Multicellularity Makes Somatic Differentiation Evolutionarily Stable

Multicellularity makes somatic differentiation evolutionarily stable

Mary E. Wahla,b,1 and Andrew W. Murraya,b,2

aDepartment of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138; and bFAS Center for Systems Biology, Harvard University, Cambridge, MA 02138

This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected in 2014.

Contributed by Andrew W. Murray, June 15, 2016 (sent for review September 13, 2015; reviewed by Harmit S. Malik and Boris I. Shraiman) Many multicellular organisms produce two cell lineages: germ cells, through lineage sequestration (12) or reduced oxidative stress whose descendants produce the next generation, and somatic cells, (13), are thus likely accessible to unicellular species. which support, protect, and disperse the germ cells. This germ-soma We propose the alternative hypothesis that unicellular somatic demarcation has evolved independently in dozens of multicellular differentiation can offer fitness benefits in a population of ge- taxa but is absent in unicellular species. A common explanation holds netically identical cells but remains rare because it is not an that in these organisms, inefficient intercellular nutrient exchange evolutionarily stable strategy (14). Commonly occurring mutants compels the fitness cost of producing nonreproductive somatic cells that do not differentiate (“cheats”) could take advantage of somatic to outweigh any potential benefits. We propose instead that the cell products in the shared media without paying the reproductive absence of unicellular, soma-producing populations reflects their costs of differentiation, thus increasing in frequency until their ge- susceptibility to invasion by nondifferentiating mutants that ulti- notype prevails. We also posit that if multicellularity results from mately eradicate the soma-producing lineage. We argue that multi- cells of a single lineage failing to disperse (rather than cells aggre- cellularity can prevent the victory of such mutants by giving germ gating from different lineages), differentiating populations can cells preferential access to the benefits conferred by somatic cells. The outcompete cheats: although cheats initially arise through mutation absence of natural unicellular, soma-producing species previously in a group with somatic cells (which the cheats can exploit), lineage- prevented these hypotheses from being directly tested in vivo: to restricted propagation forces the cheat’s descendants to be confined overcome this obstacle, we engineered strains of the budding yeast to multicellular groups composed entirely of cheats, which thus Saccharomyces cerevisiae that differ only in the presence or absence cannot benefit from the local accumulation of somatic cell products of multicellularity and somatic differentiation, permitting direct com- (15–17). This hypothesis invokes the demonstrated ability of pop- parisons between organisms with different lifestyles. Our strains im- ulation structure to maintain altruistic traits (15, 18, 19). plement the essential features of irreversible conversion from germ To test the evolutionary stability of germ-soma differentiation, line to soma, reproductive division of labor, and clonal multicellularity we designed strains of the budding yeast Saccharomyces cerevisiae while maintaining sufficient generality to permit broad extension of that produce soma, are multicellular, or combine both traits: one our conclusions. Our somatic cells can provide fitness benefits that strain is a multicellular, differentiating organism and the other two exceed the reproductive costs of their production, even in unicellular represent both possible intermediates in its evolution from a strains. We find that nondifferentiating mutants overtake unicellular nondifferentiating, unicellular ancestor (Fig. 1A). Using synthetic populations but are outcompeted by multicellular, soma-producing strains that differ from one another at only a few, well-defined loci strains, suggesting that multicellularity confers evolutionary stability to somatic differentiation. Significance

evolution | multicellularity | differentiation | synthetic biology | yeast Unicellular species lack the nonreproductive somatic cell types that characterize complex multicellular organisms. We consider omatic differentiation, a permanent change in gene expression two alternative explanations: first, that the costs of lost re- ’ Sinherited by all of a cell s descendants, produces somatic cells productive potential never exceed the benefits of somatic cells in from a totipotent germ line. Although somatic cells may divide unicellular organisms; and second, that somatic cells may profit a indefinitely, they cannot beget the complete organism and are thus unicellular population but leave it vulnerable to invasion by considered nonreproductive. Generating such sterile cells has clear common mutants. We test these hypotheses using engineered fitness costs that must be offset by somatic functions that improve yeast strains that permit direct comparisons of fitness and evo- the viability or fecundity of germ cells. The absence of a soma in lutionary stability between lifestyles. We find that the benefits of unicellular species (1), as well as the persistence of undifferentiated somatic cell production can exceed the costs in unicellular strains. multicellular groups among the volvocine algae (2) and cyano- Multicellular, soma-producing strains resist invasion by nondifferentiating mutants that overtake unicellular populations, bacteria (3), has fueled speculation that multicellularity must arise – supporting the theory that somatic differentiation is stabilized by before somatic differentiation can evolve (4 7). It has been argued population structure imposed by multicellularity. that somatic differentiation is not observed in unicellular species because the fitness benefits of somatic cells can never exceed the Author contributions: M.E.W. and A.W.M. designed research; M.E.W. performed research; cost of making them (6–8): although soma can contribute motility M.E.W. analyzed data; and M.E.W. and A.W.M. wrote the paper. and protective structures to multicellular organisms, somatic cells Reviewers: H.S.M., Fred Hutchinson Cancer Research Center; and B.I.S., University of Cal- ifornia, Santa Barbara. in a unicellular species can only benefit the germ line by secreting The authors declare no conflict of interest. useful products into a shared extracellular milieu. However, nutrient exchange between members of microbial consortia (9, Freely available online through the PNAS open access option. 1Present address: Microsoft New England Research and Development Center, Cambridge, 10) demonstrates the potential for productive interactions be- MA 02142. tween cell types in the absence of physical adhesion. Benefits 2To whom correspondence should be addressed. Email: [email protected]. associated with somatic differentiation, including reproductive This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. division of labor (11) and suppression of germ-line mutations 1073/pnas.1608278113/-/DCSupplemental.

