MuLTEERatcliff Lab · Georgia Tech

Vocabulary

Glossary

Terms used in our work on the evolution of multicellularity. Where a term is used more narrowly here than in the wider literature, the entry says so.

A

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  • ACE2
    The yeast transcription factor whose loss leaves daughter cells attached, producing the snowflake phenotype.
    ACE2 regulates the genes that degrade the bud neck septum so a daughter cell can separate from its mother after mitosis. Loss-of-function mutations leave the cells attached, and that one change produces the entire snowflake package: clonal clusters, a multicellular life history, a unicellular genetic bottleneck at every reproduction, and high heritability of cluster traits. Non-synonymous ACE2 mutations turned up in 5 of 10 independently evolved lineages, with both alleles made identical by gene conversion. This is our clearest case of a microevolutionary change with macroevolutionary consequences.
    In the MuLTEE The multicellular starting point for the MuLTEE comes from disrupting this single gene, and Pineau et al. (2024) extended the argument across deeply diverged fungi by showing that ace2 loss-of-function also drives multicellular group formation in Schizosaccharomyces pombe.
    See also Snowflake yeast · Clonal multicellularity · Heritable variation in multicellular traits · Unicellular bottleneck · Major evolutionary transitions
  • Aneuploidy
    Carrying odd numbers of particular chromosomes, treated here as a lasting adaptation rather than noise.
    Aneuploidy is a karyotype in which one or more chromosomes are present at copy numbers off the euploid baseline. It is conventionally read as a temporary fix for a novel or stressful environment, and as background genomic noise otherwise. Here it refers to the convergent, evolutionarily conserved chromosome copy-number changes that appeared in anaerobic tetraploid snowflake yeast, arising from the instability of the maintained tetraploid genome and then held in place by sustained selection for larger multicellular size.
    In the MuLTEE Tong et al. (2025, Nature) showed that extensive aneuploidy evolved mainly in the anaerobic tetraploid MuLTEE lines, tracked the evolution of macroscopic size, reversed direction in derivatives that lost macroscopic multicellularity, and persisted for thousands of generations, extending aneuploidy's known repertoire from stress tolerance to morphological innovation.
    See also Whole-genome duplication · Snowflake yeast · Morphological entanglement

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  • Biophysical scaffolding
    Physics doing a job for a young multicellular body before any adaptation evolves to do it.
    Biophysical scaffolding is the use of emergent physical processes or material properties to support a multicellular function before that function is stabilized by dedicated, genetically encoded adaptations. It answers a recurring problem in major-transitions biology: how can simple groups perform apparently sophisticated tasks with no organs, no developmental program, and no transport system? The scaffold is not the endpoint. It opens phenotypic space and creates a functional foothold that later evolution can refine, stabilize, or replace.
    In the MuLTEE Narayanasamy et al. (2025, Science Advances) named the concept after finding that sufficiently large MuLTEE clusters generate metabolically driven buoyant flows that advect nutrients through the body, allowing exponential growth even at macroscopic size without any dedicated transport structure.
    See also Morphological entanglement · Oxygen diffusion limitation · Snowflake yeast · Cell packing · Complex multicellularity

