The transition to multicellularity is a major step in the evolution of complex life. The first steps in this transition are poorly understood because multicellularity evolved long ago, and transitional forms have been lost to extinction. Previous studies developed a novel microbial model system in which simple multicellularity could be evolved de novo (Ratcliff et al., 2012). By evolving our snowflake yeast to undergo sexual reproduction we hypothesized that sex created variation in key multicellular traits, which spurs multicellular adaptation. In our 'snowflake yeast' model system, two traits are of central importance: cluster size, and programmed cell death (apoptosis). Apoptosis previously evolved to regulate cluster size, by acting as break points within clusters, allowing them to modify the size and number of multicellular propagules they produce. In prior experiments, this only develops after yeast have evolved to form large clusters. Prior experiments in the lab demonstrated that the longer snowflake yeast have been evolving, the greater the fitness benefit provided by sex. Here we examine whether this is due to sex creating greater amounts of diversity in the traits of post-sex offspring in more highly evolved multicellular yeast, allowing post-recombination offspring to 'fine tune' their multicellular traits. By using flow cytometry, we collected data on our multicellular traits. By gathering the biomass mean of the cluster size in each population and staining the cells with propidium iodide to determine the apoptotic tendencies of our cells we were able to compare our outcomes to the pre-sex ancestor, and we determined there was no increase in variation. Although apoptosis did not have an increase in variation due to sex, it created a variation in cluster size; the variation was seen in the population W8. This still supports our hypothesis that sex creates variation in multicellular traits, which allows for rapid adaptation.

Sexual selection is ubiquitous in all sexually reproducing species and a powerful evolutionary force. The effect of sexual selection on population fitness has caused wide debate and has been proposed to both enhance adaptation rates, but also possibly increase extinction risk. Using experimental evolution, the strength of sexual selection was altered by biasing adult sex ratios in replicated populations of the Indian meal moth, Plodia interpunctella. Under increasing temperatures strong sexual selection and polyandry was associated with increased population fitness. Suggesting sexual selection could provide a buffer against climate change by increasing adaptation rates. However, no effect on male mating success was observed. In contrast, under stable temperatures male mating success was increased by strong sexual selection, however, this did not translate to increased population fitness. These results indicate that female choice is a potentially strong selective pressure in P. interpunctella. Moreover, under stabilising and directional selection the costs and benefits of sexual selection change. In a field study, dung beetle species richness and abundance were compared across a gradient of habitat disturbance, ranging from old-growth rainforest to oil palm plantation. Species persistence within altered habitats was positively associated with expressing horns and relative horn lengths, suggesting male-male competition increases a species ability to persist within modified landscapes. There was not a strong effect of relative testes mass or sexual size dimorphism on the abundance of species. Additionally, by examining the expression of sexually selected traits within species, horn length and testes mass appear to be condition dependent, but only the expression of horns was effected by habitat change. Overall, from both laboratory and field studies it was found that sexual selection can increase adaptation rates and the persistence of species within altered and changing environments. Moreover, both sets of studies suggest pre-copulatory sexual selection to be an important aspect of sexual selection in driving this adaptation.

Resource conditions in nature can fluctuate markedly and how organisms adapt to survive in these conditions is of great interest in the fields of evolutionary biology and ecology. Experimental evolution using microbes has been shown to be effective in answering general evolutionary questions. Using this technique, I studied the bacterium Escherichia coli adapting to fluctuating environments to investigate the evolution of growth traits and the dynamics of adaptation. My results have provided general insight into bacterial adaptation which may allow for better prediction of growth trait evolution in a range of conditions. (1) I have shown that evolution in both predictable and unpredictable environments resulted in the evolution of a reduced lag phase, an increased growth rate and a higher maximum population size. My results suggest that bacteria do not adapt to conditions by anticipating the timing of the resource renewal. (2) I found that a trade-off exists for evolved populations between a reduced lag phase and a higher mortality rate in all environments, and propose this as an explanation as to why some bacteria retain a lag phase. (3) I show that the dynamics of adaptation do not differ between populations adapted to conditions which involved varying periods of time in stationary phase between transfers. There seem to be different mutations for different traits, with mutations to the lag reducing first, followed by growth rate, and finally population size. These findings highlight the dynamics of growth trait evolution in environments in which a complex interplay exists between reproducing and growing faster than competitors, and being able to survive in starvation conditions.