8362–8367 | PNAS | July 26, 2016 | vol. 113 | no. 30 www.pnas.org/cgi/doi/10.1073/pnas.1608278113 Downloaded by guest on September 30, 2021 ensures that no undesired variables confound the direct comparison sucrose minimal media. In nature, differentiation is often achieved

of fitness and evolutionary stability and permits the study of a uni- through multiply redundant gene regulatory networks that stabilize INAUGURAL ARTICLE cellular, differentiating lifestyle not found in nature. Experiments on cell fate (22): to simplify our system, we instead made differenti- living organisms also avoid the potential pitfall of biologically un- ation permanent and heritable by making the expression of somatic realistic parameter regimes in simulations or purely analytical cell-specific genes depend on a site-specific recombinase that ex- models. Our results show that production of soma can be advan- cises a gene needed for rapid cell proliferation (Fig. 1C). tageous even in unicellular populations, although these are sus- Germ and somatic cells must be present at a suitable ratio for ceptible to invasion by nondifferentiating cheats, which multicellular growth on sucrose: germ cells have the higher maximum growth populations effectively repel. We therefore speculate that multi- rate, but monosaccharides become limiting when somatic cells are cellularity facilitates the evolution of somatic differentiation prin- rare. We predicted that the ratio between cell types would reach a cipally by providing stability against common mutants that prevent steady-state value reflecting the balance between unidirectional differentiation. conversion of germ cells into somatic cells and the restricted division of somatic cells. We designed tunable differentiation and Construction of a Synthetic Differentiation System division rates to allow us to regulate the ratio between cell types We mimicked somatic differentiation by engineering fast-growing and thus control the growth rate of the culture as a whole. “germ” cells that can give rise to slower dividing, differentiated Both features depend on a single, genetically engineered locus “somatic” cells that secrete invertase (Suc2), an enzyme that hy- (Fig. 1C). In germ cells, this locus expresses the fluorescent protein drolyzes sucrose (which laboratory yeast strains cannot take up mCherry and a gene that accelerates cell division, the cycloheximide r directly) into the monosaccharides glucose and fructose, which any resistant (cyh2 ) allele of ribosomal protein L28 (23); in somatic cell in the shared medium can then import (20, 21) (Fig. 1B). The cells, the locus expresses a different fluorescent protein (mCitrine) somatic cells thus perform a digestive function: liberating mono- and invertase. The germ-line form is converted to the somatic form saccharides, the sole importable carbon source during growth in by a version of Cre recombinase engineered by Lindstrom et al. (24)

A Unicellular Multicellular B C licatio nversi Proteolytically- p n o o EVOLUTION e c n r = r PENO2 loxP mCherry cyh2 Term loxP mCitrine SUC2 Term cleaved linker

Cre recombinase-mediated E Conversion rate (per generation) ? gene excision 100 Germ Soma ? 10-1 Unlimited growth Limited growth P mCitrine SUC2 Term Secretes invertase ENO2 loxP 10-2 Differentiated Undifferentiated

D Bright-field mCherry mCitrine Composite 10-3

10-4

10-5 0 0.3 1 3 10 30 100 1000 [β-estradiol] (nM)

- β -estradiol F Relative growth rate of soma 1.00 0.95 0.90 0.85 0.80 0.75

+ β -estradiol 0.70 0 50 100 150 200 250 300 10 μm [Cycloheximide] (nM)

