Whole-genome duplication in the Multicellularity Long Term Evolution Experiment

Abstract

Whole-genome duplication (WGD) is widespread across eukaryotes and can promote adaptive evolution^1-4. However, given the instability of newly-formed polyploid genomes^5-7, understanding how WGDs arise in a population, persist, and underpin adaptations remains a challenge. Using our ongoing Multicellularity Long Term Evolution Experiment (MuLTEE)⁸, we show that diploid snowflake yeast (Saccharomyces cerevisiae) under selection for larger multicellular size rapidly undergo spontaneous WGD. From its origin within the first 50 days of the experiment, tetraploids persist for the next 950 days (nearly 5,000 generations, the current leading edge of our experiment) in ten replicate populations, despite being genomically unstable. Using synthetic reconstruction, biophysical modeling, and counter-selection experiments, we found that tetraploidy evolved because it confers immediate fitness benefits in this environment, by producing larger, longer cells that yield larger clusters. The same selective benefit also maintained tetraploidy over long evolutionary timescales, inhibiting the reversion to diploidy that is typically seen in laboratory evolution experiments. Once established, tetraploidy facilitated novel genetic routes for adaptation, playing a key role in the evolution of macroscopic multicellular size via the origin of evolutionarily conserved aneuploidy. These results provide unique empirical insights into the evolutionary dynamics and impacts of WGD, showing how it can initially arise due to its immediate adaptive benefits, be maintained by selection, and fuel long-term innovations by creating additional dimensions of heritable genetic variation.

Extended Data Fig. 1 |. Images of the ancestors and evolved isolates in the MuLTEE.

a,b, Representative cluster-level (a) and cell-level (b) images of PM/PA t0 and PM/PA1-5 t200, t400, t600, and t1000 isolates. The images of PM/PA t0 are reused for five replicate populations. Scale bars, 200 μ,m (a) and 10 μ,m (b).

Extended Data Fig. 2 |. Phenotypic characterization of the ancestors and evolved isolates in the MuLTEE.

a-d, Violin plots showing the distributions of cluster radius (a,b, where b is weighted by cluster volume), cell volume (c), and cell aspect ratio (d) in PM/PA t0 and PM/PA1-5 t200, t400, t600, and t1000 isolates (on average, n = 789 clusters (a,b) and 1288 cells (c,d) measured per sample). The distributions of PM/PA t0 are reused for five replicate populations. For a,b, we measured cluster radius at 4 hours (exponential phase) and 24 hours (stationary phase) after transferring the culture to fresh media, and the 24-hour measurements (corresponding to the state of the cultures right before settling selection) are used throughout the paper unless otherwise noted. For b, filled circles show biomass-weighted mean cluster radius (the 24-hour values are the same as the values in Fig. 1c). For c,d, boxes, IQR; center lines, median; whiskers, values within 1.5 × IQR of the first and third quartiles.

Extended Data Fig. 3 |. Imaging-based method for measuring ploidy level of snowflake yeast.

a, Overview of imaging and image analysis workflow. Snowflake yeast clusters are crushed into a single cell layer and imaged at the brightfield channel and fluorescent channel, with the latter showing the nuclear DNA stained by propidium iodide (PI). The nuclei in the fluorescent image are segmented and filtered to get single round nuclei, outlined in cyan. The fluorescent image is also nuclei-cleared and brightness/contrast-enhanced to show the background fluorescence in the cytoplasm, and the cytoplasm is segmented, shown in white, for background subtraction. The total fluorescence intensity of PI in each nucleus is quantified and background-subtracted. Scale bar, 20 μm. b, Distribution of the nuclear PI intensity (arbitrary unit) of the engineered diploid and tetraploid mixotrophic clusters (n = 14276 and 10031 nuclei, respectively), as a validation for this ploidy measurement method. Since asynchronous, exponential-phase cultures are used for ploidy measurements, each strain shows two peaks that correspond to G1- and G2-phase nuclei of the actively-dividing cells, and the G2 peak has double of the fluorescent intensity of the G1 peak. Also, the G2 peak of diploid clusters aligns nicely with the G1 peak of tetraploid clusters.

