intensities and an experimental estimation of the durability of a major resistance gene showed that strong genetic drift during the early stages of plant infection led to an increase in resistance durability. These results open new perspectives for more durable management of plant resistance, by breeding plant varieties inducing strong genetic drift on pathogen populations

The aim of this thesis is to investigate aspects of molecular evolution and enzyme engineering using the experimental evolution of Tobacco Etch Virus cysteine protease (TEV) as a model. I map key features of the local fitness landscape and characterise how they affect details of enzyme evolution. In order to investigate the evolution of core active site machinery, I mutated the nucleophile of TEV to serine. The differing chemical properties of oxygen and sulphur force the enzyme into a fitness valley with a >104-fold activity reduction. Nevertheless, directed evolution was able to recover function, resulting in an enzyme able to utilise either nucleophile. High-throughput screening and sequencing revealed how the array of possible beneficial mutations changes as the enzyme evolves. Potential adaptive mutations are abundant at each step along the evolutionary trajectory, enriched around the active site periphery. It is currently unclear how seemingly neutral mutations affect further adaptive evolution. I used high-throughput directed evolution to accumulate neutral variation in large, evolving enzyme populations and deep sequencing to reconstruct the complex evolutionary dynamics within the lineages. Specifically I was able to observe the emergence of robust enzymes with improved mutation tolerance whose descendants overtake later populations. Lastly, I investigate how evolvability towards new substrate specificities changed along these neutral lineages, dissecting the different determinants of immediate and long-term evolvability. Results demonstrate the utility of evolutionary understanding to protease engineering. Together, these experiments forward our understanding of the molecular details of both fundamental evolution and enzyme engineering.

Ecological processes have the potential to influence evolution through their effects on selection. This thesis explores the effects of two ecological factors - competition and ecological opportunity. Intraspecific (within-species) competition is often expected to drive adaptation and diversification by increasing selection for the use of novel resources, thereby alleviating the detrimental effects of competition. However, this is not always the expected outcome; theory suggests that intraspecific competition can also drive convergent evolution. On the other hand, interspecific (between-species) competition is usually expected to impede adaptation and diversification because competitor species occupy potential available niches, preventing the focal species from diversifying to do so. In this thesis, I review previous experimental studies exploring the effects of competition on adaptive diversification, and then directly test these effects using experimental evolution of the bacterium Pseudomonas fluorescens. I confirm that intraspecific competition drives adaptive diversification, while the effects of interspecific competition are varied. Strong interspecific competition impedes adaptation and diversification, while the presence of weak, non-diversifying interspecific competitors drives diversification through increased resource competition. The presence of ecological opportunity is essential for adaptation and diversification, and so variation in attributes of those opportunities is expected to have important effects on the dynamics of adaptive evolution. In another evolution experiment with P. fluorescens, I tested the effects of variation in ecological opportunity on adaptive evolution and found that the type and arrangement of ecological opportunities drives adaptation but, in this system, not diversification. I also show that ecological opportunity drives differences in the degree of parallel evolution at the phenotypic and genotypic level. Finally, I explore some unexpected genetic changes identified in one of these evolved populations - two synonymous mutations that conferred fitness benefits, and show that the observed fitness improvements are the result of increased gene expression. I have shown that ecological processes can play an important role in shaping the evolutionary trajectories taken by populations. Understanding the interactions between ecological and evolutionary processes is vital for our understanding of evolutionary dynamics as a whole, and the studies laid out in this thesis represent valuable contributions to this field of study.

Estimating selection is of key importance in evolutionary biology research. The recent price drop in sequencing and advances in NGS data analysis have opened up new avenues for novel methods that estimate selection quantitatively from time-series allele frequency data. However, it is not yet well understood which method performs best given specific model systems and experimental designs. Here, using popular quantitative metrics, we compared the performance of four prominent methods on a series of simulated data sets and on data from real biological experiments. We identified in three out of four methods the experi- mental conditions best suited for estimating selection. We also explored the limitations of these methods when estimating selection from complex patterns of allele frequency change in some relevant evolutionary scenarios. Our findings highlight the need for modification of population genomics models that are still used in inference of model parameters with the goal to develop new, more accurate methods for the quantitative estimation of selection in time-series genomic data.

