Figure 2. In simulated communities, a spatial environment is required to promote heterotypic cooperation.

The $R_{\to A}^{\leftarrow L}$:$C^{\leftarrow L}$ ratios of simulated (‘Sim’) spatial cooperating and cheating communities were grown either unperturbed (purple circles) or periodically mixed (orange squares). To simulate periodic mixing, the arrangement of cells was completely randomized every 12 hr. In each mixing event, the concentration of adenine and lysine throughout the community was assigned to be the average value over the entire community. Error bars show the standard deviation of ratios in six independent communities. The solid black line shows the ratio in a simulated well-mixed liquid coculture using the same parameters as simulated communities on agarose (Figure 2—source data 1). The fitness advantage of cheater over cooperator was either 2% (top panel) or 8% (bottom panel).

DOI: http://dx.doi.org/10.7554/eLife.00960.006

Figure 2—figure supplement 1. Stochastic cheater outcomes in periodically mixed communities of ancestral cooperators, cheaters, and partners.

The $R_{\to A}^{\leftarrow L}$:$C^{\leftarrow L}$ ratios diverged in six replicate experimental communities periodically mixed during growth. Cell migration during mixing allowed the mutant type most adapted to the low-lysine environment to sweep through the entire community. The most adaptive mutation could occur in either $R_{\to A}^{\leftarrow L}$ or $C^{\leftarrow L}$, which led to stochastic outcomes similar to those observed in well-mixed liquid cocultures (Waite and Shou, 2012). We started these communities from a spot at a density of ∼8 × 10⁴ total cells/mm² (Figure 1—figure supplement 1B) and physically mixed them daily (approximately every two generations) using a glass rod. The cells attached to the glass rod (estimated to be ∼10% of the community population) were suspended in water and analyzed using flow cytometry. Exp: experiment.

DOI: http://dx.doi.org/10.7554/eLife.00960.008

Figure 2—figure supplement 2. A spatial environment is required to favor heterotypic cooperation over cheating.

(A) To preadapt 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 and Shou, 2012). The initial stochastic phase was indicative of additional rounds of adaptive races (Waite and Shou, 2012) between rsp5 $R_{\to A}^{\leftarrow L}$and rsp5 $C^{\leftarrow L}$. After 250 hr, the $R_{\to A}^{\leftarrow L}$:$C^{\leftarrow L}$ ratios showed steady trends, suggesting no additional rapid adaptive races. Two of these cultures (brown) that had $R_{\to A}^{\leftarrow L}$:$C^{\leftarrow L}$ ratios close to 1:1 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. The broken axis indicates the period of time elapsed during which a small revived inoculum (30 μl frozen stock into 200 μl minimal medium and then expanded to 2 ml minimal medium) grew to detectable densities. (C) When preadapted communities were grown unperturbed on agarose pads, cooperators were favored as communities grew (purple circles). In contrast, cheaters were favored when the spatial aspect of the environment was either disrupted (daily mixing after day 5, orange squares) or absent (well-mixed liquid cocultures, black diamonds). We started all spatial communities at ∼8 × 10⁴ total cells/mm² in a ∼2 mm-diameter spot (Figure 1—figure supplement 1B) and liquid communities at 5 × 10⁵ total cells/ml in 3 ml SD. Exp: experiment.

DOI: http://dx.doi.org/10.7554/eLife.00960.009