(
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
,
rsp5
, and the ancestral
(‘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
and
rsp5
. After 250 hr, the
:
ratios showed steady trends, suggesting no additional rapid adaptive races. Two of these cultures (brown) that had
:
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 ”,
:
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 × 10
4 total cells/mm
2 in a ∼2 mm-diameter spot (
Figure 1—figure supplement 1B) and liquid communities at 5 × 10
5 total cells/ml in 3 ml SD. Exp: experiment.