Abstract
While early multicellular lineages necessarily started out as relatively simple groups of cells, little is known about how they became Darwinian entities capable of sustained multicellular evolution1,2,3. Here we investigate this with a multicellularity long-term evolution experiment, selecting for larger group size in the snowflake yeast (Saccharomyces cerevisiae) model system. Given the historical importance of oxygen limitation4, our ongoing experiment consists of three metabolic treatments5—anaerobic, obligately aerobic and mixotrophic yeast. After 600 rounds of selection, snowflake yeast in the anaerobic treatment group evolved to be macroscopic, becoming around 2 × 104 times larger (approximately mm scale) and about 104-fold more biophysically tough, while retaining a clonal multicellular life cycle. This occurred through biophysical adaptation—evolution of increasingly elongate cells that initially reduced the strain of cellular packing and then facilitated branch entanglements that enabled groups of cells to stay together even after many cellular bonds fracture. By contrast, snowflake yeast competing for low oxygen5 remained microscopic, evolving to be only around sixfold larger, underscoring the critical role of oxygen levels in the evolution of multicellular size. Together, this research provides unique insights into an ongoing evolutionary transition in individuality, showing how simple groups of cells overcome fundamental biophysical limitations through gradual, yet sustained, multicellular evolution.
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Data availability
Underlying data used to generate figures and raw data are available at GitHub (https://github.com/ozanbozdag/de_novo_evolution_of_macroscopic_multicellularity). Raw Illumina sequencing reads are available at the NIH Sequence Read Archive under accession number PRJNA943273. All microscopy images used to generate data are archived in the Ratcliff laboratories Dropbox and are available on request.
Code availability
Codes used in this study are available at GitHub (https://github.com/ozanbozdag/de_novo_evolution_of_macroscopic_multicellularity). Code for the simple 3D biophysical simulation are provided in Supplementary File 1; these simulations were adapted from ref. 35 (further information can be requested from T.C.D. or P.J.Y.).
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Acknowledgements
We thank J. T. Pentz for teaching us Illumina library preparation; S. Biliya at the High Throughput DNA Sequencing Core at Georgia Tech for sequencing the genomes of evolved strains; K. A. Boateng at Core Facilities at the Carl R. Woese Institute for Genomic Biology for the SEM imaging; S. Cao for helping with microscopy during the early stages of this project; and C. Orlic, L. Nagy, and all of the members of the Ratcliff group for comments on the manuscript. This work was supported by NIH grants R35-GM138030 to W.C.R. and R35-GM138354 to P.J.Y., Human Frontiers in Science Grant RGY0080/2020 to W.C.R., and a Packard Fellowship for Science and Engineering to W.C.R.
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Contributions
G.O.B., S.A.Z.-D., P.J.Y. and W.C.R. conceived the project. G.O.B. and W.C.R. designed the MuLTEE. G.O.B. performed the evolution experiment. G.O.B., S.A.Z.-D., P.C.K. and T.C.D. designed and collected data. S.A.Z.-D. generated SBF-SEM images. S.A.Z.-D., T.C.D. and P.J.Y. performed the yeast biophysical simulations. E.L.D. and A.H.B. assisted G.O.B. and S.A.Z.-D. with image analysis. A.J.B. genetically engineered large snowflake yeast. K.T. performed life-cycle experiments. D.T.L. measured the number of generations. P.L.C. performed unicellular reversion experiments. G.O.B., S.A.Z.-D., T.C.D., W.C.R. and P.Y. analysed the data. G.O.B. made the figures. G.O.B., W.C.R. and P.J.Y. wrote the first draft of the paper, and all of the authors contributed to the revision.
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Extended data figures and tables
Extended Data Fig. 1 Temporal dynamics of size evolution in each population and treatment group.
Data points show the weighted average radius of cluster size for the entire population. This was calculated by measuring the size of an average of 1150 snowflake yeast clusters per sample population (3 ancestors + 3 treatment groups x 5 replicate populations x 12 time points = 183 samples, all data is publicly available under the raw data file). Please see the Methods section for details on how weighted average radius was calculated.
Extended Data Fig. 2 Characterizing the life-cycle of the ancestral (microscopic) and evolved (macroscopic) snowflake yeast.
a, During the ~24-hour growth cycle, snowflake yeast compete for growth and reproduction in 10 mL of YPED (250 RPM at 30 °C). At the end of the growth phase, we select for larger group size via settling selection. While there is a theoretical maximum survival rate of 15% (that is, if all of the cells survived settling selection), we only transfer the bottom 50 µl of pellet biomass regardless of how many cells settle, creating an arms race that favours the fastest groups within the population. Our measurements of the number of cellular generations per day in Fig. 1a suggests about 3% of the cells survive from one day to the next on average. b, Both the microscopic (ancestral) and macroscopic (t600) snowflake yeast clusters have a life cycle, reproducing during the growth phase. c, Consistent with entanglement producing tough groups, macroscopic snowflake yeast release mostly microscopic propagules, possibly from branch tips at the exterior of the group, where the opportunity for entanglement is minimal. Despite the presence of many small propagules, most of the biomass in the population is contained within macroscopic clusters. The open circles represent the biomass-weighted mean size, which is the average sized group the mean cell finds itself in. A total of 14,313 clusters were analysed for the t0 time point, and 1,603 clusters were analysed for the t600 time point, across 0, 3, 6, 12, and 24-hour time points.
