Spreadsheet: 49c59426-77eb-45a7-b3b6-e243209985d9
Sheet: TableS5-ProtocolsPerFigure

Main Figure | strains used | Media / preconditions | YP or SC | growth measurement / media conditions / protocol | Data analysis | Statistical test used | N
1a | YS4 | overnight growth in YPD until high turbidity | YP | protocol from 1c; growing only in 0.5% gluose + 2.5% galactose | GrowthCurves.R script was used to smoothen data and these data were plotted in Excel as a function of population size. A line equal to the maximal growth rate tangent to the point in th growth curve is plotted as the MaxR. | NA | Representative trace from 1 sample
1b | YS4 | overnight growth in YPD until high turbidity | YP | protocol from 1c; growing only in 0.5% gluose + 2.5% galactose | GrowthCurves.R script was used to smoothen data and growth rates calculated in Excel. OD600 of the sample was plotted as a function of these growth rates. | NA | Representative trace from 1 sample
1c and d | AN63 | overnight growth in YPD until high turbidity | YP | turbid samples diluted to a density of 6.67*10-4 the original turbid culture in 150 ul YP media in bioscreen plates. YP media had 0.5% glucose alone or supplemented with 2.5% glucose (3% total) or 5% maltose or 2.5% galactose, and population sizes were monitored in the Bioscreen C machine controlled by the "EZ experiment" software, with continuous medium amplitude shaking at 30˚C and population size measurements every 15 minutes. | GrowthCurves.R script was used to smoothen data and transform growth rates as a function of population density. The average of the maximal 3 values of these were used for [NAME], and the GMR, (ln(0.85)-ln(0.25))/t, was calculated for each growth curve using the transformed values (see supplementary data table).  Reported are the standard deviations of GMRs for strains growing in the four carbon source conditions. | error bars represent the S.D. of 2-6 biological replicates. | 4 biologiclal replicates
1e and f | UWOPS83-787.3 | overnight growth in YPD until high turbidity | YP | As in 1c | As in 1c | error bars represent the S.D. of 2-6 biological replicates. | 3 biological replicates
1g | LitiEtAlStrains and AN148 | overnight exponential growth in 20% maltose | YP | exponentially growing cultures in 20% maltose were transferred to 10% glucose for 6 hours and then transferred back to 20% maltose agar pad for lag measurements. Each transfer was made by 2 washing centrifugations at 1250xg. Time lapse photomicrographs were acquired every 15 minutes for 20-24 hours. | Single-cell lag phases were determined by eye as the time at which a bud began to form, or for an already-formed bud began to resume growth. The amount of time after transfer to maltose was added to this time for the total lag time of each individual cell. Histograms were made with 0.25 hour binnings. | Shown are cumulative distributions of >150 cells. | 3 biological replicates
1h | LitiEtAlStrains and AN148 | NA | YP | the data from 1g and population level experiments as in 1c-f are plotted here | Each data point representing the average lag is plotted against the geometric mean of normalized (GMR/MaxR) GMR for each strain in the four media condtions measured for the 2-4 biological replicates. | Cox proportional hazards regression | 2-6 biological replicates
2 | AN73 | overnight exponential growth in 20% maltose | SC | exponentially growing cultures in 20% maltose were transferred to 10% glucose for 6 hours and then transferred back to 20% maltose for 7.5 hours and then transferred back to 10% glcuose on an agar pad for lag measurements. An intial fluorescence photomicrograph in the YFP channel was acquired to determine the state of committment of the cells to maltose growth, and time-lapse brightfield microscopy followed to alllow subsequent determination of single-cell growth rates. | Single-cell doubling times were determined by eye as the time it took for cell-cycles (as determined by budding). If a partially mature bud was already present on the mother cell, we waited for this bud to complete and sever from the mother before recording the first cell cycle length. Whenever cell cycle budding events were unclear due to e.g. crowding of cells by other cells, the sample was discarded. The average of 2 cell cycles are reported for each mother cell. For each initial fluorescence intensity - growth rate pariing, a sliding window across the distribution nearby the clearly OFF and ON states was drawn, and these two populatoins' growth rates were compared using the U-test. The histogram reported is randomly chosen in this window where an sample size for ON and OFF cells was equal. | [NAME] for samples having "ON" vs. "OFF" values greater than or less than a certain fluorescence intensity. One of these groups always included at lesat 35% of observations. Shown is the distribution where 50% of cells were classified as ON and 50% were OFF, around 2900 A.U. | 168 initial cells
3a | Isolate 1, 2, 3, 4 and 5, ancestral-like long lag phased strain (pop#1.1), and the ancestral AN296 | overnight exponential growth in 10% glucose | YP | stationary phase cultures in 20% maltose were transferred to 10% glucose for 20 hours and then transferred back to 20% maltose agar pads for single-cell lag measurements. | same as 1g | Logrank scores were determined using R's survdiff() function.