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  • Cell packing
    How densely cells fill a cluster, which sets how fast internal stress builds as it grows.
    Packing has a local sense, how daughter cells arrange around a parent, and a global one, the fraction of cluster volume occupied by cells rather than empty space. As cells crowd into shared volume, intercellular stress rises, clusters fracture sooner, and maximum size is capped. We use packing to argue that early multicellular evolution was shaped by hard physical limits as much as by genetics: a lineage can usually get bigger more efficiently by repacking than by evolving stronger adhesion. Day et al. (2022) added that local free space follows a maximum-entropy k-gamma law, so packing also makes group traits reproducible before regulated development exists.
    In our work Over seven weeks of selection, snowflake yeast cells elongated by 8 percent in aspect ratio and cluster volume fraction fell from 0.32 to 0.22, with a geometric simulation seeded only on measured aspect ratios reproducing the change at r² = 0.94 (Jacobeen et al., 2018, Nature Physics); across 23 populations, aspect ratio explained 92 percent of the variation in multicellular size (Bozdag et al., 2021).
    See also Mechanical fracture · Morphological entanglement · Snowflake yeast · Oxygen diffusion limitation · Coordinated cell division · Proteostatic tuning · Biophysical scaffolding · Heritable variation in multicellular traits
  • Clonal multicellularity
    Groups built by cells staying attached after division, so every cell in the group is genetically identical.
    Clonal multicellularity produces clusters from a single progenitor by postdivision adhesion. It contrasts with aggregative multicellularity, in which genetically distinct free-living cells come together, as in Dictyostelium slime molds or bacterial biofilms. Because all cells in a clonal group share a genome, there is nothing for a cheater cell to gain, and cell fitness is aligned with group fitness. That alignment is what makes cooperation, division of labor, and the shift from cell-level to organism-level selection theoretically possible.
    In our work Snowflake yeast were confirmed by time-lapse microscopy and calcofluor staining to form clonally rather than by aggregation (Ratcliff et al., 2012), and Pentz et al. (2023, eLife) later engineered a matched pair of yeast life cycles and found that only the clonal one converted selection on settling into efficient group-level adaptation.
    See also Snowflake yeast · Unicellular bottleneck · Higher-level individuality · Multilevel selection · Effective population size · Division of labor · Settling selection · Complex multicellularity
  • Complex multicellularity
    Large, integrated bodies with many specialized cell types, the rare far end of the transition.
    Complex multicellularity means more than being multicellular. It marks organisms that are large, often relatively long-lived, and built from many specialized cell types integrated into a coherent higher-level body. Few lineages ever reached it, and all extant examples develop clonally. The concept supplies the long-range benchmark for our work: the reason the earliest steps matter is that they show how a lineage might eventually become a large differentiated organism, and the rarity of that outcome is treated as a puzzle needing explanation rather than a trivial fact.
    In our work We use complex multicellularity as the distant benchmark against which early traits are judged, noting that transitions to differentiated cell types took millions of years while the first steps evolve remarkably quickly; Bingham & Ratcliff (2024) propose reduced effective population size as a hidden macroevolutionary filter, and Zhang et al. (2026) argue that large size itself lowers the fecundity cost of germ-soma specialization.
    See also Division of labor · Unicellular bottleneck · Clonal multicellularity · Effective population size · Major evolutionary transitions · Oxygen diffusion limitation · Morphological entanglement
  • Coordinated cell division
    Mothers and daughters dividing on the same schedule instead of daughters lagging behind.
    Coordinated division means division timing is aligned among related cells, without the strong age-specific delay seen in the ancestor. In snowflake yeast the change is specific: newly produced daughter cells lose a prolonged first cell cycle. That one timing shift alters the branching network's topology, and topology determines where stress concentrates, how large a cluster grows before it fractures, how big the propagules are, and therefore how well they settle. Developmental coordination in this system begins as a change in age-specific doubling times, not as a regulatory program.
    In the MuLTEE Cedeño-Pérez et al. (2025, PLOS Computational Biology) used time-lapse microscopy and single-cell tracking on MuLTEE lineages and found synchronous division emerging by day 200 and persisting through day 1000, providing benefits at both levels through faster cell growth and larger group size.
    See also Cell packing · Mechanical fracture · Snowflake yeast · Settling selection · Multilevel selection

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  • Division of labor
    Cells within a group specializing in different tasks that complement one another.
    Division of labor means different cells perform complementary functions for the collective. It is distinct from differentiation, which is only cells adopting different states. The familiar form is reproductive: germ cells reproduce while somatic cells give that up to do something else. In early multicellularity it can be rudimentary, with a small fraction of cells taking a non-reproductive role while the rest stay fertile. The hard question is how the first somatic cells arose at all, since they had no pre-existing organizational template to follow.
    In our work Tong, Bozdag & Ratcliff (2022) argue that division of labor can drive group formation rather than only follow it, since cross-feeding segregates incompatible tasks across cells in space instead of switching between them in time; Conlin et al. (2023) added the second role, showing in digital evolution that specialization also locks multicellularity in against reversion.
    See also Higher-level individuality · Complex multicellularity · Multicellular entrenchment · Ratcheting traits · Clonal multicellularity · Major evolutionary transitions · Snowflake yeast