Listeria monocytogenes the causative agent of foodborne listeriosis is a facultative pathogen that lives as a saprophyte in soil and as an intracellular parasite in host tissues. A regulatory protein, the transcriptional activator PrfA, plays a key role in the “saprotroph to parasite” conversion of L. monocytogenes by selectively activating key virulence genes essential for infection when the bacteria enter host cells. Central to this conversion is the plastic ability of PrfA allosterically shift between two states, weakly active (“ON-OFF”, outside in the environment) and strongly active (“ON”, intracellular compartment). In this thesis, I have used the PrfA “ON-OFF” virulence switch to understand the role of virulence regulators in the adaptability of facultative parasites to a wide range of niches. Using the PrfA model, I have also examined the trade-offs between the saprotroph and virulent states of facultative pathogens and the role of plasticity in maintaining adaptation to multiple environments. Using soil as a natural environment model, I have shown that overexpression of the PrfA-dependent virulence regulon has a negative impact on environmental survival of L. monocytogenes. Then I investigated the fitness consequences of losing PrfA switchability in non-host environments. The results in in-vitro growth conditions with isogenic strains with PrfA locked in the “ON” state and in which all the genes of the virulence regulon were deleted, showed that PrfA-dependent gene overexpression causes a reduction in fitness. Our data indicate this was directly attributable to the costs associated with the overproduction of an array of unneeded proteins and not to indirect effects of hyperactive PrfA in Listeria metabolism. Finally, I used experimental evolution studies in in-vitro only conditions and in alternate in-vitro and intracellular conditions with bacteria with wild-type or “ON-locked” PrfA alleles to visualize the selective pressures acting on the PrfA switch. The results of the selection experiments showed that adaptation to the different conditions involves a rapid evolution of PrfA with mutations changing its activity according to the specific environment in which selection occurred. The findings from this thesis highlight the importance of plastic ability, evolution of properly regulated genetic systems and the role of these genetic systems in enabling organisms to maximise their fitness during the adaptation process to a specific niche.

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.

Experimental evolution has been used to investigate both general and specific evolutionary processes. More recently, it has also been used to resolve protein-protein interactions. Viruses assemble through a series of protein-protein interactions which must remain more favorable than any competing off-pathway reaction. By constructing chimeric viruses with genes or segments of genes from another species, foreign elements are introduced into this system of assembly. Characterization of the resulting chimeras provides information about which proteins interact, the protein-protein interacting interface, the role of particular domains, and the importance of specific residues. Chimeric viruses often exhibit a reduction in fitness, as the foreign element is unable to interact as efficiently in the system as the native element. Through experimental evolution, mutations accumulate that affect interacting partners in the system, leading to a more optimal assembly pathway. The microviruses are well-characterized single-stranded (ss) DNA bacteriophages. They are divided into three clades, represented by φX174, G4, and α3. Incidences of horizontal gene transfer between microvirus clades are unusually rare and may be due to a complex assembly pathway with multiple stages: a foreign element has the potential to disrupt a multitude of morphogenetic steps. In this study, we exchanged major spike genes between the two microvirus species φX174 and G4, then monitored the evolutionary recovery. Results can be interpreted within this thermodynamic paradigm. Although the G4-φXG chimera could only form plaques at low temperature and exhibited reduced fitness, its evolutionary recovery was relatively straightforward. The other chimera, φX-G4G, could only form plaques when complemented with two wild-type φX174 genes. Isolating a complementation-independent chimera required the passaging of mutants through a series of different environments. The first selection yielded mutations of the largest effects. First, the truncation of a protein involved in DNA synthesis was recovered, resulting in a global decrease in gene expression. Next, a recombination event at the 3' end of the foreign gene resulted in a modification of the protein’s C-terminus. These mutations were subjected to further analysis to determine why they were so critical at this early stage of experimental evolution. Subsequent passages of the φX-G4G chimera eventually yielded viable strains, with additional mutations affecting stages of late assembly. Overall, results indicate how gene exchange can drastically affect flux through the pathway. When the system is initially perturbed, the process of experimental evolution allows the pathway to return to a normalized state. The mutations isolated during this recovery stage indicates how the flux was initially altered, and how it can be restored.

No description available.

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.

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.

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.