3b | Isolate ancestor AN296, 1, 2, 3 (4 and 5 have labeled MAL genes and therefore are difficult to compete against a labeled reference strain) | stationary phase growth in 20% maltose | YP | Stationary phase (20% malltose YP) cultures of query strains were mixed 1:1 with labeled reference strain AN74 (MAL12-mCherry), and diluted into 5 ml of 10% glucose at densities of 0.8-1.1*103 cells per ml (determined by dilution directly from the glucose mixture and plating to single colonies on solid YPD media). The rest of the cell mixture was frozen in glycerol at -80 for future analysis by flow cytometry. The competition then proceeded the same as the selection procedure: samples were allowed to grow exponentially in glucose media for 20 hours, then exponentially growing cultures were centrifuged at 1250 x g for 5 minutes, resuspended in 5 ml 20% maltose, centrifuged again, resuspended in 5 ml 20% maltose, and then allowed to grow for 48 more hours. At the end, samples were diluted and plated to single colonies to determine final population size (generally 5*108 cells per ml) | Based upon flow cytometric analysis, the logarithm of the final population density * fraction of query strain - the initial population density * fraction of query strain was taken as the competitive fitness against AN74. This value divided by the value of the ancestral strain is the fitness value that we report in figure 3c. Error bars account for the error of both the query strain and the ancestor. | error bars represent the S.D. of 2-4 biological representatives. Arithmetic mean is reported. | 6 biological replicaes
4a | AN296 | as per 1c | as per 1c | as per 1c | as per 1c | as per 1c | as per 1c
4b | Isolate 1 | as per 1c | as per 1c | as per 1c | as per 1c | as per 1c | as per 1c
4c | Isolate 1 vs AN296 | these data are the same as 4a, 4b |  | these data are the same as 4a, 4b | The Isolate 1's instantaneous growth rates with respect to optical density were divided by those of AN296 to illustrate parts of the grwoth curve where Isolate 1 performs best. | NA | as per 1c
4d | AN148 and all descendant populations. | as per 1c | as per 1c | as per 1c | The geometric mean of GMR values or the average MaxR for each of the evolved isolates or AN148 are plotted against the average lag phase of each strain. | NA
5a | All strains for which [NAME] and STD1 was sequenced (see table) | NA | NA | Samples genomic DNA was used as a template to generate PCR products of the HXK2 gene and promoter and terminator regions. These were sent for dye-terminator [NAME] sequencing. | Samples were aligned to the reference [NAME] HXK2 sequence and those clones with mutations are indicated in this figure. | NA
5b | AN296, isolate 1 and isolate 2, and strains AN401, [NAME], AN415, AN414, AN404, [NAME], AN416 | as per 3a | as per 3a | as per 3a | as per 3a | NA | 2 biological replicates
5c | AN296, Isolate 3, AN411, AN413 | as per 3a | as per 3a | as per 3a | as per 3a | NA | 2 biological replicates
6a | pop1#2, pop3#1, pop11#1, pop6#1 | overnight late-exponential growth in 20% maltose YP | YP | 100 ul of a 150ul culture (in a microwell plate) of late-exponential phase samples growing in 20% maltose were frozen in glycerol for analysis by flow cytometry. Another aliquot from these same wells were diluted to a density of 2*10-4 of the original density into 2 ml of 10% glucose media to simulate the long glucose leg of the evolution experiment or 2% glucose + 5% maltose and allowed to grow exponentially for 20 hours. BD Influx flow cytometer was used to analyze samples in the YFP and RFP channels. | SSC and FSC outliers -- about 20 to 30% of events -- were filtered out in flo jo software and the rest of the events exported to a spreadsheet, where they were binned further to yield these histograms. | NA | 1 biological replicate per isolate