Thesis advisor: Marc-Jan Gubbels / The two type I genotype T. gondii strains, RH, a lab-adapted strain, and GT1, a non-lab-adapted strain, have a genetic difference of only 0.002%, but show remarkable phenotypic differences in vitro. For example, it has long been known that RH’s in vitro virulence (i.e. plaquing capacity) and extracellular survival is far superior to that of GT1, likely due to several decades of adaptation to the in vitro environment (i.e. lab-adaptation). The genetic basis of these phenotypes, however, remains largely unknown despite previous allele-swapping experiments, thus inspiring two hypotheses: 1) epistatic interactions between two or more alleles and/or 2) gene regulatory mechanisms are responsible for lab-adaptive phenotypes. Uncovering the molecular basis underlying lab-adaptive phenotypes will support our growing understanding of T. gondii virulence and suggest therapeutic targets that affect the parasites lytic cycle in a host-independent manner. To answer this question, we applied Evolve and Resequencing (E&R) of GT1 during the first 1500 generations of its lab-adaptation in order to chronologically identify emerging genotype-phenotype correlations. Indeed, lab-adaptation augmented GT1’s in vitro virulence by improving its extracellular survival and reinvasion capabilities- both extracellular phenotypes of the lytic cycle. DNA-sequencing of parallel GT1 populations at multiple evolutionary timepoints (i.e. passages) identified a polymorphic phospholipid flippase gene whose gene expression is critical for in vitro virulence but, unfortunately, the evolved mutations could not be functionally characterized due to technical limitations. RNA-seq of both intracellular and extracellular parasites across several passages identified hundreds of “pro-tachyzoite” differentially expressed genes (DEGs), but only in extracellular parasites, paralleling our phenotypic observations. Interestingly, several upregulated DEGs are connected to fatty acid biosynthesis. Lastly, genetic KO of five seemingly non-related DEGs indicates that GT1’s lab-adaptive in vitro virulence is a complex and polygenic phenotype that is largely controlled by mechanisms independent of genomic mutations. / Thesis (PhD) — Boston College, 2020. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Le phytopathogène Agrobacterium tumefaciens est l'agent causal de la maladie appelée galle du collet, et est capable d'infecter plus de 90 familles de plantes dicotylédones. Cette ∝-protéobactérie appartient à la famille Rhizobiaceae. A. tumefaciens est un complexe de différentes espèces regroupées en 10 génomovars (G1 à G8 et G13). A. tumefaciens C58 appartient au groupe du G8. Son génome est constitué de 4 réplicons : 1 chromosome circulaire, 1 chromosome linéaire et des 2 plasmides dispensables : pAt (pour A.tumefaciens) et pTi (pour Tumor inducing, qui est requis pour la virulence). Pour explorer de nouveaux aspects du mode de vie d’A. tumefaciens, et en particulier l'interaction entre la bactérie et sa plante hôte, deux approches différentes ont été utilisées pour identifier, caractériser et analyser les gènes qui pourraient jouer un rôle dans l'adaptation des bactéries à la tumeur. Une expérience de l'évolution par des passages en série de trois souches différentes de l'agent pathogène sur la plante hôte Solanum lycopersicum a été effectuée afin de clarifier la dynamique évolutive du génome au cours de l'infection. Parallèlement, une étude de différents transcriptomes (in planta et in vitro) a été réalisée et étudiée pour élucider des gènes bactériens candidats impliqués dans l'interaction de la bactérie avec la plante et divers composés produits dans la tumeur. Ce travail tente de donner une vue plus générale du processus d'adaptation de la bactérie à la niche écologique qui est la tumeur. / Agrobacterium tumefaciens is the causal agent of the plant disease called crowngall, and it’s able to infect more than 90 families of dicotyledonous plants. It is an α-Proteobacterium and belongs to the Rhizobiaceae family. A. tumefaciens is a complex of different species grouped in 10 genomovars (G1 to G8, and G13). A. tumefaciens C58 belongs to the G8 group. Its genome consists in 4 replicons: 1 chromosome circular, 1 chromosome linear and 2 dispensable plasmids: pAt (for A. tumefaciens) and pTi (for Tumor inducing), which is required for virulence. To explore new aspects of the A. tumefaciens lifestyle, and in particular the interaction between the bacteria and its plant host, two different approaches have been used to identify, characterize and analyze genes that could play a role in the adaptation of the bacteria to tumor lifestyle. An evolution experiment by serial passages of three different strains of thepathogen on the host plant Solanum lycopersicum has been carried out to clarify the evolutionary dynamics of the genome during the course of infection. In parallel, a study of different transcriptomes (in planta and in vitro) was performed and studied to elucidate bacterial candidate genes involved in the interaction of the bacteria with the plant and various compounds produced in the tumor. This work attempts to give a more general view of the process of adaptation of the bacteria to the ecological niche that is the tumor.

Ecological opportunity—the availability of open niche space to an evolving lineage—has long been thought to modulate the extent of adaptive diversification. Many microbial evolution experiments have confirmed that ecological opportunity drives diversification of initially homogeneous populations into communities of ecologically distinct sub-lineages (ecotypes). Interactions among ecotypes are crucial for both community function and the maintenance of the ecological diversity produced during adaptive diversification, however the factors influencing the evolution of these interactions remain unexplored. We assessed the influence of ecological opportunity on this process by studying communities of the bacterium Pseudomonas aeruginosa that were evolved in either nutritionally complex (COM) or simple (SIM) environments. We measured the net ecological interactions in these communities by comparing the cellular productivity and competitive fitness of whole communities from each environment to that of their component isolates in both complex and simple media. On average, COM communities had both higher productivity and fitness than their component isolates in complex media, indicating that the components of these communities share net positive interactions. The same was not true of SIM communities, which did not differ in either measure from their component isolates. Follow-up experiments revealed that high fitness in two COM communities was driven by rare variants (frequency < 0.1%) that secrete compounds during growth which inhibit PA14, the strain used as a common competitor for fitness assays. Taken together, our results suggest that environments with high levels of ecological opportunity drive diversification into ecotypes that share net positive ecological interactions. The strong effect of diversity on productivity and fitness we found in newly diversified communities has a number of implications for evolutionary ecology as well as the treatment of P. aeruginosa infections.