William C. Ratcliff

Many organisms exhibit branching morphologies that twist around each other and become entangled. Entanglement occurs when different objects interlock with each other, creating complex and often irreversible configurations. This physical phenomenon is well studied in nonliving materials, such as granular matter, polymers, and wires, where it has been shown that entanglement is highly sensitive to the geometry of the component parts. However, entanglement is not yet well understood in living systems, despite its presence in many organisms. In fact, recent work has shown that entanglement can evolve rapidly and play a crucial role in the evolution of tough, macroscopic multicellular groups. Here, through a combination of experiments, simulations, and numerical analyses, we show that growth generically facilitates entanglement for a broad range of geometries. We find that experimentally grown entangled …

The evolution of multicellular life spurred evolutionary radiations, fundamentally changing many of Earth’s ecosystems. Yet little is known about how early steps in the evolution of multicellularity affect eco-evolutionary dynamics. Through long-term experimental evolution, we observed niche partitioning and the adaptive divergence of two specialized lineages from a single multicellular ancestor. Over 715 daily transfers, snowflake yeast were subjected to selection for rapid growth, followed by selection favouring larger group size. Small and large cluster-forming lineages evolved from a monomorphic ancestor, coexisting for over ~4,300 generations, specializing on divergent aspects of a trade-off between growth rate and survival. Through modelling and experimentation, we demonstrate that coexistence is maintained by a trade-off between organismal size and competitiveness for dissolved oxygen. Taken together …

Whole-genome duplication (WGD) is widespread across eukaryotes and can promote adaptive evolution. However, given the instability of newly-formed polyploid genomes, understanding how WGDs arise in a population, persist, and underpin adaptations remains a challenge. Using our ongoing Multicellularity Long Term Evolution Experiment (MuLTEE), 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.

The evolution of multicellularity paved the way for the origin of complex life on Earth, but little is known about the mechanistic basis of early multicellular evolution. Here, we examine the molecular basis of multicellular adaptation in the multicellularity long-term evolution experiment (MuLTEE). We demonstrate that cellular elongation, a key adaptation underpinning increased biophysical toughness and organismal size, is convergently driven by down-regulation of the chaperone Hsp90. Mechanistically, Hsp90-mediated morphogenesis operates by destabilizing the cyclin-dependent kinase Cdc28, resulting in delayed mitosis and prolonged polarized growth. Reinstatement of Hsp90 or Cdc28 expression resulted in shortened cells that formed smaller groups with reduced multicellular fitness. Together, our results show how ancient protein folding systems can be tuned to drive rapid evolution at a new level of …

The Type VI Secretion System (T6SS) is a widespread and highly effective mechanism of microbial warfare; it confers the ability to efficiently kill susceptible cells within close proximity. Due to its large physical size, complexity, and ballistic basis for intoxication it has widely been assumed to incur significant growth costs in the absence of improved competitive outcomes. In this study, we precisely examine the fitness costs of constitutive T6SS firing in the bacterium Vibrio cholerae. We find that, contrary to expectations, constitutive use of T6SS has a negligible impact on growth, reducing growth fitness by 0.025 ± 0.5% (95% CI) relative to a T6SS- control. Mathematical modeling of microbial populations demonstrates that, due to clonal interference, constitutive expression of the T6SS will often be neutral, with little impact on evolutionary outcomes. Our findings underscore the importance of precisely measuring the fitness costs of microbial social behaviors, and help explain the prevalence of the T6SS across Gram negative bacteria.

“Complex multicellularity,” conventionally defined as large organisms with many specialized cell types, has evolved five times independently in eukaryotes, but never within prokaryotes. A number of hypotheses have been proposed to explain this phenomenon, most of which posit that eukaryotes evolved key traits (e.g., dynamic cytoskeletons, alternative mechanisms of gene regulation, or subcellular compartments) which were a necessary prerequisite for the evolution of complex multicellularity. Here, we propose an alternative, nonadaptive hypothesis for this broad macroevolutionary pattern. By binning cells into groups with finite genetic bottlenecks between generations, the evolution of multicellularity greatly reduces the effective population size (Ne) of cellular populations, increasing the role of genetic drift in evolutionary change. While both prokaryotes and eukaryotes experience this phenomenon, they have …

Macroalgae are multicellular, aquatic autotrophs that play vital roles in global climate maintenance and have diverse applications in biotechnology and eco-engineering, which are directly linked to their multicellularity phenotypes. However, their genomic diversity and the evolutionary mechanisms underlying multicellularity in these organisms remain uncharacterized. In this study, we sequenced 110 macroalgal genomes from diverse climates and phyla, and identified key genomic features that distinguish them from their microalgal relatives. Genes for cell adhesion, extracellular matrix formation, cell polarity, transport, and cell differentiation distinguish macroalgae from microalgae across all three major phyla, constituting conserved and unique gene sets supporting multicellular processes. Adhesome genes show phylum- and climate-specific expansions that may facilitate niche adaptation. Collectively, our study …

Phototrophic metabolism, the capture of light for energy, was a pivotal biological innovation that greatly increased the total energy available to the biosphere. Chlorophyll-based photosynthesis is the most familiar phototrophic metabolism, but retinal-based microbial rhodopsins transduce nearly as much light energy as chlorophyll does,1 via a simpler mechanism, and are found in far more taxonomic groups. Although this system has apparently spread widely via horizontal gene transfer,2,3,4 little is known about how rhodopsin genes (with phylogenetic origins within prokaryotes5,6) are horizontally acquired by eukaryotic cells with complex internal membrane architectures or the conditions under which they provide a fitness advantage. To address this knowledge gap, we sought to determine whether Saccharomyces cerevisiae, a heterotrophic yeast with no known evolutionary history of phototrophy, can function as …

Long-term experimental evolution in brewer's yeast reveals how the transition to simple multicellularity can drive ecological divergence and maintain diversity.