6b | AN355 and AN74 | exponential phase cultures either growing in 10% glucose or 20% maltose containing media. | YP | An aliquot of an exponentially growing (< 5*106 cells/ml) culture was diluted to to a density of 2*103 cells per ml in 2 ml of 10% glucose media to simulate the long glucose leg of the evolution experiment or 2% glucose + 5% maltose and allowed to grow exponentially for 24 hours. BD Influx flow cytometer was used to analyze samples in the YFP and RFP channels. | This is a representative sample from 3 biological replicates performed on the same day (the other two displayed identical behavior). SSC and FSC outliers -- about 20 to 30% of events -- were filtered out in flo jo software and the simple "cell count" histogram representation of this software program was used to represent the curves. Although  that the relative density for each of these samples is different for each of the curves, by making all curves appear to have equal density, it illustrates the large differences in fluorescence distribution between samples. | NA | Representative traces of 3 biological replicates
Supplemental Figures | strains used | x | YP or SC | growth measurement / media conditions / protocol | Data analysis | Statistical test used | N
S1a | [NAME] MAL+, SK1 ,YPS128, Y55 | overnight growth in YPD until high turbidity | YP | protocol from 1c | GrowthCurves.R script was used to smoothen data and transform growth rates as a function of population size. The values from the [NAME] and LG+galactose conditions were divided by the instantaneous growth rates of the cultures growing in LG alone to yield these traces. | NA | Representative traces from 1 sample
S1b | SGRP strains + S288c MAL+ | overnight growth in YPD until high turbidity | YP | protocol from 1c | Calculated GMRs for each strain growing in HG conditoins were divided by the GMRs of the same strain growing in the diauxic shift conditions (LG, [NAME], LG+galactose). Thus, large values reflect that the strain grew relatively more quikcly in HG media, and reflect how severe the strain's lag phases in the other media were. | NA | Representative traces from 1 sample
S1c through f | All strains and conditoins reported in this study |  |  |  | The fractoin surviving, minimal lag phase and standard deviation of cell survival were plotted against the average lag phase for each strain/condition. The standard deviation divided by the average single-cell lag phase are shown as a histogram in panel f.
S2a | AN73 | overnight exponential growth in 20% maltose | SC | exponentially growing cultures in 20% maltose were transferred to 10% glucose for times indicated. | Gene expression was determined by measuring signal of 50,000 single-cell events in the flo-jo cytometer. Outliers in the SSC/FSC scatterplot were excluded from analysis (excluding 20-30% of events). Single-cell lag phases were determined by eye as the time at which a bud began to form, or for an already-formed bud began to resume growth. The amount of time after transfer to maltose was added to this time for the total lag time of each individual cell. Average single-cell lag values are reported, with standard deviations of all cells reported to give an idea of the heterogeneity in single-cell lag phases. | NA | 3 biological replicates
S2b | AN63 (WT), [NAME] (MAL11 OE), [NAME] (MAL12 OE), [NAME] (MAL11/MAL12 dual OE) | overnight exponential growth in 20% maltose | SC | exponentially growing cultures in 20% maltose were transferred to 10% glucose for 24 hours of exponential growth then trasnferred back to 20% maltose agar pads for single-cell lag measurements. | Single-cell lag phases were determined by eye as the time at which a bud began to form, or for an already-formed bud began to resume growth. The amount of time after transfer to maltose was added to this time for the total lag time of each individual cell. Histograms were made with 0.25 hour binnings. | NA | 3 biological replicates for each genetic background.