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  • Effective population size
    How many genetically distinct lineages evolution actually has to work with, once bottlenecks are counted.
    Effective population size, or Ne, is the size of an idealized population that would experience the same strength of genetic drift as the real one. Multicellular life cycles partition cells into discrete reproducing groups and force each generation through a bottleneck, so evolution operates on far fewer independent lineages than raw cell counts suggest. This is the flip side of the usual argument for bottlenecks: the same life-cycle architecture that builds individuality and suppresses conflict also amplifies drift and the stochastic loss of beneficial mutations.
    In our work Pentz et al. (2023, eLife) showed by genome sequencing and modeling that the genetic bottlenecks in a clonal life cycle drive much higher rates of drift, and Bingham & Ratcliff (2024) report that measured Ne is typically two to three orders of magnitude lower in multicellular than in unicellular taxa, proposing this as part of why complex multicellularity is repeatedly eukaryotic and never prokaryotic.
    See also Clonal multicellularity · Unicellular bottleneck · Complex multicellularity · Major evolutionary transitions · Higher-level individuality

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  • Heritable variation in multicellular traits
    Whether cluster-level traits pass reliably from parent group to offspring group.
    Darwinian evolution at the multicellular level requires that differences between clusters be transmitted to their offspring, and broad-sense heritability (H², the fraction of phenotypic variance that is genetic) measures it. Nothing guarantees this when a multicellular phenotype first arises: random variation among clusters, mixing of lineages, and growth noise could all erode it. The finding that none of them do in snowflake yeast, because deterministic branching growth leaves little variation among clusters of the same clone, is our central quantitative result on the point.
    In our work Ratcliff et al. (2015) measured H² = 0.84 for cluster size at reproduction, which the paper calls extraordinarily high even by the standards of extant clonal multicellular organisms; Herron et al. (2018) then showed mathematically that collective-level heritability usually exceeds particle-level heritability and comes 'for free' as a byproduct of group formation, and Zamani-Dahaj et al. (2023) confirmed that in engineered snowflake yeast.
    See also Snowflake yeast · Higher-level individuality · Multilevel selection · Cell packing · Mechanical fracture · ACE2 · Clonal multicellularity
  • Higher-level individuality
    The status of a group as a genuine Darwinian individual in its own right.
    A collective attains higher-level individuality when it reproduces, varies heritably in its own traits, and that variation affects its fitness. We ground this in Okasha's MLS2 framework and the Lewontin conditions, and treat individuality as a spectrum rather than a switch: collectives range from barely individual to fully individual, with complete fitness reorganization and division of labor at the far end. The research program is organized around one question, how incipient collectives acquire these properties, and the snowflake yeast system was built to answer it experimentally.
    In the MuLTEE Bozdag et al. (2023, Nature) is the first experimental demonstration that a simple clonal multicellular lineage undergoes sustained adaptation and evolves distinct group-level size and toughness traits under long-term selection, and Montrose et al. (2024, Science Advances) traced one of those group-beneficial phenotypes back through the Hsp90-Cdc28 axis into cell biology.
    See also Major evolutionary transitions · Multilevel selection · Heritable variation in multicellular traits · Clonal multicellularity · Unicellular bottleneck · Division of labor · Multicellular entrenchment · Morphological entanglement