Extended Data Fig. 3 Cluster size and aspect ratio distribution.
a, Biomass distribution as a function of cluster size for the ancestral snowflake yeast (dotted line) and 600 day evolved populations of PA1-PA5. The ‘weighted mean size’ used in Figs. 1, 2 and 4 is the mean of the biomass distribution. b, Distribution of aspect ratios for ancestral and 600-day evolved populations of anaerobic snowflake yeast.
Extended Data Fig. 4 Cell shape is not substantially affected by location within macroscopic yeast.
a and b show cell volume and cell shape (aspect ratio) measured for 10 cells from the interior of a macroscopic cluster and 10 cells from the exterior of a cluster (measured in t600 macroscopic clusters). Average cell volume for exterior and interior are 110.8 µm3 and 113.1 µm3 (p = 0.88, t = 0.15 df = 17.55, Welch’s t-test), and average cell shape for exterior and interior are 2.9 and 2.8 (p = 0.51, t = 0.68, df = 14, Welch’s t-test). Individual measurements are marked as points, the mean and one standard deviation are indicated by the bar plot.
Extended Data Fig. 5 Parallel evolution of elongated cell shape across all five replicates of each PA population.
For each evolutionary time point and population, five different cells are shown (organized vertically from left to right: PA1 on the further left and PA5 on the further right in each box). Scale bar is 5 µm (under the ancestral cell). This is a more detailed version of the plot shown in Fig. 2c.
Extended Data Fig. 6 Macroscopic snowflake yeast are monoclonal, growing via permanent mother-daughter cellular bonds, not aggregation.
We co-cultured GFP and RFP-tagged genotypes of a macroscopic single strain isolate (PA2, strain ID: GOB1413-600) for 5 days, then imaged 70 clusters on a Nikon Ti-E. Shown are a composite of 11 individual clusters, which all remain entirely green or red. Individual clusters were compressed with a coverslip for imaging, resulting in their fragmentation into multiple modules. Scale bar (top-left) is 100 µm.
Extended Data Fig. 7 Quantifying entanglement via analysis of the topology and geometry of a snowflake yeast cluster.
a, We measured entanglement of individual components by fitting a convex hull around each component, and determining whether the other component overlaps with the space bounded by this convex hull. Here we just show the convex hull for the blue component, which overlaps with the red component. These components are thus part of the same entangled component. b, Using this approach, we identified the components within a sub-volume of a macroscopic snowflake yeast, and used a percolation analysis to examine the fraction of the biomass that is part of the same entangled component (coloured in red).
Extended Data Fig. 8 Cell stiffness and stress-strain curve.
a, Individual cells do not change their stiffness over 600 rounds of selection (average cell stiffness for the ancestor and t600 isolates are 0.019 and 0.020, respectively. p = 0.77, t = 0.31, df = 8, Welch’s unequal variances t-test). Single-cell stiffness values measured from atomic force microscopy (AFM) of individual cells. Error bars are one standard deviation. b, Macroscopic snowflake yeast fractured into small modules prior to compression do not show strain stiffening behaviour. Shown here is an AFM trajectory of cantilever deflection vs displacement for one t600 cluster that has been crushed into small, unentangled pieces.
Extended Data Fig. 9 Representative confocal images show chimeric clusters that are formed after growth in liquid culture followed by entanglement on agar plates.
a,b, Close-up view of clusters, highlighting the tangled red and green branches. Each frame is 139.64 x 139.64 x 34.50 µm in X, Y, and Z axes, respectively.
Extended Data Fig. 10 Dimensions of bud scars connecting cells in microscopic, ancestral (t0, grey) and macroscopic, evolved snowflake yeast clusters (PA2 t600, blue).
Macroscopic t600 yeast had 2.4x larger bud scar cross-sectional area (a; p < 0.001, t = 5.3, df = 24, t-test), 2.8x greater bud scar height (b; p < 0.001, t = 12.5, df = 24, t-test), resulting in bud scars with 5.8-fold greater volume (c; p < 0.001, t = 7.3, df = 24, t-test) than the microscopic ancestor. Error bars are one standard deviation. b, Histogram of pixel intensities for bud scars stained with chitin stain calcofluor white, isolated from ancestor (t0, microscopic) and t600 (macroscopic) bud scars. The t600 strain has a 27% higher mean fluorescence intensity, suggesting that they may have evolved moderately higher chitin density in the bud scar. c, The size differences in bud scars is readily visible. Shown are the side view of buds from the ancestor (left) and t600 evolved (right), imaged at the same microscope settings. The scale bar is 0.5 µm.
Supplementary information
Supplementary Table 1
A list of strain isolates, primers and plasmids used in the study.
Supplementary Video 1
Comparison of the ancestor and a population of macroscopic snowflake yeast (PA2 t600, on the right).
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Bozdag, G.O., Zamani-Dahaj, S.A., Day, T.C. et al. De novo evolution of macroscopic multicellularity. Nature 617, 747–754 (2023). https://doi.org/10.1038/s41586-023-06052-1
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DOI: https://doi.org/10.1038/s41586-023-06052-1
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