S1c | AN148 | as per 1g | YP | as per 1g, except fluorescence images acqruied every half hour in the YFP channel to track MAL11-yECITRINE levels | NA | NA | Representative XY position from this experiment
S2d | AN73 | as per fig 2 | as per fig 2 | as per fig 2 | this is a histogram of initial fluorescence intensities for results reported in Figure 2. | as per fig 2 | as per fig 2
S2e | AN73 | as per fig 2 | as per fig 2 | as per fig 2 | as per fig 2 | [NAME] for samples having "ON" vs. "OFF" values greater than or less than a certain fluorescence intensity. One of these groups always included at lesat 35% of observations. Shown is the distribution where 50% of cells were classified as ON and 50% were OFF, around 2900 A.U. | 168 initial cells
S3a | AN296-descendant genetically heterogeneous evolving populations + AN296 control ancestral clone. | 10% glucose YP; after 6 rounds of glucose > maltose selection cycles. The genetically heterogenous samples were resurrected from the freezer (10 ul of frozen stock directly into 150 ul of 0.5% glucose + 5% maltose). | YP | Populations were resurrected overnight: 10 µl (~5*106 cells) of the frozen maltose-grown samples from the previous selection were resurrected into 150 ul of 0.5% glucose + 5% maltose, allowed to reach high turbidity over night, then the entire 150 ul culture was transferred to 2 ml of 20% maltose YP for overnight growth. Then the samples were diluted into glucose and allowed to grow 22 hours as per the normal selection experiment. Samples were then transferred as in the experimental evolution protocol for 22 hours of exponential growth in 10% glucose. Time lapse photomicrographs were acquired every 15 minutes for 20 hours. | as per figure 1g. | NA | The 12 evolving populations (100-150 cells each) and three duplicates of the ancestral clone (for a total of ~500 cells)
S3b | AN148-descendant genetically heterogeneous evolving populations | 10% glucose YP; after 0, 2, 6 and 8 rounds of glucose > maltose selection cycles. The samples were always in the middle of the glucose leg of the selection (going into the next round of maltose selection) before they were recorded. | YP | This experiment was recorded in the middle of the experimental evolution of strain AN148. Samples had grown previously in glucose leg of the selection cycle. As they were washed into 20% maltose for the final round of selection, small aliquots were also transferred to 20% maltose agar pad for lag measurements. Time lapse photomicrographs were acquired every 15 minutes for 20-24 hours. Due to technical reasons, sometimes we could not record all populations or even an entire round would be lost. In this case, 10 µl (~5*106 cells) of the frozen maltose-grown samples from the previous selection were resurrected into 150 ul of 0.5% glucose + 5% maltose, allowed to reach high turbidity over night, then the entire 150 ul culture was transferred to 2 ml of 20% maltose YP for overnight growth. Then the samples were diluted into glucose and allowed to grow 22 hours as per the normal selection experiment. | as per figure 1g | NA | 50-100 cells each population / timepoint.
S3c | AN148 and all descendant populations. | turbid growth in 20% maltose | YP | Stationary phase cultures of isolated mutatns were grown in 20% maltose were transferred to 10% glucose for 6 hours and then transferred back to 20% maltose agar pads for single-cell lag measurements. A 6 hour growth period in glucose provided less variable results for samples with very long lag phases (compared to 20 hours' growth, see figure S2a). | as per figure 1g | NA | 50-100 cells/isolate
S4a and b | AN148 and all descendant populations. | as per 1c | as per 1c | as per 1c | The GMR and MaxR measurements for each strain in population growth experiments reported in figure 4 are shown. These numbers were used to generate figures S4.
