Experimental evolution can be used to increase survival of yeast under freeze-thaw stress.

A Experimental design of selection for freeze-thaw tolerance. B Survival fraction of cells after freeze-thaw over the course of evolution. Inset: Number of viable cells after every freeze-thaw-growth cycle. Purple - evolving population. Pink - control population. C Basal intracellular concentrations of trehalose relative to the control measured over the course of evolution.

Freeze-thaw tolerant cells undergo reduced membrane damage and survive membrane damage.

A Flow cytometry measurements on Wild Type cells. Each point represents a single cell and the plot contains data for 10000 cells. The x-axis is the fluorescence intensity in the 5-CFDA channel. The y-axis is the fluorescence intensity in the propidium iodide channel. The contour lines represent changes in probability density of cells. The cluster on the top left comprises membrane damaged cells and the cluster at the bottom right comprises cells with intact membranes. B Flow cytometry measurements on freeze-thaw tolerant cells. Each point represents a single cell and the plot contains data for 10000 cells. The x-axis is the fluorescence intensity in the 5-CFDA channel. The y-axis is the fluorescence intensity in the propidium iodide channel. The contour lines represent changes in probability density of cells. The cluster on the top left comprises membrane damaged cells and the cluster at the bottom right comprises cells with intact membranes. C The 2-dimensional data from A and B are used to compute a 1-dimensional distribution of “membrane damage” in cells. D Viability of cells measured after sorting the two clusters identified from the data in A and B.

Cells of the population adapted to survive freeze and thaw are smaller, denser and exhibit reduced budding.

A Brightfield image of cells. Scale bar - 10µm. B Projected area of cells measured from images as shown in A. Sample size: Control = 1107, Evolved = 4281, P = 1.387 ∗ 102. C Single cell mass density measured using a percoll gradient column. P = 1.7867 ∗ 107. D Fraction of cells budding in stationary phase measured from images as shown in A. Sample size: Control = 309, Evolved = 422, P = 1.2079 ∗ 106. E Distribution of normalized fluorescence intensities of DNA in cells stained with SYTOX Green measured using flow cytometry. Sample size: Control = 199682, Evolved = 399113. F Design of the microfluidic channel through which cells are flowed to measure deformation with an image of cells leaving the microfluidic channel. Scale bar - 15µm. G Eccentricity and area of cells measured in the narrow channel. Flow rate: Control - 5.5cm/s, Evolved - 5.6cm/s. H Fraction of intact cells over time measured by turbidity of cells placed in water after Zymolyase treatment. I Image of Genetically Encoded Multimers (GEMs) in the cytoplasm of cells. Scale bar - 5µm. J Mean squared displacement (MSD) of GEMs tracked in the cytoplasm of cells, plotted on logarithmic axes. Inset: MSD plotted on linear axes. K Distribution of the exponent of time, α.

Selection based on trehalose linked entry into a qui- escence-like state recapitulates the adaptation dynamics.

A Optical density of the cell cultures inoculated into fresh media after a freeze-thaw. B Average doubling time of cells in the population calculated from the growth curves in A. C Average lag phase duration of cells in the population calculated from the growth curves in A. D Survival fraction of cells after freeze-thaw simulated over the course of evolution. E Basal intracellular concentrations of trehalose relative to the control simulated over the course of evolution.

The mechano-chemical adaptation to freeze-thaw tolerance is convergent.

A Experimental design to measure survival after refreezing and for the evolution of refreezing tolerance. B Number of viable cells after consecutive freeze-thaws. C Basal amounts of intracellular trehalose measured in the stationary phase. D Fraction of cells budding measured in the stationary phase. E Mean squared displacement (MSD) of GEMs tracked in the cytoplasm of cells, plotted on logarithmic axes. Inset: MSD plotted on linear axes. F Distribution of the exponent of time, α.

A listing of the genetic mutations across all replicates including both the kFT = 1 and kFT = 3 selection regimes, coloured boxes indicate the identified mutations.

Replicates of populations of cells adapted to freeze-thaw stress do not have any specific genetic mutation in common. FS - Frame Shift, SC - Stop Codon, NS - Non-Synonymous, S - Synonymous, IG - Inter-genic region.

Parameters for spin coating

Parameter values for simulations.

Sequenced samples

Replicates of populations of cells adapted to freeze-thaw stress do not have any duplicated genes in common

List of duplicated genes across all replicates including both the kFT = 1 and kFT = 3 selection regimes. Coloured cells denote a duplication.

Survival fraction of cells measured over different freeze durations

Design of the deformability cytometry device

Sketch of the experimental design to probe the digestion of the cell wall.

BME - beta-mercaptoethanol.

Map of cloned GEMs Plasmid

Sketch of the analysis of sequencing reads

A Representation of samples collected for sequencing. Sample collection points are displayed on a plot of the the number of viable cells in the control and evolving population across freeze-thaw-growth cycles. B Mapping of reads to a reference genome followed by variant calling. C A few representative variants filtered and clustered based on their frequency across all samples. Error bars denote 95% confidence intervals. D Trajectories of frequencies of representative variants from parent strain till the end point of selection. Each line represents a sample.