M

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  • Major evolutionary transitions
    Rare events in which independent biological entities merge into a new, higher level of individuality.
    Formalized by Maynard Smith and Szathmary in 1995, the major transitions are the handful of events in which formerly independent entities became integrated into a higher-level unit: prokaryotic symbionts into eukaryotic cells, single cells into multicellular organisms, solitary insects into eusocial colonies. Their hallmark is a reorganization of fitness from the lower level to the higher one. This framework provides the theoretical basis for our work, and it supplies the questions we ask: how selection shifts from cells to organisms, how genetic conflict is resolved, and how division of labor evolves.
    In the MuLTEE The MuLTEE was built inside this framework rather than as a test of it. The experiment exists because the origin of multicellularity is one of the canonical transitions, and it provides a real-time observation of that transition happening de novo rather than a reconstruction from fossils and phylogenies.
    See also Higher-level individuality · Multilevel selection · Snowflake yeast · Clonal multicellularity · Unicellular bottleneck · Ratcheting traits · Complex multicellularity · Effective population size
  • Mechanical fracture
    Snowflake yeast reproduce by splitting apart under stress built up by their own growth.
    Fracture here is not accidental breakage. Cells multiplying inside a space-constrained cluster compress one another until accumulated stress exceeds the strength of a single intercellular chitin bond, and because the body is a branching tree, one bond failure is enough to divide the cluster. The smaller piece is the propagule, with a mean radius about 61 to 62 percent of the pre-fracture cluster, which means fracture happens deep in the interior where crowding is worst. Since one bond sets the threshold, the force at fracture stays roughly constant across cluster sizes, unlike bulk materials where it scales with cross-sectional area.
    In our work Because fracture is driven by growth-induced internal stress rather than external load, evolution can enlarge clusters by changing packing geometry instead of adhesion machinery, and that is the route snowflake yeast took; understanding fracture is therefore equivalent to understanding multicellular reproduction in this system (Jacobeen et al., 2018, Nature Physics).
    See also Cell packing · Snowflake yeast · Morphological entanglement · Heritable variation in multicellular traits · Coordinated cell division · Settling selection
  • Morphological entanglement
    Branches that grow into each other and become mechanically trapped, holding a body together.
    Entanglement is the physical interlocking of growing branched structures into configurations that cannot be reached or undone by translation and rotation alone. We use the term for entanglement produced by growth in living systems, rather than the engineered entanglement of nonliving materials. It gives branching organisms tough, fracture-resistant bodies with no developmental program and no specialized adhesive, turning growth itself into a source of higher-level material innovation.
    In the MuLTEE Anaerobic MuLTEE lineages evolved entangled branches that keep large groups intact even after many cell-cell bonds have fractured (Bozdag et al., 2023, Nature), and Day et al. (2024, Physical Review X) then showed the effect is largely insensitive to the geometry of branched trees and depends instead on how long growth continues in contact.
    See also Cell packing · Snowflake yeast · Mechanical fracture · Biophysical scaffolding · Higher-level individuality · Complex multicellularity
  • Multicellular entrenchment
    The evolutionary locking-in of multicellularity against reversion to single cells.
    Entrenchment is narrower than persistence. A lineage becomes entrenched when later mutations change the reversibility of the transition itself, making unicellular revertants rarer, less competitive, or both. Conlin et al. (2023) introduced the term in this sense, importing it from molecular evolution, where entrenchment describes substitutions that grow harder to reverse as epistatic context accumulates. The point is that originating a higher-level unit is not enough: a multicellular form that dissolves easily makes for a shallow transition, so stability becomes a criterion alongside variation, heredity, and group-level fitness.
    In the MuLTEE Conlin et al. (2023) showed in a digital-evolution system that reproductive division of labor causes entrenchment directly; whether long-term experimental evolution produces measurable entrenchment before obvious cell-type differentiation appears is listed as an open question for the MuLTEE.
    See also Ratcheting traits · Division of labor · Higher-level individuality · Major evolutionary transitions · Clonal multicellularity
  • Multilevel selection
    Natural selection acting at more than one level at once, on cells and on the groups they form.
    The framework distinguishes two cases. In MLS1 the trait of interest belongs to the particle, usually the cell, and group membership only shapes particle fitness. In MLS2 the collective itself is the unit whose traits, reproduction, and fitness matter, and collective-level heritability governs the response to selection. MLS2 is the mode relevant to major transitions. The distinction is not merely terminological: it determines which heritability measure is the one that predicts evolutionary change.
    In the MuLTEE Whether the balance shifts from MLS1 toward MLS2 as the MuLTEE organism becomes more integrated is still an open question for the experiment; the firmest anchors so far are H² = 0.84 for cluster-level traits (Ratcliff et al., 2015) and Pentz et al. (2023, eLife), where snowflake yeast gained fitness mainly during the settling phase while aggregative floc yeast gained theirs during growth.
    See also Higher-level individuality · Major evolutionary transitions · Heritable variation in multicellular traits · Clonal multicellularity · Snowflake yeast · Coordinated cell division

