Phenotypic correlations are products of genetic and environmental interactions, yet the nature of these correlations is obscured by the multitude of genes organisms possess. My dissertation work focused on using 12 populations of Escherichia coli from Richard Lenski's long-term evolution experiment (LTEE) to understand how genetic correlations facilitate or impede an organism's evolution. In chapter 1, I describe how ancient correlations between aerobic and anaerobic metabolism have maintained - and even improved - the capacity of E. coli to grow in an anoxic environment despite 50,000 generations of relaxed selection for anaerobic growth. I present genomic evidence illustrating substantially more mutations have accumulated in anaerobic-specific genes and show parallel evolution at two genetic loci whose protein products regulate the aerobic-to-anaerobic metabolic switch. My findings reject the "if you don't use it, you lose it" notion underpinning relaxed selection and show modules with deep evolutionary roots can overlap more, hence making them harder to break. In chapter 2, I revisit previous work in the LTEE showing that the fitness increases measured for the 12 populations positively correlated with an increase in cell size. This finding was contrary to theory predicting smaller cells should have evolved. Sixty thousand generations have surpassed since that initial study, and new fitness data collected for the 12 populations show fitness has continued to increase over this period. Here, I asked whether cell size also continued to increase. To this end, I measured the size of cells for each of the 12 populations spanning 50,000 generations of evolution using a particle counter, microscopy, and machine learning. I show cell size has continued to increase and that it remains positively correlated with fitness. I also present several other observations including heterogeneity in cell shape and size, parallel mutations in cell-shape determining genes, and elevated cell death in the single LTEE population that evolved a novel metabolism - namely the ability to grow aerobically on citrate. This last observation formed the basis of my chapter 3 research where my collaborators and I fully examine the cell death finding and the associated genotypic and phenotypic consequences of the citrate metabolic innovation.
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