Fig. 1. Engineering yeast as a model for somatic differentiation. (A) Alternative evolutionary trajectories for the evolution of development. Multicellularity and somatic differentiation have separate biological bases and thus probably evolved sequentially. In some clades, the persistence of likely evolutionary intermediates suggests that multicellularity arose first (solid arrows); no examples of evolution through a unicellular differentiating intermediate (dashed arrows) are known. Somatic cells are colored yellow. (B) Schematic of germ-soma division of labor model engineered in our differentiating strains. (C) Cell type-defining locus in the differentiating strains. Each pair of cell type-specific proteins (mCherry and Cyh2r for germ cells, mCitrine and Suc2 for somatic cells) is initially expressed as a single polypeptide with a ubiquitin linker (red triangles). Cellular deubiquitinating enzymes cleave this linker posttranslationally at its C terminus (34), allowing the two resulting peptides to localize and function independently: for example, Suc2 enters the secretory pathway, whereas mCitrine remains in the cytoplasm. A transcriptional terminator (Term) blocks expression of the somatic cell proteins in germ cells. Cre recombinase-mediated gene excision between loxP sites removes the terminator, as well as the germ cell-specific genes. (D) The unicellular differentiating strain (yMEW192) during growth in yeast extract-peptone-dextrose media (YPD) before (Top) or 5 h after addition of 1 μM β-estradiol (Bottom). The white arrow indicates a budded cell that has recently undergone conversion and contains both mCherry and mCitrine proteins and thus fluoresces in both channels (cf Fig. S1). (E) Conversion rates estimated by flow cytometry during growth in YPD containing β-estradiol. Error bars represent 95% confidence intervals (CIs) of the mean, determined using data obtained from three biological replicates. (F) The growth disadvantage of somatic cells relative to germ cells was measured by growing them in coculture in YPD plus cycloheximide and determining the change in the ratio between cell types over multiple rounds of cell division. Error bars represent 95% CIs of the mean, determined using data obtained from three biological replicates.

Wahl and Murray PNAS | July 26, 2016 | vol. 113 | no. 30 | 8363 Downloaded by guest on September 30, 2021 ABCApproach to steady-state cell type ratio Fraction of somatic cells at steady-state Culture growth rate in 0.5% sucrose media containing 150 nM cycloheximide 1.0 1.0 0.25 25 nM CHX 100 nM CHX 0.8 0.8 300 nM CHX 0.20

0.6 0.6 0.15

0.4 0.4 0.10

0.2 0.2 0.05 Fraction of somatic cells Fraction of somatic cells Growth rate (doublings/hr.) 0.0 0 5 10 15 20 25 30 35 0.0 0.00 1 10 100 1000 0.0 0.2 0.4 0.6 0.8 1.0 Culture doublings [β-estradiol] (nM) Fraction of somatic cells

Fig. 2. Stable maintenance of both cell types facilitates growth in sucrose + cycloheximide media. (A) Representative time courses of the fraction of somatic cells in cultures of the unicellular differentiating strain (yMEW192 and yMEW192 convertant) initiated at various cell type ratios and passaged in YPD containing 10 nM β-estradiol and 600 nM cycloheximide. (B) The steady-state fraction of somatic cells was determined as in A for various cycloheximide and β-estradiol concentrations. Error bars represent 2 standard deviations, calculated using data obtained from three biological replicates. (C) Dependence of growth rate on fraction of somatic cells in cultures of the unicellular differentiating strain growing in 0.5% sucrose minimal media containing 150 nMcy- cloheximide and β-estradiol concentrations of 0, 0.3,1, 3, 10, and 50 nM, respectively. Error bars represent 95% CIs of the mean, determined using data obtained from three biological replicates.

