Evolution de la diversification adaptative bactérienne : origine et maintenance à long-terme. – EvoDiv

1-Scientific background and objectives Natural selection has shaped all the extraordinary diverse phenotypes of living organisms, while random mutations provide the raw material for this process. One of the most fundamental issues of evolution is to understand the forces driving the origin and maintenance of diversity: how can two living types diverge from a common ancestor and co-exist at the same spatial location? What kind of interactions made the co-existence stable? It is usually difficult to dissect evolutionary changes underlying both the evolution of diversity and the subsequent maintenance of phenotypic and genetic variation, mainly because the relevant events happened in the distant past. Moreover, it is difficult to link phenotypic and genotypic traits. However, we can efficiently reproduce similar events in the laboratory by experimental evolution strategies, allowing analysis and comparison over tens of thousands of generations. The use of micro-organisms, with short generation times and large populations, makes it possible to study the dynamics of evolutionary processes in the laboratory, under controlled conditions and with high replication. Ancestors and evolved individuals isolated during evolution can be frozen and revived, providing a frozen fossil record of sorts. The use of Escherichia coli offers additional power because the tremendous amount of information available on this organism helps to analyze and interpret the genetic bases of phenotypic changes. During the longest-running evolution experiment, where an ancestral cell of E. coli has been used to propagate twelve populations in a defined environment for tens of thousands of generations, we have observed a unique event of divergence: two phenotypically-differentiated ecotypes have emerged from a common ancestor, and have co-existed for tens of thousands of generations by negative frequency-dependent selection. Our objective is to understand the successive genetic events leading to the divergence of the two bacterial types, and to their stable and dynamic co-existence. To accomplish this we have assembled an interdisciplinary team of physicist and evolutionist. 2-Description of the project, methodology This project combines the development of an automated technology and mathematical modelling with genomic and phenotypic analyses of evolutionary processes during experimental evolution of E. coli. Twelve populations, derived from a common ancestor, have been propagated in a defined environment during more than 40,000 generations. Very early in the evolutionary history of one population, an ancestor gave rise to two ecotypes, which subsequently have been shown to co-exist while they compete and co-adapt. Mutations have been selected in each ecotype during this co-adaptation. We have detected many events in which one ecotype becomes predominant, without however being able to eliminate the other. These population switches denote successive selective sweeps (winning strategies) by each one of the ecotypes. Both ecotypes are characterized by few population dynamics parameters that determine how much each benefits/suffers from the presence of the other: growth rate on the primary resource; differential death rates during stationary phase. Using an interdisciplinary approach, we will evaluate these parameters reliably for any evolutionary time point. To produce the quality high-throughput data we need, these analyses will be automated. We can then establish for each population switch event which parameter was modified and which strategy adopted. The next step will be to correlate the parameter changes to a set of specific genetic expression modifications and ultimately, to single mutations. Global expression profiles will be performed both for evolved clones isolated at key evolutionary stages and for a constructed set of isogenic strains, except for specific mutations. We will analyze these transcriptional profiles by multi-level comparisons (across and within each ecotype, at different time points and growth stages), to identify key mutations during the process of adaptive diversification. All these phenotypic and genetic parameters will be available for any evolutionary time point, including the period of divergence and of long-term co-existence. The predictions inferred by these correlations will then be experimentally tested to dissect the connections between these co-evolving biological systems. 3-Expected results This project will analyse for the first time the entire evolutionary stages underlying the emergence of diversity from a monomorphic state and the subsequent maintenance of the phenotypic and genetic variation. Finally, the objective of the project will be to link genetic, biochemical and physiological changes to evolutionary processes leading to bacterial adaptive diversification. More generally, this will improve our knowledge about the dynamics of phenotypic and genomic evolution.