Extended Data Fig. 4 |. Copy number variation of the ancestors and evolved isolates in the MuLTEE.

a,b, Estimated copy number of each 1kb non-overlapping bin in each chromosome in PM/PA t0 and PM/PA1-5 t200, t400, t600, and t1000 isolates (a, PM; b, PA). Estimated bin copy numbers above 12 are shown as 12, indicated by little triangles. Red horizontal line, baseline ploidy of each strain (i.e., 2 for PM/PA t0 and 4 for all evolved isolates). Red arrowhead, incidence of segmental aneuploidy.

Extended Data Fig. 5 |. Genetic construction and phenotypic characterization of diploid and tetraploid clusters.

a, Procedure for engineering isogenic grande diploid and tetraploid clusters, from each of which four independent petite mutants were isolated. Isolating multiple petite mutants is important because petite mutations are not isogenic and may confound ploidy-phenotype map. Grande and petite clusters correspond to mixotrophic and anaerobic conditions, respectively. b-e, Violin plots showing the distributions of cluster radius (b,c, where c is weighted by cluster volume), cell volume (d), and cell aspect ratio (e) in engineered diploid and tetraploid clusters under mixotrophic and anaerobic conditions (on average, n = 922 clusters (b,c) and 2458 cells (d,e) measured per sample). Four biological replicates were measured for the mixotrophic condition, and the four independent petite mutants (each with one biological replicate) were measured for the anaerobic condition. For b,c, we measured cluster radius at 4 hours (exponential phase) and 24 hours (stationary phase) after transferring the culture to fresh media, and the 24-hour measurements are used throughout the paper unless otherwise noted. For c, filled circles show biomass-weighted mean cluster radius (the 24-hour values are the same as the values in Fig. 3e). For d,e, boxes, IQR; center lines, median; whiskers, values within 1.5 × IQR of the first and third quartiles. f,g, Comparison of the biomass-weighted mean cluster radius (f) and mean cell aspect ratio (g) of the engineered petite tetraploid clusters (mean of the four independent petite mutants, the same values as those in Fig. 1c,e) to the PA t0 and PA1-5 t50, t100, t150, and t200 populations (data from Bozdag et al. 2023).

Extended Data Fig. 6 |. Label-free method for distinguishing engineered diploid and tetraploid clusters in competition assays.

a, Brightfield image of a snowflake yeast cluster (an engineered tetraploid mixotrophic cluster is shown) (top), whose bright cells in the cluster edges are detected (bottom). Scale bar, 30 μm. b, Mean area of the five largest cells detected in a cluster can be used to distinguish between the engineered tetraploid and diploid clusters under both mixotrophic and anaerobic conditions, with the dashed line indicating the manually-chosen decision boundary.

Extended Data Fig. 7 |. Experimental evolution of the MuLTEE ancestors and t1000 isolates with selection against larger size.

a, Experimental setup. We evolved PM/PA t0 and PM/PA1-5 t1000 isolates, with four replicate populations (A, B, C, D), under selection against larger size for 70 days (~500 generations) by growing them on agar in 24-well plates with daily dilution. b, Distributions of cluster radius (weighted by cluster volume) in the ancestral (“anc”) and evolved populations (on average, n = 406 clusters measured per population). Vertical thick solid line, biomass-weighted mean cluster radius of each population. Vertical dashed line, biomass-weighted mean cluster radius of PM/PA t0. Color code for the level of ploidy reduction in each population is the same as that in Fig. 3i, and the ancestral populations are colored in gray.

Extended Data Fig. 8 |. Point mutation changes in donut-to-spread transitions.