to be active only in the presence of β-estradiol. Adding β-estradiol CTS1 deletion thus allowed us to produce strains exhibiting all of to a growing culture induced conversion of germ to somatic the life strategies needed to compare the evolutionary stability of cells, apparent as the onset of mCitrine expression and slow loss unicellular and multicellular differentiation. of mCherry fluorescence by dilution (Fig. 1D, Fig. S1, and Movie Somatic cells in unicellular strains can only benefit germ cells by S1). As the β-estradiol concentration increased, conversion rates secreting useful products into a shared medium; nondifferentiating −3 − ranged from undetectable levels (<10 conversions per cell per cheats (e.g., Cre germ cells) and germ cells in well-mixed media generation) to ∼0.3 conversions per cell per generation (Fig. 1E have equal access to somatic cell products, but cheats do not pay A and Fig. S2 ). Expression of the codominant, cycloheximide- the reproductive toll of differentiation. We therefore predicted CYH2 sensitive WT allele of from its native locus permitted that cheats would enjoy a fitness advantage over germ cells, cyh2r continued growth following excision, but at a reduced rate allowing them to invade unicellular, differentiating populations that depended on cycloheximide concentration. The growth rate (Fig. 4A, Top). In multicellular species, however, significant local deficit of somatic cells ranged from undetectable (<1%) to nearly 30% as the cycloheximide concentration increased (Fig. 1F and Fig. S2B). A Bright-field mCherry mCitrine Composite The combination of irreversible differentiation and restricted somatic cell division caused cultures to approach a steady-state ratio between the two cell types over time (Fig. 2A). The steady- state fraction of somatic cells increased with the conversion rate and decreased with the somatic cells’ growth disadvantage, as B expected (Fig. 2 ). The steady-state ratio between cell types 10 μm could be tuned over four orders of magnitude by altering the cycloheximide and β-estradiol concentrations (Fig. 2B). BCClump fluorescence distribution Clump composition by cell type 76.4% 1.0 To investigate the ability of invertase secretion from somatic 18.2% 10 cells to support germ cell proliferation, we determined how the 0.8 ’ 8 culture s growth rate on sucrose depended on the ratio between Somatic only 0.6 cell types. In the presence of cycloheximide, cultures containing 6 Both types 0.4 both cell types at an intermediate ratio grew more quickly in 4 Germ only sucrose media than cultures of either cell type alone (Fig. 2C), confirming that somatic cells benefit their germ line through 2 Fraction of clumps 0.2 5.4% mCitrine Fluorescence (A.U.) 0 0.0 invertase secretion. 012345 300 pM 1 nM 3 nM 10 nM Clonal multicellularity arises through the maintenance of mCherry Fluorescence (A.U.) [β-estradiol] contact between daughter cells following cytokinesis (25). In bud- Δ ding yeast, clonal multicellularity can be produced, either through Fig. 3. cts1 strains form multicellular clumps containing both cell types. (A) Representative image of clump size and cell type composition in a well-mixed engineering (26) or evolution (16, 27), by mutations that prevent liquid culture of the Δcts1 multicellular differentiating strain (yMEW208) at a degradation of the septum, the specialized part of the cell wall that steady-state cell type ratio in sucrose minimal media containing 10 nM β-estra- holds mother and daughter cells together after their cytoplasms diol and 150 nM cycloheximide. (B) Representative distribution of clump have been separated by cytokinesis (28). Deletion of CTS1,achi- mCherry and mCitrine fluorescence for the multicellular differentiating strain tinase gene required for septum degradation, causes formation of (yMEW208) at a steady-state cell type ratio in sucrose minimal media containing “clumps” (groups of daughter cells attached through persistent 10 nM β-estradiol and 150 nM cycloheximide. Gating of clumps by cell type septa) that typically contain 4–30 cells during growth in well-mixed composition (lower sector: germ cells only; middle sector: both cell types; top liquid medium (Fig. 3A) (29). In the presence of β-estradiol, dif- sector: somatic cells only) is shown in red. (C) The fraction of clumps containing CTS1 Δcts1 one or both cell types was determined as in B for cultures of the multicellular ferentiating strains that lack ( ) produced clumps that differentiating strain (yMEW208) at a steady-state cell type ratio in sucrose frequently contained both germ and somatic cells, as shown by minimal media containing 150 nM cycloheximide at the indicated β-estradiol fluorescence microscopy (Fig. 3A) and flow cytometry (Fig. 3 B and concentrations. Error bars represent 95% CIs of the mean, determined with data C). Combining our gene excision-based differentiation system with obtained from six biological replicates.

8364 | www.pnas.org/cgi/doi/10.1073/pnas.1608278113 Wahl and Murray Downloaded by guest on September 30, 2021 A media, where somatic cells should confer no fitness advantage to

