Inducible Mutability and Components of Adaptation Figure 7. Average percent reduction in doubling time. Doubling time of UVR lineage population samples, UVR lineage isolates, and non-UVR lineage isolates after (A) 250 and (B) 500 generations of experimental evolution was measured under non-UVR (open bars) and UVR (shaded bars) conditions. Values represent the average percent improvement compared to the P. cichorii 302959 ancestor doubling times of 1.4560.09 hrs and 1.6860.10 hrs under non-UVR and UVR conditions, respectively. Error bars represent standard error of the mean. n = 8 for UVR lineage population samples and non-UVR lineage isolates but n = 16 for UVR lineage isolates. Values are significant by two-tailed independent t-test (a = 0.05) where indicated (* P,0.05, ** P,0.01, *** P,0.001). P-paired values correspond to two-tailed paired t-tests (a = 0.05) between non-UVR and UVR conditions. doi:10.1371/journal.pone.0015975.g007 transcriptional regulators that tightly control the temporal activity of its gene products [8,24,25]. Beneficial adaptations targeted to any of these regulators that contribute to the timing or efficiency of the SOS network could be observable as a reduction in lag time particularly under UVR conditions. We explored changes in UVR tolerance as an obvious candidate for improved fitness and lag time under UVR conditions but only observed increased UVR tolerance concur- rently with fitness gains in a limited number of instances. In general, there was no pattern to suggest that greater UVR tolerance was primarily responsible for increased relative fitness observed under UVR conditions. Alternatively, improved fitness under UVR conditions could result from enhanced nutrient scavenging. Approximately 90% of the population died as a result of UVR exposure during each cycle of the UVR lineage regime. The death of these individuals could release additional nutrients in the form of cell lysate that is transferred with surviving cells to fresh medium for the next cycle of growth. Any mutation that would enhance the utilization of these nutrients could lead to greater fitness exclusively under UVR conditions. In evolution experiments with E. coli, adaptive mutations that contribute to survival through catabolism of cellular detritus [26] or secreted metabolites [27] have been reported. However, we were unable to observe growth by the ancestral P. cichorii 302959 or any evolved lineages in glucose-free Davis Minimal medium amended with a bacterial suspension killed either by prolonged heat or UVR exposure (data not shown). Therefore, increased relative fitness under UVR conditions and the adaptive specificity of UVR lineages that is reflected in improved growth likely did not result from either increased UVR tolerance or a heightened ability to scavenge nutrients as a carbon source. We previously observed the reproducible emergence of an F morphotype that coexisted with the ancestral, R morphotype in all UVR lineages but never in non-UVR lineages [12]. In the current study, single-colony representatives of both morphologies have been examined for changes in relative fitness and physiology at generation 250 and 500 under both UVR and non-UVR conditions. We have found no evidence to suggest that either morphotype possesses an inherent adaptive advantage observable as improved relative fitness, growth dynamics, or UVR tolerance. In our analyses we found only subtle differences in doubling times between the two groups. The results of the current study are consistent with our previous observations of the stochastic fluctuations of F morphotype abundance [12] suggesting the F determinant itself likely does not carry a strong selective advantage. Therefore, measurements of the two groups can be combined and interpreted as additional sampling of the genetic diversity present in the UVR lineages. It is clear from our relative fitness measurements that adaptive changes in both non-UVR and UVR lineages were concentrated in the first 250 generations, confirming both the strong selection pressure of the experimental environments and the high adaptive potential of the ancestral P. cichorii 302959 genotype. Furthermore, the early fitness improvements by UVR lineages are consistent with the emergence of F morphotypes and spontaneous rifampi- cin-resistant colonies reported previously [12]. For a more detailed view of adaptation, we tracked the acquisition of relative fitness under UVR and non-UVR conditions in four randomly selected PLoS ONE | www.plosone.org 9 January 2011 | Volume 6 | Issue 1 | e15975