S4c | AN148 and all descendant populations. | as per 1c | as per 1c | as per 1c | The MaxR and GMR for each of the 4 carbon source conditions were divided by those of the ancestral strain AN148 to illustrate the relative GMR and MaxR gains or tradeoffs experienced by the populations. Error bars represent the square-root of the sum of the squared standard deviations for both the query strain and AN148. 2-4 biological replicates per measurement. | error bars represent the S.D. of 2-4 biological representatives. | as per 1c
S4d | AN148 and all descendant populations. | as per 1c | as per 1c | as per 1c | The normalized GMR measurements for the 4 carbon source environments are plotted against lag phases for each isolate and the ancestral strain AN148. Same idea as that in figure 1h. | student's 2-tailed t-test assuming unequal variance
S5a | AN148 ancestor and non-flocculent descendant populations | As in 6a. | As in 6a. | As in 6a. | As in 6a. | for each isolate, the mean fluorescence per cell in the mCherry channel (corresponding to MalS expression) and the YFP channel (corresponding to the MalT signal) are shown as a scatterplot. | 2-4 replicates for growth; 1 sample only for fluorescence
S5b | AN355 | exponential phase culture growing in 10% glucose containing media. | SC | An aliquot of an exponentially growing (< 5*106 cells/ml) 2 ml culture was diluted to to a density of 2*103 cells per ml in 2 ml of 2% glucose + 5% maltose containing media and allowed to grow exponentially for 24 hours. A 2 ul aliquot of the sampple was spotted onto a 2% glucose and 5% maltose SC-containing agar pad for growth rate and gene expression analysis. Recording proceeded in brightfield every 5 minutes, and in the mCherry channel every hour. | These representative images are an overlay between fluorescence and brightfield images. LUTs were linearly adjusted to appropriate levels to remove background signal and improve contrast. | NA | Representative image of 3 biological replicates
S5c | AN74 and AN355 | as in S5b | as in S5b | as in S5b | as in S5b | as in S5b | as in S5b
[NAME] | AN296, AN443 and AN445 | [NAME] cultures were inoculated into 3% glucose for 6 hours, then split into 5 ml of media in tubes with either 2% glucose 5% maltose or sorbitol + 2% glucose of equivalent osmolarity to the maltose+ glucose condition. | YP | Iniital cell density in the 3% glucose condition was determined by a Biorad TC20 automated cell counter, and cells were inoculated to low cell densities such that they would be approximately 2.5-10*10^6 cells per ml 20 hours later. Cultures were incubated at 30C on a roller wheel for 20 hours. Cell densities were determined, the log of the fold change in population density used to calculate growth rates as reported in figure S8b, and the rest of the culture was iced, centrifuged to form a cell pellet, and resuspended in 1x PBS + glycerol for analysis by microscopy. A 2 ul aliquot of the concentrated cells was spotted onto an agar+1xPBS pad in the lag chambers and cells dried onto the pad. This worked fine so the frozen cells were never used. Cells were imaged in our microscope in the mCherry channel. The exposure time and aperature of the shutter was chosen such that maximal pixel intensities of the brightest cells was below saturation. | ROIs were drawn in NIS elements, background per slide was drawn, and these numbers were in downstream data and statistical analyses. | R's [NAME] package was used to make plots grouped and colored based upon genotype and growth condition.Three independent transformants were measured, however no clone-specific effect was found so they were all just grouped together. [NAME] were used to test significance. P-values are bonferonni corrected for the multiple tests. | 50-100 cells per clone were quantified.
S6 | AN148, p8#1, [NAME], [NAME] and p11#1 | Turdid YP 2% mal or YPD overnights were inoculated into either 5% maltose or 3% glucose respectively and allowed to grow exponentially for 8 hours. | YP | Two independent clones were used. Cultures were inoculated into a 5% maltose + 2% glucose mixture and grown on a wheel at 30 degrees. Every 1-2 horus an aliquot was taken from the growing cultures, centrifuged, and resuspended in 1x PBS + 25% glycerol. Growth was concurrently monitored in a plate reader. After the experiment the fluorescence of the cells were analyzed at the flow cytometer and population doublings calculated based upon population density measurements. | R's Flowcore package was used to process data as in figure 6a. About 30% of events were discarded after SSC and FSC filtering. | nothing. This is representative of single clones. | 2 independent cultures; one shown.
