Inducible Mutability and Components of Adaptation Figure 1. Evolutionary history of isolates and population samples derived from experimental lineages of P. cichorii 302959 and characterized in this study. Sixteen parallel lineages of P. cichorii 302959 were derived from a single colony and propagated by serial transfer. Eight UVR lineages received daily UVR exposure and diversified into mixtures of ‘round’ and ‘fuzzy’ colony morphotypes. Eight non-UVR lineages did not receive UVR exposure and contained only the ancestral, ‘round’ colony morphotype. All 16 lineages were propagated for 500 generations and samples from each were periodically preserved. Isolates and population samples in this study were derived from the ancestor following either 250 or 500 generations of selection in the evolution experiment. doi:10.1371/journal.pone.0015975.g001 samples were removed for OD measurement at 600-nm in 96-well microtiter plates using a Tecan Safire (Tecan US, Inc., Durham, NC). Samples were removed for measurement at 0 hr and at 1 hr intervals from 4 hr to 15 hr. Growth curves for each lineage isolate and population sample were repeated in triplicate under both UVR and non-UVR conditions. OD600 values from growth curves were standardized by dividing by the initial OD600 (0 hr) and then log2 transformed. The transformed values were plotted, and the window of exponential growth was identified by linear regression [20]. Lag time was calculated simply as the x-intercept of the linear equation. Doubling time (g) was determined by the equation: g~3:3( log 10(N2){ log 10(N1))7t where N1 and N2 are raw, untransformed OD values corres- ponding to the window of exponential growth observed in the log2 transformation plot and t is the time interval between them [21]. UVR tolerance assays Lineage isolates and population samples were grown overnight in LB broth and 2 ml of culture were pelleted, washed with 1 ml saline, resuspended in 1 ml saline, and held on ice. The cell suspensions were mixed with 9 ml saline in a glass petri dish and exposed to a single dose of approximately 140 J m22 UVC as described above. Cell suspensions were mixed continuously while receiving UVR doses to eliminate survival as a result of shading. Following irradiation, surviving cells were enumerated by dilution plating conducted under dark conditions. Results Relative fitness of evolved lineages The relative fitness of all lineages was determined by competition with the P. cichorii 302959 ancestor under both UVR and non-UVR conditions to examine adaptation during experimental evolution. We evaluated the relative fitness of each lineage by competing both population samples (a subset of the entire lineage maintaining the assemblage of diverse genotypes) and single-colony isolates. In our previous study, we analyzed changes in relative fitness in replicate lineages after 500 generations [12]. In this current study, we report on alterations in relative fitness occurring after 250 generations with a focus on underlying mechanistic changes responsible for enhancement of relative fitness. Isolates from UVR lineages expressed two different morphol- ogies, R and F, and single-colony representatives of each from generations 250 and 500 were characterized. Neither morphotype group exhibited a significant fitness advantage under either UVR or non-UVR conditions (all P.0.320). Therefore, measurements of the two groups have been combined when comparing isolates from UVR and non-UVR lineages but are also presented individually to provide additional sampling of the genetic diversity present in UVR lineages. Relative fitness measurements were also conducted using population samples to observe any influence of genotype community structure in UVR lineages. We have combined the relative fitness values determined at generation 250 in this study with previously published measurements at generation 500 [12] in our data analysis and illustrations to highlight differences in adaptive improvements at the two time points. The average relative fitness of all isolates and population samples is summarized in Figure 2 and individual measurements of each lineage are reported in Figures 3, 4, 5, 6. Each relative fitness change was analyzed by an independent t-test against the hypothesis of mean fitness equal to that of the ancestral P. cichorii 302959 (W = 1) and is significant where indicated in the figures. Paired t-tests were also used to determine significant differences in relative fitness between growth environments (UVR vs. non-UVR conditions) or growth intervals (generations 250 vs. 500) and these P values are listed at the bottom of each figure. Population samples of ,108 cells from UVR lineages at generation 250 exhibited increased relative fitness under non- UVR conditions and further improvements under UVR condi- PLoS ONE | www.plosone.org 3 January 2011 | Volume 6 | Issue 1 | e15975