Article Figures & Data
Figures
- FIG 1
The majority of evolved replicates improved growth in the evolutionary environment after 100 generations of evolution. Fitness was measured as growth (optical density) in YPD + 1 μg/ml fluconazole, the evolutionary environment after 24 h (top) and 72 h (bottom). Strains are ordered by parental fitness in the evolutionary environment at 72 h. Parental fitness (the median growth among 12 parental replicates) is indicated for each strain by a gray bar. Each colored point represents one of 12 evolved replicates, while the colored bars indicate median evolved growth for visual comparison to parental growth.
- FIG 2
Significant association between parental fitness and change in mean fitness (upper graphs) and replicate variability (lower graphs). Fitness was measured as optical density in YPD + 1 μg/ml fluconazole after 24 h (left panels) and 72 h (right panels). The colors are as in Fig. 1, based on parental fitness at 72 h (low parental fitness = yellow, high parental fitness = purple). The regression line is for visualization purposes to illustrate the one-way relationship between parental fitness and change in fitness or change in variability, which was significant (P < 0.05) in multiway models that also take into account clade and mating locus (P > 0.05 in all cases; see text for details).
- FIG 3
Evolved variation in clinical resistance and tolerance. Strains are arranged on the x axis by parental MIC, with each panel separating the three MIC-based classes of strains. (A) The majority of evolved replicates did not acquire clinical resistance. Clinical drug resistance was measured as MIC50 using broth microdilution assays (1). The black lines indicate parental MIC50s. (B) Tolerance was variable among the replicates. Tolerance was measured as the average growth observed in supra-MIC levels of fluconazole normalized to the growth in a very low level of drug after 72 h. The black lines indicate the range of tolerance values measured among parental replicates. Each point represents an individually evolved replicate line, colored as in Fig. 1, based on fitness in the evolutionary environment.
- FIG 4
Genome size and variation in genome size increased after evolution to low fluconazole. (a and b) Median genome size (a) and coefficient of variation (CV; i.e., variability among replicates) (b) in each strain background. A dashed line indicates a nonsignificant change between parental and evolved replicates. (c) Flow cytometry traces of each replicate evolved line, ordered and grouped by parental MIC50. Box color indicates parental fitness in the evolutionary environment.
Tables
- TABLE 1
Strains used in this studyh
↵a Original reference, 79.
↵b Original reference, 73.
↵c Original reference, 80.
↵d Original reference, 81.
↵e Original reference, 82.
↵f Original reference, 83.
↵g Original reference, 84.
↵h Fitness was measured as the optical density (A600) in YPD + 1 μg/ml FLC, the evolutionary drug environment. Drug resistance was measured as MIC50 in μg/ml at 30°C by broth microdilution assay. Tolerance was measured as the average optical density at 72 h in the measured drug concentrations of drug above the MIC divided by optical density in the lowest measured drug level.
- TABLE 2
Welch two-sample t tests to compare parental and evolved genome size
TABLE S1
The majority of strains evolved an increased growth ability in the evolutionary environment. The test column indicates the test that was run: a t test when both parental and evolved replicate groups were normally distributed with equal variance, a t test with Welch approximation for degrees of freedom when variances were unequal, or the Wilcoxon rank sum test when the data from at least one group were not normally distributed. Equal variance was assessed with an F test, and normality was assessed with the Shapiro-Wilk test (assumption test results not shown). Download Table S1, PDF file, 0.5 MB.
Copyright © 2020 Gerstein and Berman.
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FIG S1
Raw broth microdilution (MIC) data at 24 h. Strains are arranged based on parental MIC50 (calculated as the drug concentration at which a 50% reduction of growth is observed after 24 h compared to growth in no drug), indicated by the horizontal gray line in each panel. The thick black line indicates the mean parental trace from 12 replicates. The other lines indicate evolved replicates that had increased MIC50 (thick blue lines) or decreased MIC50 (thick red lines) or did not change (gray lines). Download FIG S1, PDF file, 0.01 MB.
Copyright © 2020 Gerstein and Berman.
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FIG S2
Tolerance of evolved strains measured at 24 h (a), 48 h (b), and 72 h (c). Tolerance was measured as the growth observed in supra-MIC levels of fluconazole (as appropriate to each replicate) normalized to the growth in the lowest level of drug. Strains are arranged on the x axis (and colored) by initial growth in the evolutionary environment. Each point represents an individually evolved replicate line. The black lines indicate the range of tolerance values measured among parental replicates. Download FIG S2, PDF file, 0.05 MB.
Copyright © 2020 Gerstein and Berman.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
FIG S3
Raw broth microdilution data at 72 h. Strains are arranged based on parental MIC50 (calculated as the drug concentration at which a 50% reduction of growth is observed after 24 h compared to growth in no drug), indicated by the horizontal gray line in each panel. The thick black line indicates the mean parental trace of optical density measurement at 72 h from 12 replicates. The other lines indicate evolved replicates that had increased MIC50 (thick blue lines) or decreased MIC50 (thick red lines) or did not change (gray lines). Download FIG S3, PDF file, 0.01 MB.
Copyright © 2020 Gerstein and Berman.
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FIG S4
Evolved fitness and evolved tolerance are not consistently correlated among replicates. Fitness was measured as optical density after 72 h in 1 μg/ml fluconazole. Tolerance was measured as the average growth observed in supra-MIC levels of fluconazole normalized to the growth in a very low level of drug after 72 h (“SMG”). The black line indicates the correlation among all replicates; a solid line indicates a significant correlation (P < 0.05). Download FIG S4, PDF file, 0.01 MB.
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This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
FIG S5
The majority of evolved replicates increased genome size. Filled circles indicate the most prominent evolved genome size peak; when multiple G1 peaks are present, this is indicated with an open circle. Strains are ordered based on parental MIC. The gray lines in each panel indicate the range of genome size values measured for the 12 parental replicates from each strain at the first transfer. Download FIG S5, PDF file, 0.02 MB.
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FIG S6
Evolved genome size and change in fitness are not correlated. Evolved genome size indicates the most prominent G1 peak; when multiple G1 peaks are present, this is indicated with a triangle. Fitness was measured as optical density after 72 h in fluconazole. Each point represents an independently evolved replicate line. Filled points are those lines that have an MIC50 of >1. Strains are ordered based on parental fitness. A correlation was not assessed for the five strains that had parentally high MIC levels above the evolutionary environment and did not evolve variation in genome size. Download FIG S6, PDF file, 0.01 MB.
Copyright © 2020 Gerstein and Berman.
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FIG S7
Evolved genome size and change in tolerance are not generally correlated. Evolved genome size indicates the most prominent G1 peak; when multiple G1 peaks are present, this is indicated with a triangle. Tolerance was measured as the average growth observed in supra-MIC levels of fluconazole normalized to the growth in a very low level of drug after 72 h (“SMG”). Each point represents an independently evolved replicate line. Strains are ordered based on parental fitness. A solid line indicates a significant (P < 0.05) Spearman correlation; the dashed lines are provided for visualization. A correlation was not assessed for the five strains that had parentally high MIC levels above the evolutionary environment and did not evolve variation in genome size. Download FIG S7, PDF file, 0.01 MB.
Copyright © 2020 Gerstein and Berman.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
TABLE S2
The block design for the replicate MIC experiments that were conducted. Download Table S2, PDF file, 0.5 MB.
Copyright © 2020 Gerstein and Berman.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
