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Figure S3. Comparison of lasB expression under different culture conditions. The expression values of lasB gene in each culture were normalized to that of LB on day 1. Data shown are the mean values ± SD of three independent experiments. Statistical significance by two-tailed unpaired t test in comparison to 0.5% casein of each column is indicated as: ***P

Figure S2. Phenotypic identification of cooperator, conditional defector, and defector. (A) Growth curves of mono-cultured cooperation strain, conditional defection strain, and defection strain in 1.0 ml of M9-casein (0.5%) broth. Cultures were started from equal number of cells (1 × 105 CFUs, which was diluted from OD600 = 0.005). Data are mean values ± SD (log10 of CFUs) of three individual experiments. (B) Percentage elastase production of conditional defection and defection strains compared with a cooperation strain pure culture. The liquid supernatants (10 μl) of each strain from the experiment in panel (A) were conducted for elastin-Congo red based protease production assay. All the absorbance values at 495 nm were normalized to that of a cooperation strain at the 12 h time point. Data shown are the mean values ± SD of three independent experiments. Statistical significance by two-tailed unpaired t-test is indicated: *P

Analysis of mathematical models. (PDF 359 kb)

Figure S5. Population sizes of the punishment deficient strain (ΔrhlI) and parental WT PAO1 in 1.0 ml of M9-casein (0.5%) broth at the end of each cycle. The punishment mechanism of ΔrhlI can be repaired by the supplementation of C4HSL (C4, 20 μM). Data shown are the mean values ± SD (log10 of CFUs) of three independent experiments. (PDF 164 kb)

Figure S4. Population sizes of repeatedly subcultured P. aeruginosa in 1.0 ml of different media at 24-h intervals. (A) M9-casein (0.5%). (B) Blank M9. (C) LB. (D) 1/4-LB. (E) M9-casamino acids (CAA, 0.5%). (F) M9-CAA (0.1%). Data shown are the mean values ± SD (log10 of CFUs) of three independent experiments. (PDF 175 kb)

Figure S7. Evolutionary dynamics of cooperation, conditional defection, and defection in the simplex for different exclusion probabilities (p = 0.2 for top row, p = 0.8 for middle row, and p = 1 for bottom row). Panels (A), (C), and (E) depict the time series of frequencies of cooperator (black lines), defector (red lines), and conditional defector (blue lines). Panels (B), (D), and (F) depict the evolutionary trajectories in the simplex, where filled circle represents stable fixed point and open circles represent unstable fixed points. When the exclusion probability is relatively small, the system converges to the full defection state finally (A and B), indicating that defectors dominate the whole population. Whereas when the exclusion probability is relatively high, the periodic oscillations among the three strategists appear (C–F), leading to the stabilization of cooperators in the pool. Other parameters: N = 5, r = 3, c = 0.3, δ = 0.3, ∆ = 0.35, and q = 0.1. C, cooperator. CD, conditional defector. D, defector. (PDF 423 kb)

Figure S6. Evolutionary dynamics of cooperation, conditional defection, and defection in the simplex with moderately high exclusion probability. Panel (A) depicts the time series of frequencies of cooperator (black lines), defector (red lines), and conditional defector (blue lines). Panels (B) depicts the evolutionary trajectories in the simplex. Parameters: group size N = 5, multiplication factor r = 3, contribution cost c = 0.3, cost of exclusion δ = 0.3, probability of exclusion p = 0.8, transfer rate of conditional defectors q = 0, and observation cost ∆ = 0.35. C, cooperator. CD, conditional defector. D, defector. (PDF 303 kb)

Table S1. Characterization of lasR mutants during the in vitro evolution of wild type P. aeruginosa in 1.0â ml of M9-casein (0.5%) broth. (PDF 200 kb)

Figure S1. The gradient of selection in dependence on the fraction of cooperators for different exclusion probabilities in infinite populations. Stable equilibria are described by solid circles, while unstable equilibria are described by open circles. Arrows indicate the expected direction of evolution. Cooperation is more favored by natural selection when the arrow points to the right. (A) When the exclusion probability is significantly small, full defection is the only stable equilibrium in the population. (B) With a larger exclusion probability, a coordination game with full cooperation and full defection as the two stable equilibria appears. (C) When the exclusion probability is further increased, full cooperation is the only stable equilibrium. The values of exclusion probability are (A) p = 0.1; (B) p = 0.6; (C) p = 0.8. Other parameters: N = 5, r = 3, c = 0.3, and δ = 0.3. (PDF 262 kb)