Two donut-to-spread transitions with near-triploidization were excluded, and mutation allele frequency refers to the corrected allele frequency, calculated by dividing estimated allele copy number with copy number of the chromosome that carries the mutation. LOH, loss of heterozygosity. a,b, Number of mutations in each category of change in allele frequency in all donut-to-spread transitions combined, colored by change in allele copy number (a), and how change in allele frequency is associated with change in chromosome copy number (b). a,b share the color code. c, Number of mutations in each category of change in allele frequency in each donut-to-spread transition, colored by mutation impact. d, Percentage of mutations in each mutation impact category in each donut background, whose total number of mutations is indicated in the brackets. c,d share the color code. e, Comparison of the distribution of mutation impacts, between the mutations that were gained in all donut-to-spread transitions combined and the mutations randomly introduced into yeast genome, as well as between the mutations that underwent loss, LOH, increase, decrease, or maintenance in terms of allele frequency in all donut-to-spread transitions combined and the mutations in all donut backgrounds combined. Number of mutations is indicated in the brackets. P values were calculated by chi-squared test. f, For each donut-to-spread transition, the percentage of high/moderate-impact mutations in the mutations that increased or decreased in allele frequency is on average not significantly larger than the percentage of high/moderate-impact mutations in the donut background (for increase and decrease, respectively, P = 0.891 and 0.442, t₂₂ = −1.27 and 0.147, one-tailed one-sample t-test), and is largely explained by random sampling of mutations in the donut background (simulation with random seed = 1). Values are mean± s.d. (n = 23 donut-to-spread transitions).

Fig. 1 |. Increased cell volume and aspect ratio drive the evolution of larger multicellular groups in the 1000-day MuLTEE.

a, Experimental setup of the MuLTEE. b, Representative cluster-level (top two rows) and cell-level (bottom two rows) images of the ancestor (t0) and day-1000 (t1000) isolates from the five replicate populations evolved under mixotrophic (PM) and anaerobic (PA) conditions. Scale bars, 200 μm (cluster-level images) and 10 μm (cell-level images). c-e, Evolutionary dynamics of biomass-weighted mean cluster radius (Methods) (c), mean cell volume (d), and mean cell aspect ratio (e), showing the values of PM/PA t0 and PM/PA1-5 t200, t400, t600, and t1000 isolates (on average, n = 886 clusters (c) and 1288 cells (d,e) measured for each of the 42 strains), with the gray dotted and dashed lines representing artificially-constructed diploid and tetraploid strains (mean value of the four biological replicates in Fig. 3c-e), respectively. f, Relationship of mean cell volume and mean cell aspect ratio of strains in c-e. g-i, Heat maps showing the biophysical simulations of how cell volume and cell aspect ratio affect the mean cluster radius (g), cell number per cluster (h), and cell packing fraction (fraction of the cluster volume occupied by cells) (i) of clusters at fragmentation (n = 50 simulated clusters per pair of parameter values).

Fig. 2 |. Tetraploidy rapidly emerged and was maintained in all PMs and PAs; PAs subsequently evolved extensive aneuploidy.

a, Violin plot of the distribution of allele frequencies (each determined as the fraction of the read number of the mutant allele in the read number of all alleles) of novel point mutations identified in PM/PA1-5 t200, t400, t600, and t1000 isolates, with each dot representing one point mutation. b, Evolutionary dynamics of DNA content, showing that of PM/PA t0 and the strains used in a (on average, n = 11456 cells measured for each of the 42 strains). c, Ridge plot of the distribution of cellular DNA contents in the PM/PA t0 and PM/PA1-5 t50 and t100 populations (on average, n = 12922 cells measured for each of the 22 populations). Since asynchronous cultures were used for ploidy measurements, G1- and G2-phase cells of a single genotype in a population could form two distinct DNA-content peaks. d, Heat maps of the karyotypes of PM/PA t0 and the strains used in a, determined based on chromosome coverages from the whole-genome sequencing data.