clumps (Fig. 4B). Moreover, the growth advantage of differentiating INAUGURAL ARTICLE strains depended strongly on the conversion rate (β-estradiol concentration): higher conversion rates were advantageous only in the multicellular differentiating case (Fig. 4B), where they increased the Unicellular fraction of somatic cells overall, as well as the fraction of clumps containing at least one somatic cell (Fig. 3C). In unicellular cultures grown on sucrose, or in cultures grown on glucose (unicellular or multicellular), increasing the conversion rate increased the growth advantage of cheats by reducing the growth rate of the germ cell Multicellular population (Fig. 4B). Legend: Germ cell Somatic cell Non-differentiating cheat Our results show that unicellular, soma-producing strains are evolutionarily unstable to invasion by nondifferentiating cheats. This B 0.10 finding is unlikely to depend on the specific molecular mechanisms 0.05 that effect differentiation or allow somatic cells to assist germ cells: any form of differentiation in a single-celled species would require that the two cell types exchange resources through a shared medium. 0.00 − From the spontaneous mutation rate in budding yeast [∼ 3 × 10 10 -0.05 per base pair per cell division (30)] and the size of the recombinase gene, we estimate mutations that inactivate our engineered re- -0.10 combination system would occur in about one out of every 107 cell divisions; this is likely an underestimate of the frequency of inacti- -0.15 vating mutations in natural differentiation, which typically requires -0.20 300 pM β-estradiol more loci and thus presents a larger target for mutation (22). Be- 1 nM β-estradiol cause this frequency is high relative to typical microbial population -0.25 3 nM β-estradiol sizes, cheats would thus likely arise and reach high frequencies β Growth advantage of differentiating strain 10 nM -estradiol shortly after the appearance of unicellular somatic differentiation, -0.30 EVOLUTION Δcts1 Δcts1 CTS1 CTS1 explaining the absence of extant species with this life strategy. The 0.5% sucrose 0.5% glu/fru 0.5% sucrose 0.5% glu/fru short persistence time of unicellular differentiating species makes them an unlikely intermediate in the evolution of development rel- − Fig. 4. Multicellular differentiating strains resist invasion by Cre cheats. ative to undifferentiated multicellular species, which have persisted (A) Hypothesized evolutionary outcomes for novel nondifferentiating mu- for hundreds of millions to billions of years in some clades (1–3). We tants (red) in unicellular (Top) and multicellular (Bottom) populations. Germ cannot, however, rule out the possibility that the transient existence cells are blue and somatic cells are yellow. (B) Growth advantages of dif- of a unicellular differentiating species might suffice for the secondary ferentiating strains (unicellular: yMEW192, multicellular: yMEW208) relative to cheaters (unicellular: yMEW193, multicellular: yMEW209) in minimal evolution of multicellularity, which has been observed experimentally media containing 150 nM cycloheximide. Error bars represent 95% CIs of in small populations on the timescale of weeks (16, 31). In any case, mean growth advantages, determined using data obtained from three bi- the differentiating phenotype cannot be stably maintained against ological replicates. Growth advantages of the multicellular strain growing in cheats until clonal multicellularity evolves. sucrose minimal medium at 1, 3, and 10 nM β-estradiol are significantly Our study demonstrates that synthetic biology can directly test − greater than zero (P < 10 3, one-tailed t test); growth advantages of the hypotheses about evolutionary transitions, complementing retro- unicellular strain growing in sucrose are significantly less than zero at all spective inference through comparison of existing species, exper- −6 β-estradiol concentrations (P < 10 , one-tailed t test). imental evolution, mathematical modeling, and simulation. We note that other major evolutionary transitions, including the appearance of body plans (spatially ordered arrangements of cell accumulation of somatic cell products (in our experiment, mono- types) and life cycles (temporal sequences of growth and dis- saccharides) within multicellular groups can give differentiating persion), could be studied through experimental evolution or lineages an advantage over cheats as long as the benefit of better further engineering of the strains described above. Furthermore, nutrition overcomes the cost of producing slower-replicating, so- our differentiating strain provides an experimentally tractable matic cells (26). This benefit is present even in well-mixed cultures version of a common simplifying assumption in population ge- because of the unstirred liquid within and surrounding the clump. In netics: independent deleterious mutations are modeled as having Δcts1 r clonally multicellular species, such as our strain, novel cheats the same fitness cost (32). In our strains, cyh2 excision is a form arising by mutation will eventually be segregated into cheat-only of irreversible mutation that always produces the same fitness groups by cell division and group fragmentation. We hypothesized disadvantage, but the magnitude of the fitness cost can be ex- that cheats would then experience reduced access to somatic cell perimentally varied by altering the cycloheximide concentration. products, potentially negating their growth advantage over germ cells Thus, engineered organisms, including those we have developed, A Bottom (Fig. 4 , ). can permit robust experimental testing of a wide variety of To test the prediction that cheats could overcome unicellular but outstanding hypotheses in evolutionary biology. not multicellular differentiation, we introduced cheats into unicellular or multicellular differentiating cultures and investigated their Materials and Methods fate. We mixed differentiating cultures expressing a third fluores- Conversion Time Courses and Conversion Rate Assays. We followed the dynamics cent protein, Cerulean, with cheats that lack this third color and of expression of fluorescent proteins during conversion from germ line to soma cannot differentiate because they lack the recombinase whose ac- by adding β-estradiol at the indicated concentration to cultures of pure germ − tion gives rise to somatic cells (Cre ). We monitored the relative cells (yMEW192), which were pregrown in log phase in YPD [yeast extract, frequency of the two strains over a series of growth and dilution peptone, dextrose (glucose)]. Cultures were maintained in log phase for additional growth while samples were collected for analysis by flow cytometry. At cycles. In sucrose media, cheats invaded unicellular, differentiating the indicated time point, cultures were washed twice with PBS to remove populations but were outcompeted in multicellular, differentiating β-estradiol and resuspended in YPD for additional growth before a final flow populations (Fig. 4B). The growth advantage of multicellular, dif- cytometry analysis. More details of the design and data from this experiment ferentiating strains was nullified in monosaccharide-containing are shown in Fig. S1.