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  • Oxygen diffusion limitation
    Oxygen cannot diffuse fast enough to reach the interior cells of a large body.
    The constraint comes in two forms we keep separate. Environmental limitation is too little oxygen in the surrounding medium, so even peripheral cells are underfed. Anatomical limitation is plenty of oxygen outside but too long a diffusion path through metabolically active tissue to supply the interior, and it worsens geometrically as an organism grows. We call oxygen a double-edged sword, metabolically advantageous and geometrically punishing. Counterintuitively, abundant environmental oxygen can make matters worse, because more aerobic growth produces denser respiring tissue.
    In the MuLTEE Oxygen sets the direction of selection on multicellular size and not merely its ceiling: intermediate oxygen suppresses the evolution of large bodies (Bozdag et al., 2021, Nature Communications), under aerobic conditions competition for dissolved oxygen keeps small and large specialists coexisting (Pineau et al., 2024), and Wong et al. (2025) found that oxygen-binding proteins relieve the anatomical form of the limit most powerfully in large clusters at high oxygen.
    See also Size-niche diversification · Cell packing · Biophysical scaffolding · Complex multicellularity · Snowflake yeast

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  • Proteostatic tuning
    Adjusting the cell's protein-folding machinery to produce new heritable multicellular traits.
    Proteostatic tuning treats chaperone systems as something selection can adjust rather than as passive housekeeping. Reducing Hsp90 destabilizes the cyclin-dependent kinase Cdc28, which delays mitosis and elongates the cell, and elongated cells build larger and tougher groups. The argument broadens our major-transitions case: early multicellular innovation can come from tuning ancient conserved systems that reshape genotype-phenotype relationships, not only from mutations in structural genes or developmental circuits.
    In the MuLTEE Montrose et al. (2024, Science Advances) traced a repeatable MuLTEE route from Hsp90 down-regulation to Cdc28 to delayed mitosis to elongated cells to larger and tougher multicellular groups, showing that ancient protein-folding systems can be tuned to drive rapid evolution at a new level of biological individuality.
    See also Cell packing · Morphological entanglement · Snowflake yeast · Higher-level individuality · Coordinated cell division

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  • Ratcheting traits
    Traits that help cells inside a group but handicap them outside it, making reversion harder.
    A ratcheting trait raises fitness in the group context and lowers it in the ancestral free-living state. Once such traits accumulate, going back to unicellularity stops being one mutation away and becomes a many-step problem. Libby et al. (2016) separated two routes to the same outcome: mutations can make revertants less fit, or they can make reversion less accessible by mutation, and the two reinforce each other. The general rule Libby & Ratcliff state is that the more a trait makes cells in a cluster mutually reliant, the more it works as a ratchet.
    In our work The canonical example comes from the snowflake system: apoptosis is favored because it improves group reproduction, and the same trait makes an individual cell worse at surviving on its own (Libby & Ratcliff, 2014, Science).
    See also Multicellular entrenchment · Division of labor · Higher-level individuality · Major evolutionary transitions · Snowflake yeast

