Inducible Mutability and Components of Adaptation Figure 5. Relative fitness of ‘fuzzy’ (F) isolates from UVR lineages 25-32. Relative fitness was measured by direct competition with the ancestor under (A) non-UVR and (B) UVR conditions. Single isolates of P. cichorii 302959 exhibiting the ‘fuzzy’ colony morphology were taken after 250 (open bars) and 500 (shaded bars) generations of experimental evolution. Fitness values are means and error bars represent standard error of the mean. Values are significant by two-tailed independent t-test (d.f. = 5, a = 0.05) where indicated (* P,0.05, ** P,0.01, *** P,0.001). P-paired values correspond to two-tailed paired t-tests (d.f. = 5, a = 0.05) between relative fitness values for isolates from generation 250 and 500. doi:10.1371/journal.pone.0015975.g005 until additional fitness gains were later achieved under UVR conditions by generation 250 (Figure 9A). UVR lineage 30 displayed improved fitness only under UVR conditions for a much longer period until later gaining fitness under non-UVR conditions (Figure 9B). However, even after gaining fitness under non-UVR conditions, UVR lineage 30 continued to exhibit significantly higher fitness under UVR conditions. Similarly, non-UVR lineages 36 and 39 exhibited fitness gains under their native, non-UVR conditions first (Fig. 9C and D). Neither lineage 36 nor 39 exhibited a significant increase in non- UVR fitness following the initial improvements observed in the first 100 generations. However, additional fitness improvements under UVR conditions were achieved before generation 500 in both non-UVR lineages examined (Figure 9C and D). Discussion Our results suggest that adaptive specificity in laboratory evolved populations of the highly UVR-mutable P. cichorii 302959 emerged early and correlated with discrete growth improvements but was not dependent upon MDR activation. In a previous study, we maintained parallel cultures of P. cichorii 302959 for 500 generations with daily MDR activation in the form of UVR exposure and observed higher relative fitness particularly under UVR conditions [12]. In the present study, changes in relative fitness and underlying physiological characteristics were analyzed at intermediate points (in particular, at generation 250) in the experimental evolution of P. cichorii 302959 to evaluate this observed adaptive specificity. The relative fitness of all 16 lineages was determined at the generation 250 midpoint of our evolution experiment with the intent to gain further information on the acquisition of fitness observed previously at the generation 500 endpoint. Fitness measurements at the midpoint can offer some indication of the timing, number, magnitude, and parallel nature of adaptive improvements achieved by each lineage. Measurements of relative fitness at generation 250 reflected the same patterns of adaptive specificity observed previously at generation 500 [12]. The interaction between sympatric genotypes within UVR lineages was previously shown to contribute to relative fitness under UVR conditions [12]. Such ecological interactions were established in UVR lineages by generation 250 and population samples exhibited greater fitness gains than individual isolates. A comparison of relative fitness values at different time points PLoS ONE | www.plosone.org 7 January 2011 | Volume 6 | Issue 1 | e15975