(A) Recurrent mutations are self-serving. We measured growth rates of mutant and ancestral strains in minimal SD medium with various lysine concentrations, using a calibrated fluorescence microscopy assay (Hart et al., 2019c). Briefly, for each sample, total fluorescence intensity of image frames was tracked over time, and the maximal positive slope of ln(fluorescence intensity) against time was used as the growth rate. Evolved strains grew faster than the ancestor in community environment (the gray dotted line corresponds to the lysine level supporting a growth rate of 0.1/hr as observed in ancestral Cooperation that is Synthetic and Mutually Obligatory [CoSMO] Hart et al., 2019a). Measurements performed on independent days (≥3 trials) were pooled and the average growth rate is plotted. Fit lines are based on Moser’s equation , where b(L) is the cell birth rate at metabolite concentration L, bmax is the maximum birth rate in excess lysine, KL is the lysine concentration at which half maximum birth rate is achieved, and n is the cooperitivity cooefficient describing the sigmoidal shape of the curve (Hart et al., 2019c). Evolved strains are marked with evo; engineered or backcrossed mutants are marked with the genotype. Data for DISOMY14 are reproduced from Hart et al., 2019b as a comparison. Data can be found in Figure 2—source data 1. (B) Self-serving mutations stabilize Lyp1 localization on cell membrane. We fluorescently tagged Lyp1 with GFP (green fluorescent protein) in ancestor (WY1620), ecm21Δ (WY2355), and rsp5(P772L) (WY2356) to observe Lyp1 localization. We imaged each strain in a high lysine concentration (164 µM) as well as after 4 and 10 hr incubation in low lysine (1 µM). Note that low lysine was not consumed during incubation (Hart et al., 2019c). During prolonged lysine limitation, Lyp1 was stabilized to cell membrane in both mutants compared to the ancestor. Images contain samples from several fields of view so that more cells can be visualized.