The path to re-evolve cooperation is constrained in Pseudomonas aeruginosa

Background: A common form of cooperation in bacteria is based on the secretion of beneficial metabolites, shareable as public good among cells within a group. Because cooperation can be exploited by cheating mutants, which contribute less or nothing to the public good, there has been great interest in understanding the conditions required for cooperation to remain evolutionarily stable. In contrast, much less is known about whether cheats, once fixed in the population, are able to revert back to cooperation when conditions change. Here, we tackle this question by subjecting experimentally evolved cheats of Pseudomonas aeruginosa, partly deficient for the production of the iron-scavenging public good pyoverdine, to conditions previously shown to favor cooperation. Results: Following approximately 200 generations of experimental evolution, we screened 720 evolved clones for changes in their pyoverdine production levels. We found no evidence for the re-evolution of full cooperation, even in environments with increased spatial structure, and reduced costs of public good production - two conditions that have previously been shown to maintain cooperation. In contrast, we observed selection for complete abolishment of pyoverdine production. The patterns of complete trait degradation were likely driven by cheating on cheats in unstructured, iron-limited environments where pyoverdine is important for growth, and selection against a maladaptive trait in iron-rich environments where pyoverdine is superfluous. Conclusions: Our study shows that the path to re-evolve public-goods cooperation can be constrained. While a limitation of the number of mutational targets potentially leading to reversion might be one reason for the observed pattern, an alternative explanation is that the selective conditions required for revertants to spread from rarity are much more stringent than those needed to maintain cooperation.