How the experiment works

The experimental protocol

Each day the yeast grow, the culture is left to settle, and only the clusters that reach the bottom first are moved into fresh medium. This is the only selection we impose, and it is applied the same way to all fifteen populations in three metabolic environments. Every method described on this page has its own protocol, starting with daily growth and settling selection. As part of our open-science policy, we aim to post each one in the Protocols section of this site as it is written up.

The organism

What a snowflake yeast is

Baker's yeast, Saccharomyces cerevisiae, reproduces by budding. A daughter cell swells off the side of a mother, and when mitosis is finished an enzyme dissolves the septum at the bud neck so the two cells come apart. Snowflake yeast are what happens when that last step fails. Mother and daughter stay joined, the daughter buds in turn, and the cluster grows outward from one founding cell into a branched, roughly radial body.

Because the cluster is built by cells staying attached after division rather than by free-living cells finding each other, every cell in it carries the same genome. This is clonal multicellularity, and it matters: with no genetic differences inside a cluster, there is nothing for a cheater cell to gain, and cell fitness and group fitness point the same way from the start.

The genetic basis is a single transcription factor. ACE2 regulates the genes that degrade the bud neck septum, so a loss-of-function mutation leaves daughters attached. When we sequenced ten independently evolved snowflake lineages, five carried non-synonymous ACE2 mutations, and in every case both copies were identical, most likely through gene conversion (Ratcliff et al. 2015).

Loss of ACE2 has arisen independently many times in yeast evolution experiments that favour multicellular groups, including in other labs and other species: in fast-sedimenting lineages of S. cerevisiae (Oud et al. 2013), in the budding yeast Kluyveromyces lactis, where it was the main route to multicellularity (Wang, Driscoll & Travisano 2024), and in two of five lineages of the fission yeast Schizosaccharomyces pombe, which split from budding yeast hundreds of millions of years ago (Pineau et al. 2024).

All fifteen MuLTEE populations were founded from an ace2Δ strain in the Y55 background, so they were multicellular from the first day.

Confocal projection of a snowflake yeast cluster: cells outlined in bright cyan, each holding a single orange nucleus, fanning outward from a central branch point, with smaller clusters and loose cells scattered across a black field.

A single cluster. The cyan outlines are cell walls and the orange points are nuclei, one per cell. Each branch point is where a daughter cell budded and stayed attached.

The daily cycle

Growth, settling, transfer

One round of the MuLTEE takes a single day. The populations grow, they sediment under gravity, and the fraction that settles fastest is transferred by pipette into fresh medium. The regime is therefore selection for rapid growth followed by selection for larger group size.

01

Grow, for about twenty-four hours in liquid medium

The small volume carried over from yesterday expands back to a full culture. This phase is selective too: it rewards whatever divides fastest, and in the obligately aerobic populations, where the measurement has been made, that is the smaller clusters.
02

Settle, under gravity

The culture is left standing and biomass falls out of suspension. Larger and denser clusters sink faster, so within a fixed settling window they arrive at the bottom of the tube while smaller ones are still in the liquid above.
03

Transfer the fastest-settling fraction into fresh medium

Only the bottom fraction is moved forward. Everything still in suspension is discarded. Over 600 transfers this took mean cluster radius from 16 µm to 434 µm in the anaerobic populations, roughly a 20,000-fold increase in volume, and took the number of cells in a group from about 100 to about 450,000.

Interactive · Schematic, not to scale

Settling selection, simulated

Forty clusters in a tube. Press play and they fall, larger ones faster. Everything that reaches the transfer line goes into fresh medium and regrows to a full culture; everything still in suspension is thrown away. The chart beside the vessel is the size distribution of the whole culture, and the starting distribution stays on it as a dashed outline. About fifteen transfers is enough to see the whole distribution move right.

This section carries an interactive diagram of the selection step, which needs JavaScript. The mechanism it draws is the one described above: clusters of different sizes fall through a tube, larger ones sink faster and reach the bottom first, only the bottom fraction is pipetted into fresh medium, and because size is inherited the size distribution of the population shifts toward larger clusters transfer by transfer.

Transfer 1 of 20.

In the simulation, settling speed rises with cluster size by a simple rule, plus a small random term. Real clusters are branched and porous, so their shape and packing also affect how fast they sink. Sizes are shown relative to the starting mean.

The selection step is simplified too. In the widget the settling window ends when the pipette volume is full, so about twelve clusters out of forty carry forward every transfer. The real regime uses a fixed settling window, which is not quite the same thing.

Both phases are selective and they act in opposite directions. Growth favours small, fast-dividing clusters, and settling favours large ones. A trait spreads only if it pays for itself across the full 24-hour cycle, which is why the same selection regime produced macroscopic bodies in one treatment and, in another, a stable split into small growth specialists and large survival specialists that have coexisted for thousands of generations. When snowflake yeast are competed against unicellular or floc-forming yeast and the two phases are scored separately, they make most of their fitness gains during settling (Pentz et al. 2023), which means they are increasing fitness at the level of the group rather than the cell.