Wahl and Murray PNAS | July 26, 2016 | vol. 113 | no. 30 | 8365 Downloaded by guest on September 30, 2021 We measured the rate of germ line to soma conversion by adding β-estradiol cells of the differentiating strain, respectively. After an acclimation period of to pure cultures of germ cells (yMEW192) pregrown in log phase in YPD approximately 10 h, culture density measurements were taken regularly with a (Fig. S2A). Pure cultures of germ cells were maintained in parallel to permit Coulter Counter, and a 95% CI of the slope determined by linear regression of calculation of the number of germ cell generations elapsed through cell density log(culture density) vs. time. The reported growth rates (Fig. 2C) were obtained measurement with a Coulter Counter (Beckman Coulter). Samples collected at from the weighted arithmetic means of slopes and their corresponding vari- β indicated time points were washed twice in PBS solution to remove -estradiol ances estimated from three or more independent experiments. and then resuspended in YPD for continued growth, allowing recently converted somatic cells to dilute out remaining mCherry so that cell types could be Determination of the Distribution of Cell Type Composition in Clumps. Cultures unambiguously distinguished by flow cytometry. Linear regression of the log of Δcts1 strains growing in minimal media containing 0.5% sucrose and the fraction of cells that were germ cells vs. the number of germ cell generations indicated concentrations of cycloheximide and β-estradiol were analyzed by elapsed was used to determine a 95% CI of the conversion rate for each ex- flow cytometry. Clumps in these cultures typically ranged in size from 4 to 30 periment using the fit and confint functions in MATLAB (Mathworks). The clumps; forward and side scatter gating could therefore not be used to conversion rates reported in Fig. 1E correspond to the weighted arithmetic mean and corresponding variance calculated with data obtained from three or eliminate events consisting of two or more clumps. Instead, cultures were more independent experiments. More details of the design and data from this diluted and vortexed thoroughly before flow cytometry to reduce the experiment are shown in Fig. S2. probability of multiple clumps being counted as a single event. The efficacy of this strategy was checked by combining pure cultures of germ and so- Fitness Assays for Relative Growth Difference Between Germ and Somatic Cells. matic cells in the absence of β-estradiol and confirming that the fraction of + + The growth rate difference between cell types was determined using a fitness mCherry mCitrine events in the mixed culture was less than 1%. Clumps assay protocol described previously (33). Briefly, pure cultures of germ containing only germ cells had negligible fluorescence in the mCitrine (yMEW192) and somatic (yMEW192 convertant) cells pregrown in log phase in channel and thus formed a distinguishable population; likewise, clumps YPD containing cycloheximide were combined and maintained in log phase containing only somatic cells had negligible fluorescence in the mCherry through multiple growth and dilution cycles in the indicated media (Fig. S2B). channel. Clumps with nonnegligible fluorescence in both channels were Samples taken at each dilution step were used to determine the fraction of considered to contain both cell types. each cell type by flow cytometry. Pure cultures of germ (yMEW192) cells were + maintained in log phase in parallel to determine the number of germ cell Competition Assays. In competition assays, Cerulean differentiating strains − generations elapsed through cell density measurements with a Beckman (unicellular: yMEW192; multicellular: yMEW208) were mixed with Cerulean Coulter Counter. The 95% CI for the slope of the linear regression line of Cre− reference strains (unicellular: yMEW193; multicellular: yMEW209) at a log(somatic cells/germ cells) vs. the number of germ cell generations elapsed 1:1 ratio and passaged through five cycles of growth and dilution in in- was determined as described above. The growth rate differences reported in dicated media. Samples taken at each dilution step were analyzed by flow Fig. 1F represent the weighted arithmetic means and corresponding variances + − cytometry to determine the ratio between Cerulean and Cerulean events of the slopes calculated in three independent experiments. (cells or clumps). The 95% CI of the slope of the linear regression line of log(Cerulean+ events/Cerulean− events) vs. the number of culture doublings Steady-State Ratio Assays. Pure cultures of germ cells (yMEW192) and somatic elapsed was determined as described above. The growth advantages of the cells (yMEW192) were mixed to initiate cultures from a range of cell type differentiating strain reported in Fig. 4B represent the weighted arithmetic ratios. Cultures were washed with PBS and resuspended in YPD containing β-estradiol and cycloheximide at the indicated concentrations. Samples were means and corresponding variances of the slopes calculated in three or more taken at the indicated time points to determine the number of culture independent experiments. doublings since initiation (determined from Coulter Counter measurements Additional materials and methods are provided as SI Text. Strain geno- of culture density) and the fraction of somatic (mCitrine+ mCherry−) cells in types are found in Table S1, plasmid details in Table S2. the culture. Cultures typically converged on a steady-state ratio after 30–40 culture doublings. ACKNOWLEDGMENTS. We thank Derek Lindstrom and Dan Gottschling for providing the estradiol-inducible Cre construct PSCW11-cre-EBD78;AlexSchier, Growth Rate Assays in Sucrose Minimal Media. Cultures of converting strains at Michael Desai, Michael Laub, Cassandra Extavour, David Haig, and members of the A.W.M. and David Nelson laboratories for helpful discussions during man- their steady-state ratios were washed twice with PBS and resuspended at uscript preparation; and Beverly Neugeboren, Linda Kefalas, and Sara Amaral ∼ 5 10 cells/mL in minimal media containing 0.5% sucrose and the concen- for research support. This work was supported in part by National Science trations of cycloheximide and β-estradiol in which they had been growing − Foundation and Department of Defense National Defense Science and Engi- previously. Pure cultures of the Cre strain yMEW193 and the yMEW192 neering graduate research fellowships and by NIH/National Institute of General convertant were also used to represent pure cultures of germ and somatic Medical Sciences Grant GM068763.