S7a | as in S6 | as in S6 | as in S6 | as in S6 | Mean fluorescence of representative clones vs time is plotted in Excel. | as in S6 | as in S6
S7b | AN148 and pop8#1 | as in S6 | as in S6 | as in S6 | A model of exponential dilution of intitial fluorescence values based upon measured population doublings (init fluorescence divided by fold dilution) is plotted alongside actually measured mean fluorescence intensities of the ancestral strain and pop8#1. | as in S6 | as in S6
S7c | as in S6 | as in S6 | as in S6 | as in S6 | The final two timepoints were used to calculate protein dilution rates of each strain/replicate based upon the initial fluorescence intensities. The difference between expected dilution and measured values with respect to the change in population density gives this per-generation switching rate. | 2-way anova for strain and precondition are highly significant for conditions alone and strain condition interactions. T-tests for strains grouped per condition vs the ancestor return p-values less than 0.025 for each pair | 2 independent cultures per strain and condition.
S8a | AN148 ancestor and non-flocculent descendant populations | exponential phase cultures either growing in 10% glucose or 20% maltose containing media. | YP | A 150 ul aliquot of an exponentially growing (~ 4*106 cells/ml) culture was washed into 2% glucose and 5% maltose containing media and transferred immediately into the Bioscreen C.  Population sizes were monitored in the Bioscreen C machine controlled by the "EZ experiment" software, with continuous medium amplitude shaking at 30˚C and population size (OD600) measurements every 15 minutes. | GrowthCurves.R script was used to smoothen data, and the average of the maximal 3 growth rate values were used to determine MaxR of each of the cultures.  Error bars represent the s.d. of two to three biological replicates. For fluorescence measurements, the average fluorescence  was determined using FlowJo | All correlations: student's 2-tailed t-test assuming unequal variance | 2-3 biological replicates per isolate for each growth condition.
S8b | AN443 and AN445 | as in S5b | as in S5b | as in S5b | as in S5b | as in S5b | 1 replicate each of 3 independent transformants.
S8c | AN355 | exponential phase culture growing in 10% glucose-containing media. | SC | As in 6b | This chart represents quantitative analysis of the same experiment reported in figure S5c and [NAME].  An ROI was drawn around initial cells (greater than or equal to 8 cells), final microcolony (3 hours later). Growth rate was calculated as the (ln(ROIAreaFinal/ROIareaInitial))/∆t. Fluorescence intentisies are equal to the raw average intensity of the ROI measurement minus the background (determined by large rectangular ROIs drawn in three positions within the image that did not have cells). The scatter plot represents the combined data of the samples that intiially began growth with MAL genes OFF as well as the samples that were partially induced coming from the maltose-glucose mixture. | All correlations: student's 2-tailed t-test assuming unequal variance | 3 biological replicates for samples coming from 2 preconditions. ~200 microcolonies measured in total.
S8d | AN355 | exponential growth overnight in 10% glucose | YP | An aliquot of an exponentially growing (< 5*106 cells/ml) 2 ml culture was diluted to to a density of 0.5*106 cells per ml in 2 ml of 2% glucose + 5% maltose containing media and allowed to grow exponentially for 8 hours, enough time for some MAL OFF cells to begin to switch ON. Samples were washed twice in maltose media and a 2 ul aliquot of the sample was spotted onto a 5% maltose SC-containing agar pad for  lag phase measurement and and gene expression analysis. Recording proceeded in brightfield every 15 minutes, and in the mCherry channel only at the start of the recording. | Initial fluorescence intensities determined as in figure 2 and lag measurements as in figure 1g. | Log-transformed data (initial fluorescence vs lag time) display a highly significant correlation by student's t-test assuming unequal variance. | 3 biological replicates; ~300 cells recorded.
S8e | same as S5d | same as S5d | same as S5d | same as S5d | Here the same data as in 5d are shown as cumulative distributions. Cutoffs were made between samples with fluorescence intensities greater or less than 1000 A.U. to show how turning on the MAL genes in mixed mal/glu media allow cells to more rapidly adapt to depleted glucose levels. | same as S5d | same as S5d