Fig. 3 |. Tetraploidy confers immediate phenotypic effects that are beneficial under selection for larger size, driving the origin and maintenance of tetraploidy despite its genomic instability.

a,b, Cell-level (a) and cluster-level (b) images of the engineered diploid and tetraploid clusters under mixotrophic and anaerobic conditions. Scale bars, 5 μ,m (a) and 50 μm (b). c-e, Mean cell volume (c), mean cell aspect ratio (d), and biomass-weighted mean cluster radius (e) of the strains in a,b. f, Relationship of the changes in cell volume and cell aspect ratio due to tetraploidization, under mixotrophic and anaerobic conditions. Values are means of the four replicates or petite mutants in c,d. g, Per day selection rate of competing engineered tetraploid clusters against diploid counterparts with and without settling selection for three days, under mixotrophic and anaerobic conditions. For c-e,g, values are mean ± s.e.m. (n = 4 biological replicates for mixotrophic condition or 4 independent petite mutants for anaerobic condition; on average, 2458 cells (c,d) and 1077 clusters (e) were measured per replicate or petite mutant, and 381 clusters (g) were measured per sample). P values were calculated by two-tailed Welch’s t-test. h,i, Distribution of cellular DNA contents in the evolved populations, initiated with PM/PA t0 (h) and PM/PA1-5 t1000 isolates (i) with four replicate populations (A-D), after selecting against larger size for 70 days (on average, n = 14487 cells measured per population). Gray vertical line, ancestral DNA content of each strain from Fig. 2b (corresponding to G1 peak). Levels of ploidy reduction: “complete”, reduction to diploidy; “partial”, reduction to an intermediate ploidy level between diploidy and tetraploidy, or a mixture of tetraploidy and lower ploidy levels; “no”, no detectable ploidy reduction.

Fig. 4 |. Aneuploidy promotes the origin of macroscopic multicellularity in PAs.

a, Evolved macroscopic isolates (PA3 t1000 is shown) form “donut” colonies (D) and occasionally “spread” colonies (S) that cannot form macroscopic clusters. Scale bar, 5 mm. b-d, Biomass-weighted mean cluster radius (b), mean cell volume (c), and mean cell aspect ratio (d) of the 25 D-S pairs (each connected by a line) from nine macroscopic isolate backgrounds. Boxes, interquartile range (IQR); center lines, median; whiskers, values within 1.5 × IQR of the first and third quartiles. P values were calculated by two-tailed paired t-test (on average, 605 clusters (b) and 1294 cells (c,d) measured per strain). e,f, Relationship of how cell volume and aspect ratio changed in D-S transitions, with donut and spread strains separated by a linear boundary generated by support vector machine (e), and with each D-S transition connected by a line and grouped into four classes based on the signs of changes in cell volume and cell aspect ratio (P < 0.05, two-tailed Welch’s t-test with Benjamini-Hochberg correction; ns, not significant; D-S pair counts in each class are indicated in brackets) (f). g,h, Karyotypes of D-S pairs (g) and karyotype changes in D-S transitions (h). Inside the heatmaps, numbers (g) show chromosome copy number differences between donut strains and their backgrounds in Fig. 2d, and dashes (h) indicate two D-S transitions with near-triploidization that were excluded from subsequent analyses. Symbols beside the heatmap (h) indicate cases of convergent karyotype changes where two D-S pairs came from the same background (red), different backgrounds but the same PA line (green), or different lines (blue), with each case in a different symbol shape. i, For each background, chromosomes with copy number changes in the evolution (light rectangular backgrounds) and loss (dark bars, showing number of occurrences) of macroscopic size. j, Histogram of the number of chromosomes with copy number changes in each D-S pair, colored by background. k, Changes in cell volume and aspect ratio compared between D-S pairs with convergent karyotype changes, with symbol color-shape code consistent with h and line color-shape code similarly assigned. l-o, Aggregating all backgrounds in i, number of times each chromosome changes its copy number in the loss (l,m) and evolution (n,o) of macroscopic size, colored by background (l,n, share color legend with j) or direction of change (m,o).