How and why we select for size

Larger clusters sink faster, so settling selection favours larger groups. We chose it because it is easy to control: its strength can be tuned by shortening the settling time, and unlike predation it needs no second species that could co-evolve or die out. Sinking rate is also an ecologically relevant trait for aquatic organisms.

Many of the traits that evolved were not selected for directly. Cells became longer, which loosens packing and delays fracture; branches became entangled; and cell division became synchronized. These traits spread because they made clusters larger or harder to break.

Three-dimensional rendering of one snowflake yeast cluster on black, its cells coloured cyan and blue at the front through green and yellow to orange at the back, giving a spiky radial branching silhouette.

About eighteen small three-dimensional snowflake yeast clusters scattered across a black field, each depth coded from blue through green to yellow, showing a range of sizes and branching shapes.

Selection acts on a whole distribution of cluster sizes, like the one in Plate 03, and retains its upper tail every day. Cluster size is reported as a biomass-weighted radius for this reason: a population containing many small propagules and a few large adults would otherwise be summarised by its propagules.

Three metabolic treatments

Cells inside a large cluster rely on oxygen diffusing in from outside, and diffusion limits how large a solid mass of respiring cells can grow. The three treatments test whether this limit constrains multicellular size over the long run.

The treatments come from a twenty-population experiment established in 2018. Results from its first 145 transfers, published in 2021, showed that intermediate oxygen suppressed size evolution, while anaerobic conditions and high oxygen allowed it. Fifteen of those populations have been transferred ever since and form the MuLTEE.

PA1–5 Anaerobic

Cannot respire

Metabolism: Obligate fermentation
Mitochondria: Petite; respiration lost
Medium: YPD (glucose)

Petite mutants carry defective mitochondria and cannot use oxygen at all. Removing respiration also removes the diffusion limit on size, so if oxygen delivery is what constrains size, these lines were the ones expected to escape it. All five became macroscopic.

PM1–5 Mixotrophic

Can ferment or respire

Metabolism: Fermentation and respiration
Mitochondria: Intact
Medium: YPD (glucose)

Glucose with access to oxygen, which is how ordinary yeast grows. These populations stayed microscopic.

PO1–5 Obligately aerobic

Must respire

Metabolism: Obligate respiration
Mitochondria: Intact and required
Medium: YPG (glycerol)

Glycerol cannot be fermented, so growth is impossible without dissolved oxygen. That turns oxygen into something clusters compete over, and three of the five populations split into coexisting small and large lineages held together by negative frequency-dependent selection.

Population-by-population detail is on the Populations page.

The frozen fossil record

Every 25 days, roughly 125 generations, we cryopreserve all fifteen populations at −80 °C. Frozen samples neither die nor continue to evolve, so when they are thawed they resume growing in the state the population was in on the day it was frozen. This frozen fossil record now holds more than 3,000 samples.

Because frozen samples can be revived, an ancestor can be grown in the same tube as its own descendants, which is how fitness is measured (see What gets measured).

The archive also lets us test the order in which things happened. If a trait is present at day 600, we can thaw day 400 and check whether it was already segregating there, rather than inferring it. Reversibility can be tested the same way, by selecting against large size in revived evolved strains and seeing whether the trait is lost. That is how we showed that tetraploidy in these populations is maintained by ongoing selection for size rather than by genome stability.

The archive also keeps samples for methods that do not exist yet. Some of the most important results from Lenski's E. coli LTEE came from genome sequencing and genetic reconstruction, neither of which was available at scale when that experiment began in 1988.

Working with the archive

Requests for any population at any timepoint go through the strain request page.

What gets measured

Types of data we collect

Measurements are made on isolates revived from the archive, so samples from day 0, day 400 and day 1,000 can be measured side by side. Each method below has its own protocol on the Protocols page, or will once it is written up.

Morphology

Biomass-weighted cluster radius across evolutionary time, cellular aspect ratio, and bud scar geometry, which records where every past division happened.
Fitness

Evolved isolates competed head to head against the revived ancestor under the standard growth-plus-settling regime, with the growth phase and the settling phase scored separately when the question calls for it.
Mechanics

Compression assays for the stress-strain response and for multicellular toughness. As a material, these clusters went from around 100-fold weaker than gelatin to the strength and toughness of wood.
Genomes

Whole-genome sequencing of evolved isolates, plus synthetic reconstruction: candidate mutations are engineered back into a clean background to test whether they actually cause the phenotype they correlate with.
Imaging

Serial block-face scanning electron microscopy to reconstruct the inside of a cluster, time-lapse microscopy to follow division timing cell by cell, and super-resolution panoramic integration to measure subcellular shape across whole populations rather than a handful of picked cells.