1. Grosberg RK, Strathmann RR (2007) The evolution of multicellularity: A minor major 16. Ratcliff WC, Denison RF, Borrello M, Travisano M (2012) Experimental evolution of transition? Annu Rev Ecol Evol Syst 38(1):621–654. multicellularity. Proc Natl Acad Sci USA 109(5):1595–1600. 2. Herron MD, Hackett JD, Aylward FO, Michod RE (2009) Triassic origin and early ra- 17. Ratcliff WC, et al. (2013) Experimental evolution of an alternating uni- and multi- diation of multicellular volvocine algae. Proc Natl Acad Sci USA 106(9):3254–3258. cellular life cycle in Chlamydomonas reinhardtii. Nat Commun 4:2742. 3. Schirrmeister BE, Antonelli A, Bagheri HC (2011) The origin of multicellularity in cy- 18. Gilbert OM, Foster KR, Mehdiabadi NJ, Strassmann JE, Queller DC (2007) High re- anobacteria. BMC Evol Biol 11:45. latedness maintains multicellular cooperation in a social amoeba by controlling 4. Kirk DL (2005) A twelve-step program for evolving multicellularity and a division of cheater mutants. Proc Natl Acad Sci USA 104(21):8913–8917. labor. BioEssays 27(3):299–310. 19. Momeni B, Waite AJ, Shou W (2013) Spatial self-organization favors heterotypic co- 5. Bell G, Mooers AO (1997) Size and complexity among multicellular organisms. Biol J operation over cheating. eLife 2:e00960. Linn Soc Lond 60(3):345–363. 20. Greig D, Travisano M (2004) The Prisoner’s Dilemma and polymorphism in yeast SUC 6. Bonner JT (2003) On the origin of differentiation. J Biosci 28(4):523–528. genes. Proc Biol Sci 271(Suppl 3):S25–S26. 7. Solari CA, Kessler JO, Goldstein RE (2013) A general allometric and life-history model for 21. Gore J, Youk H, van Oudenaarden A (2009) Snowdrift game dynamics and facultative cellular differentiation in the transition to multicellularity. Am Nat 181(3):369–380. cheating in yeast. Nature 459(7244):253–256. 8. Willensdorfer M (2009) On the evolution of differentiated multicellularity. Evolution 22. Davidson EH (2006) The Regulatory Genome: Gene Regulatory Networks in Development 63(2):306–323. and Evolution (Elsevier/Academic Press, Amsterdam). 9. Hom EF, Murray AW (2014) Plant-fungal ecology. Niche engineering demonstrates a 23. Stöcklein W, Piepersberg W, Böck A (1981) Amino acid replacements in ribosomal latent capacity for fungal-algal mutualism. Science 345(6192):94–98. protein YL24 of Saccharomyces cerevisiae causing resistance to cycloheximide. FEBS 10. Hekstra DR, Leibler S (2012) Contingency and statistical laws in replicate microbial Lett 136(2):265–268. closed ecosystems. Cell 149(5):1164–1173. 24. Lindstrom DL, Gottschling DE (2009) The mother enrichment program: A genetic 11. Bell G (1985) The origin and early evolution of germ cells as illustrated by the vol- system for facile replicative life span analysis in Saccharomyces cerevisiae. Genetics vocales. The Origin and Evolution of Sex (MBL Lectures in Biology), eds Monroy A, 183(2):413–422. Halvorson HO (A. R. Liss, New York), pp 221–256. 25. Queller DC (2000) Relatedness and the fraternal major transitions. Philos Trans R Soc 12. Buss LW, Mayr Ea (1987) The Evolution of Individuality (Princeton Univ Press, Princeton). Lond B Biol Sci 355(1403):1647–1655. 13. Goldsby HJ, Knoester DB, Ofria C, Kerr B (2014) The evolutionary origin of somatic 26. Koschwanez JH, Foster KR, Murray AW (2011) Sucrose utilization in budding yeast as cells under the dirty work hypothesis. PLoS Biol 12(5):e1001858. a model for the origin of undifferentiated multicellularity. PLoS Biol 9(8):e1001122. 14. Smith JM, Price GR (1973) The logic of animal conflict. Nature 246(5427):15–18. 27. Koschwanez JH, Foster KR, Murray AW (2013) Improved use of a public good selects 15. Smith JM (1964) Group selection and kin selection. Nature 201(4924):1145–1147. for the evolution of undifferentiated multicellularity. eLife 2:e00367.

