Theoretical models of ecological specialization commonly assume that adaptation to one environment leads to fitness reductions (costs) in others. However, experiments often fail to detect such costs. We addressed this conundrum using experimental evolution with Escherichia coli in several constant and fluctuating environments at multiple population sizes. We found that in fluctuating environments, smaller populations paid significant costs, but larger ones avoided them altogether. Contrastingly, in constant environments, larger populations paid more costs than the smaller ones. Overall, large population sizes and fluctuating environments led to cost avoidance only when present together. Mutational frequency distributions obtained from whole-genome whole-population sequencing revealed that the primary mechanism of cost avoidance was the enrichment of multiple beneficial mutations within the same lineage. Since the conditions revealed by our study for avoiding costs are widespread, it provides a novel explanation of the conundrum why the costs expected in theory are rarely detected in experiments.

Measure of Fitness: The maximum growth rate (R), computed as the maximum slope of the 24-hr growth curve over a dynamic window of ten OD readings. 

The fitness values reported here were scaled by the corresponding ancestral growth rates (0.073 (in G), 0.076 (in M), 0.055 (in S), 0.043 (in T)). The fitness values for the TL, TS, GL and GS regimens have been reported previously in Chavhan, Y., Malusare, S. & Dey, S. (2020) Heredity, 124, 726–736.