The evolution of robustness and fragility during long-term bacterial adaptation

Theory predicts that well-adapted populations may evolve mechanisms to counteract the inevitable influx of deleterious mutations. While mutational robustness can be directly selected in the laboratory, evidence for its spontaneous evolution during general adaptation is mixed. Moreover, whether robustness evolves to include pleiotropic effects remains largely unexplored. Here, we studied the effects of point mutations in the RNA polymerase of <i>Escherichia coli</i> over a 15,000-generation adaptive trajectory. Fitness effects of both beneficial and deleterious mutations were attenuated in fitter backgrounds. In contrast, pleiotropic effects became more severe and widespread with greater adaptation. These results show that trade-offs between robustness and fragility can evolve in regulatory networks, regardless of whether driven by adaptive or nonadaptive processes. More broadly, they illustrate how adaptation can generate hidden variability, with unpredictable evolutionary consequences in new environments.