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    Experimental evolution under predation in P. fluorescens SBW25 : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Microbiology & Genetics at Massey University, Auckland, New Zealand
    (Massey University, 2022) Sadeghpour Golzar, Farhad
    Predator-prey interactions are recognised to change the pace of evolution in microbial communities, but investigations into how selection for predation accelerates antagonistic behaviours, genomic evolution and the process of coadaptation are scarce. Here we performed a 20-day and an extended 90-day evolution experiment to investigate the adaptive traits that arise in prey bacterium Pseudomonas fluorescens SBW25 on solid media in the presence and absence of a wild Acanthamoeba sp. Coevolution led to bacterial diversity, resistance to predation in coevolved bacterial lineages and evolution of predators. We show evidence of reciprocal adaptation, strong phenotypic and genotypic parallelism among prey lineages undergoing predation. We observed evolution of new colony morphotypes such as Wrinkly Spreader, Volcano and Mountain. Evolved morphotypes conferred grazing resistance and an increase in relative prey fitness that resulted in increased encystment and reduced replication of the protozoan populations. Mutation profiles of the coevolved phenotypes were associated with altered gene function in amrZ, wspF, fadD1, fadD2 and putative hypothetical protein upstream of RND transporter. RNA sequencing results of the mutants also revealed a significant increase in the number of genes that up or downregulated while interacting with Acanthamoeba sp. We investigated the degree to which these mutations affect biofilm formation, capsulation, motility, mucoid and fatty acid degradation pathways. Some of these traits are associated with virulence in pathogenic organisms. We further found evidence of mutualisms where both prey and predator increased their survival relative to their respective ancestors. On the other side, we show promoted killing performance and higher generations upon feeding on WT bacteria in coevolved Acanthamoeba compared to their ancestors. Together, our findings demonstrate the emergence of divergent colony morphologies and molecular parallelism that arise as an adaptation to predation and notably affects the fitness and evolution of predators suggesting Red Queen co-evolutionary dynamics between predators and prey. These findings suggest that protozoan predation can profoundly influence the course of genetic and phenotypic evolution in short and long-time scales.
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    Investigating the evolutionary changes in Crabtree-negative yeasts during a long-term evolution experiment : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Genetics at Massey University, Albany, New Zealand
    (Massey University, 2017) Morley, Annabel
    The Crabtree effect is a metabolic strategy that allows yeast to ferment in the presence of oxygen. This is of interest as not all yeasts display this strategy, and nearly 100 years after its discovery it is still unclear what the overall benefit is. Two key theories attempt to explain the emergence of this phenomenon, the make-accumulate-consume theory and the rate/yield trade-off theory. The aim of this thesis was to investigate whether a trade-off between rate and yield develops in Crabtree-negative yeasts over the course of 1500 generations in a high sugar environment. Chapter Two demonstrates that growth rate is more likely to increase than decrease while growth yield is more likely to decrease than increase in the isolate-derived populations of yeast. We find that species that started out relatively fast, changed little while the slower species had more significant gains in growth rate. With growth yield, the species with initially high yield lost more significantly than the already low yield species. This could suggest there is an overall optimum growth rate and growth yield, that the species are evolving towards. In Chapter Three, ethanol production was measured using colorimetric tests and no change was observed to support the development of the Crabtree effect in these populations after 1500 generations. In Chapter Four growth yield was investigated using flow cytometry and it was found that several yeast populations both increased in cell size and decreased in growth yield. This is an interesting observation that has been observed in several previous experimental evolution experiments. In Chapter Five, as cell size is often associated with ploidy changes, DNA content was measured using DAPI and SYTOX DNA stains, detected by flow cytometry. This did not provide any statistically significant conclusions but highlighted the importance of employing further techniques to analyse the DNA content of these populations. This thesis has illustrated the importance of studying the competitive behaviours of microorganisms in isolation, where selfish traits appear to thrive.
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    Genetically integrated traits and rugged adaptive landscapes in digital organisms.
    (12/05/2015) Ostrowski EA; Ofria C; Lenski RE
    BACKGROUND: When overlapping sets of genes encode multiple traits, those traits may not be able to evolve independently, resulting in constraints on adaptation. We examined the evolution of genetically integrated traits in digital organisms-self-replicating computer programs that mutate, compete, adapt, and evolve in a virtual world. We assessed whether overlap in the encoding of two traits - here, the ability to perform different logic functions - constrained adaptation. We also examined whether strong opposing selection could separate otherwise entangled traits, allowing them to be independently optimized. RESULTS: Correlated responses were often asymmetric. That is, selection to increase one function produced a correlated response in the other function, while selection to increase the second function caused a complete loss of the ability to perform the first function. Nevertheless, most pairs of genetically integrated traits could be successfully disentangled when opposing selection was applied to break them apart. In an interesting exception to this pattern, the logic function AND evolved counter to its optimum in some populations owing to selection on the EQU function. Moreover, the EQU function showed the strongest response to selection only after it was disentangled from AND, such that the ability to perform AND was lost. Subsequent analyses indicated that selection against AND had altered the local adaptive landscape such that populations could cross what would otherwise have been an adaptive valley and thereby reach a higher fitness peak. CONCLUSIONS: Correlated responses to selection can sometimes constrain adaptation. However, in our study, even strongly overlapping genes were usually insufficient to impose long-lasting constraints, given the input of new mutations that fueled selective responses. We also showed that detailed information about the adaptive landscape was useful for predicting the outcome of selection on correlated traits. Finally, our results illustrate the richness of evolutionary dynamics in digital systems and highlight their utility for studying processes thought to be important in biological systems, but which are difficult to investigate in those systems.