Using experimental evolution to evaluate diversification of Pseudomonas fluorescens SBW25 in complex environments

Understanding the ecological conditions promoting diversification of lineages has been one of the major challenges in the field of evolutionary ecology. The ecological theory of adaptive radiation identified two potential conditions to divergence; selection resulting from differences between environments and from the interaction between species. So far, the best explained factor is differences between environmental features giving rise to divergence of lineages. Most of the other cases on adaptive radiation indicated that resource competition is an important process underlying diversification, although the roles of the other species interactions are not explicitly tested. Predation, for instance, is still underexplored, despite having both ecological and evolutionary impacts on prey populations. Role of predation on diversification remained unsettled, as field studies are challenging and experimental studies showed its modifying effect when selection is driven by resource competition. Importantly, the interacting effects of different ecological processes on diversification should be evaluated in detail. The aim of this thesis is to evaluate the importance of predation, and its effect on diversification when predation and resource competition are both present. The first chapter investigated the relative contributions of predation and resource competition to diversification when they act simultaneously. In our experimental evolution study, initially isogenic populations of bacterial prey grew either in the presence or absence of predation in two different resource levels. We determined the major classes of colony morphologies (i.e. morphotypes), which we observed over time in replicate populations. Then, we used the changes in the frequency of these morphotypes to estimate phenotypic diversity over time. We found that predation was the main ecological process driving diversification of bacterial populations. In the absence of predation, resource competition did not lead to diversification. Importantly, the resource level of the environment generated an eco-evolutionary feedback. The differences in the resource level led to differences in the number of predators per prey, which changed the strength of selection, which in the end determined the level of diversity. As a result, the resource level of the environment had a significant contribution to diversification in the presence of predation leading to a higher level of prey diversity in the low resource environment. It has been challenging to disentangle the inputs of different species interactions, however, our experiment presented how the resource level of the environment can contribute both to resource competition and predation leading to differences in selection through eco-evolutionary feedbacks. The second chapter aimed to understand how predation selected for different bacterial morphotypes in environments with different resource levels. Here, we investigated ecologically relevant phenotypic traits and measured the fitness of morphotypes in the presence and absence of predation. We discovered that morphotypes, which were evolved and coexisted in the low resource environment in the presence of predation, showed clumping and cell-chaining traits. Although having these different defense strategies, the coexisting morphotypes had similar defense levels with different competitive abilities. These data suggested that difference in defense trait was not solely selected by predation, but by predation in interaction with resource competition. In the high resource environment, on the other hand, only clumping morphotypes were found. Our data demonstrated that predation generated strong directional selection for defense evolution favoring one defensive phenotype in the high resource environment, where resources were abundant and resource competition was not effecting the selection directly. This chapter provided insight into how different ecological conditions favored different traits. The third chapter examined the roles of ecological processes acting in our evolution experiment on stable coexistence. We investigated the ecological mechanisms which could potentially lead to stable coexistence of two morphotypes in the low resource environment and in the presence of predation. By measuring the fitness of morphotypes in different ecological conditions, we showed that predation operated as an equalizing mechanism and decreased the strength of resource competition and thus allowed morphotypes to coexist stably. Additionally, although identifying the degree of niche overlap experimentally is difficult in such complex ecological conditions, multiple lines of evidence in our study supported possible niche differentiation. Specifically, we presented potential niche differentiation by demonstrating how morphotypes differed in ecologically relevant traits and by following their frequency over time from the early stages of diversification on. We concluded that interacting ecological processes play an important role in stable coexistence. Importantly, our findings highlighted the importance of investigating both types of coexistence mechanisms, before inferring that high degree of niche overlap limits stable coexistence