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  • Settling selection
    Selecting for larger size by keeping only the cells and clusters that sink fastest.
    Populations are grown in liquid, allowed to settle under gravity or spun briefly in a centrifuge, and only the bottom fraction is transferred to fresh medium. Larger and denser clusters sink faster, so the regime is directional selection on size. We chose it for tractability rather than ecological realism: settling is more replicable than predation, there is no predator to co-evolve or go extinct, and the strength of selection can be tuned by changing the settling time. Tong, Bozdag & Ratcliff (2022) later argued that sedimentation rate is in fact an evolvable ecological trait in aquatic organisms.
    In the MuLTEE Settling selection is the daily regime that drives the MuLTEE; in the founding experiment the protocol shifted from 45 minutes of standing settling to 10 seconds at 100 x g, and Ratcliff, Pentz & Travisano (2013) intensified it three times over 227 days, each time roughly halving survival, to keep selection strong as populations adapted.
    See also Snowflake yeast · Clonal multicellularity · Size-niche diversification · Mechanical fracture · Coordinated cell division · Unicellular bottleneck
  • Size-niche diversification
    Getting bigger creates new ecological niches, so differently sized lineages can coexist.
    John Tyler Bonner argued that multicellular size is itself ecologically generative. Larger and smaller bodies differ in oxygen access, growth rate, resource use, and settling survival, so a lineage that becomes multicellular can split along a growth-versus-survival trade-off and occupy two niches where there was one. The claim matters because it makes early multicellularity ecologically consequential before tissues, differentiated cell types, or circulatory systems exist: the origin of size expands niche space on its own.
    In the MuLTEE Pineau et al. (2024, Nature Ecology & Evolution) found that obligately aerobic MuLTEE populations diversified into deeply diverged small and large lineages whose coexistence is stably maintained by oxygen-mediated frequency-dependent selection, a direct experimental test of Bonner's size-niche hypothesis.
    See also Oxygen diffusion limitation · Snowflake yeast · Higher-level individuality · Settling selection · Complex multicellularity
  • Snowflake yeast
    Clusters of budding yeast whose daughter cells stay attached after division; the MuLTEE's model organism.
    Snowflake yeast are multicellular clusters of ordinary baker's yeast, Saccharomyces cerevisiae, formed when mother and daughter cells fail to separate after budding. Because clusters grow outward from one founding cell rather than assembling from free-living cells, every cell in a cluster is genetically identical. The clusters have a real multicellular life history, with a juvenile phase, determinate growth, and reproduction by releasing multicellular propagules. They can be evolved from a unicellular ancestor in the laboratory, which makes the first steps of a major evolutionary transition directly observable.
    In the MuLTEE Running snowflake yeast for roughly 3,000 generations under selection on group size showed that a simple clonal collective keeps adapting as a multicellular organism, with anaerobic lineages reaching millimetre scale and gaining more than 10,000-fold in group toughness (Bozdag et al., 2023, Nature).
    See also Settling selection · Clonal multicellularity · ACE2 · Cell packing · Mechanical fracture · Morphological entanglement · Higher-level individuality · Major evolutionary transitions

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  • Unicellular bottleneck
    A life cycle in which each new individual develops from a single cell.
    Animals develop from a zygote, plants from a spore or seed, volvocine algae from one gonidial cell. Passing through a single cell each generation means every organism starts with one genome, which limits genetic conflict among its cells, and the standard explanation for the bottleneck is exactly that conflict suppression. Ratcliff et al. (2013) offered an alternative that does not need conflict at all: unicellular propagules maximize how many offspring a cluster can produce, since fewer cells per propagule means more propagules, and a life-history model shows this holds even though single cells survive size-based selection poorly.
    In our work The bottleneck evolved de novo in Chlamydomonas under settling selection and proved adaptive for fecundity with no intercellular conflict present, so a trait long read as a conflict-suppression adaptation can instead arrive early by co-opting the ancestral unicell; Herron et al. (2019) added the boundary condition that under predation some lineages abandon it and release multicellular propagules instead.
    See also Clonal multicellularity · Higher-level individuality · Major evolutionary transitions · Complex multicellularity · Effective population size · ACE2 · Settling selection

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  • Whole-genome duplication
    Doubling the entire chromosome set, which in the MuLTEE evolved as an adaptation to size selection.
    Whole-genome duplication doubles an organism's chromosome complement. Polyploid genomes are intrinsically unstable, and laboratory evolution experiments normally see reversion to the lower ploidy. We use the term for tetraploid snowflake yeast arising spontaneously from diploid ancestors, and argue that selection on the immediate phenotypic effect of doubling, namely larger and longer cells, is enough to drive both its origin and its long-term maintenance without any prior genome-stabilizing mutations.
    In the MuLTEE Tong et al. (2025, Nature) found tetraploidy evolving convergently in 10 MuLTEE populations because doubling immediately makes cells and clusters larger under settling selection, and the same benefit maintained it over long timescales rather than reverting, which then opened new genetic routes to adaptation.
    See also Aneuploidy · Snowflake yeast · Settling selection · Cell packing