Author response image 2. Well-mixed evolved cocultures of heterotypic cooperators and cheaters showed a steady change in the $R_{\to A}^{\leftarrow L}$ :$C^{\leftarrow L}$ ratio. (A) To pre-adapt cooperators and cheaters in the lysine-limited coculture environment so that no new mutations of large fitness benefits could quickly arise, we started eight well-mixed replicates (marked by different symbols) consisting of rsp5 $R_{\to A}^{\leftarrow L}$, rsp5 $C^{\leftarrow L}$, and the ancestral $G_{\to L}^{\leftarrow A}$ (Materials and methods). The rsp5 mutation was previously found to be highly adaptive for the lysine-requiring cells in a lysine-limited environment (Waite & Shou 2012). The initial stochastic phase at 150 hours is indicative of additional rounds of adaptive races²¹ between rsp5 $R_{\to A}^{\leftarrow L}$ and rsp5 $C^{\leftarrow L}$. After 250 hours, the $R_{\to A}^{\leftarrow L}$ :$C^{\leftarrow L}$ ratios showed steady trends, suggesting no additional rapid adaptive races. Two of these cultures (purple) were revived from frozen stocks. (B) In the two revived cocultures where the evolved populations were denoted with a “ ʹ ”, ${R^{\prime }}_{\to A}^{\leftarrow L}$ :${C^{\prime }}^{\leftarrow L}$ continued to decline steadily before being used for experiments in Figure 2–figure supplement 2. Broken axis indicates the period of time elapsed during which a small revived inoculum (30 μl into 200 μl minimal medium and then expanded to 2 ml minimal medium) grew to detectable densities.