8366 | www.pnas.org/cgi/doi/10.1073/pnas.1608278113 Wahl and Murray Downloaded by guest on September 30, 2021 28. Dohrmann PR, et al. (1992) Parallel pathways of gene regulation: Homologous reg- 40. Sheff MA, Thorn KS (2004) Optimized cassettes for fluorescent protein tagging in ulators SWI5 and ACE2 differentially control transcription of HO and chitinase. Genes Saccharomyces cerevisiae. Yeast 21(8):661–670. INAUGURAL ARTICLE Dev 6(1):93–104. 41. Shaner NC, et al. (2004) Improved monomeric red, orange and yellow fluorescent 29. Kuranda MJ, Robbins PW (1991) Chitinase is required for cell separation during proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 22(12): – growth of Saccharomyces cerevisiae. J Biol Chem 266(29):19758 19767. 1567–1572. 30. Lang GI, Murray AW (2008) Estimating the per-base-pair mutation rate in the yeast 42. Abremski K, Hoess R (1984) Bacteriophage P1 site-specific recombination. Purification – Saccharomyces cerevisiae. Genetics 178(1):67 82. and properties of the Cre recombinase protein. J Biol Chem 259(3):1509–1514. 31. Boraas M, Seale D, Boxhorn J (1998) Phagotrophy by a flagellate selects for colonial 43. Yoshimatsu T, Nagawa F (1989) Control of gene expression by artificial introns in prey: A possible origin of multicellularity. Evol Ecol 12(2):153–164. Saccharomyces cerevisiae. Science 244(4910):1346–1348. 32. Haldane JBS (1937) The effect of variation of fitness. Am Nat 71(735):337–349. 44. Orr-Weaver TL, Szostak JW, Rothstein RJ (1981) Yeast transformation: A model system 33. Lang GI, Botstein D, Desai MM (2011) Genetic variation and the fate of beneficial for the study of recombination. Proc Natl Acad Sci USA 78(10):6354–6358. mutations in asexual populations. Genetics 188(3):647–661. 45. Fried HM, Warner JR (1982) Molecular cloning and analysis of yeast gene for cyclo- 34. Bachmair A, Finley D, Varshavsky A (1986) In vivo half-life of a protein is a function of heximide resistance and ribosomal protein L29. Nucleic Acids Res 10(10):3133–3148. its amino-terminal residue. Science 234(4773):179–186. 46. Hauf J, Zimmermann FK, Müller S (2000) Simultaneous genomic overexpression of 35. Adams A, Cold Spring Harbor L (1997) Methods in Yeast Genetics: a Cold Spring Harbor seven glycolytic enzymes in the yeast Saccharomyces cerevisiae. Enzyme Microb Laboratory Course Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). – 36. Li MZ, Elledge SJ (2012) SLIC: A method for sequence- and ligation-independent Technol 26(9-10):688 698. cloning. Methods Mol Biol 852:51–59. 47. Voth WP, Olsen AE, Sbia M, Freedman KH, Stillman DJ (2005) ACE2, CBK1, and BUD4 – 37. Sikorski RS, Hieter P (1989) A system of shuttle vectors and yeast host strains designed in budding and cell separation. Eukaryot Cell 4(6):1018 1028. for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122(1):19–27. 48. Wach A, Brachat A, Alberti-Segui C, Rebischung C, Philippsen P (1997) Heterologous 38. Longtine MS, et al. (1998) Additional modules for versatile and economical PCR-based HIS3 marker and GFP reporter modules for PCR-targeting in Saccharomyces cerevisiae. gene deletion and modification in Saccharomyces cerevisiae. Yeast 14(10):953–961. Yeast 13(11):1065–1075. 39. Rizzo MA, Springer GH, Granada B, Piston DW (2004) An improved cyan fluorescent 49. Schindelin J, et al. (2012) Fiji: An open-source platform for biological-image analysis. protein variant useful for FRET. Nat Biotechnol 22(4):445–449. Nat Methods 9(7):676–682. EVOLUTION

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