Many scientists approach speaking as they do writing a paper: an opportunity to present their data. But data without proper context is difficult to absorb. In this article, I describe a philosophy and set of heuristics for giving an engaging, narratively driven talk, inspired by the legendary documentaries of Sir David Attenborough.

The major transitions in evolution include events and processes that result in the emergence of new levels of biological individuality. For collectives to undergo Darwinian evolution, their traits must be heritable, but the emergence of higher-level heritability is poorly understood and has long been considered a stumbling block for nascent evolutionary transitions. Using analytical models, synthetic biology, and biologically-informed simulations, we explored the emergence of trait heritability during the evolution of multicellularity. Prior work on the evolution of multicellularity has asserted that substantial collective-level trait heritability either emerges only late in the transition or requires some evolutionary change subsequent to the formation of clonal multicellular groups. In a prior analytical model, we showed that collective-level heritability not only exists but is usually more heritable than the underlying cell-level trait upon which it is based, as soon as multicellular groups form. Here, we show that key assumptions and predictions of that model are borne out in a real engineered biological system, with important implications for the emergence of collective-level heritability.

While early multicellular lineages necessarily started out as relatively simple groups of cells, little is known about how they became Darwinian entities capable of sustained multicellular evolution, –. Here we investigate this with a multicellularity long-term evolution experiment, selecting for larger group size in the snowflake yeast (Saccharomyces cerevisiae) model system. Given the historical importance of oxygen limitation, our ongoing experiment consists of three metabolic treatments—anaerobic, obligately aerobic and mixotrophic yeast. After 600 rounds of selection, snowflake yeast in the anaerobic treatment group evolved to be macroscopic, becoming around 2 × 104 times larger (approximately mm scale) and about 104-fold more biophysically tough, while retaining a clonal multicellular life cycle. This occurred through biophysical adaptation—evolution of increasingly elongate cells that initially reduced the …

The Type VI Secretion System (T6SS) is a nano-harpoon used by many bacteria to inject toxins into neighboring cells. While much is understood about mechanisms of T6SS-mediated toxicity, less is known about the ways that competitors can defend themselves against this attack, especially in the absence of their own T6SS. Here we subjected eight replicate populations of Escherichia coli to T6SS attack by Vibrio cholerae. Over ∼500 generations of competition, isolates of the E. coli populations evolved to survive T6SS attack an average of 27-fold better, through two convergently evolved pathways: apaH was mutated in six of the eight replicate populations, while the other two populations each had mutations in both yejM and yjeP. However, the mutations we identified are pleiotropic, reducing cellular growth rates, and increasing susceptibility to antibiotics and elevated pH. These trade-offs help us understand …

Multicellular organisms exhibit a fascinating diversity of life cycles, but little is known about the factors governing life-cycle evolution. New studies of wild yeast and cyanobacteria provide insight into how and why facultative multicellular life cycles arise.

The evolution of multicellularity represents a major transition in life’s history, enabling the rise of complex organisms. Multicellular groups can evolve through multiple developmental modes, but a common step is the formation of permanent cell-cell attachments after division. The characteristics of the multicellular morphology which emerges has profound consequences for the subsequent evolution of a nascent multicellular lineage, but little prior work has examined these dynamics directly. Here we examine a widespread yet understudied emergent multicellular morphology: cuboidal packing. Extinct and extant multicellular organisms across the tree of life have evolved to form groups in which spherical cells divide but remain attached, forming approximately cubic subunits. To experimentally investigate the evolution of cuboidal cell packing, we used settling selection to favor the evolution of simple multicellularity in unicellular, spherical Schizosaccharomyces pombe yeast. Multicellular clusters with cuboidal organization rapidly evolved, displacing the unicellular ancestor. These clusters displayed key hallmarks of an evolutionary transition in individuality: groups possess an emergent life cycle driven by physical fracture, group size is heritable, and they respond to group-level selection via multicellular adaptation. In 2/5 lineages, group formation was driven by mutations in the ACE2 gene, preventing daughter cell separation after division. Remarkably, ACE2 mutations also underlie the transition to multicellularity in Saccharomyces cerevisiae and C. galabrata, lineages last shared a common ancestor >300 million years ago. Our results provide …

Oxygen availability is a key factor in the evolution of multicellularity, as larger and more sophisticated organisms often require mechanisms allowing efficient oxygen delivery to their tissues. One such mechanism is the presence of oxygen-binding proteins, such as globins and hemerythrins, which arose in the ancestor of bilaterian animals. Despite their importance, the precise mechanisms by which oxygen-binding proteins influenced the early stages of multicellular evolution under varying environmental oxygen levels are not yet clear. We addressed this knowledge gap by heterologously expressing the oxygen binding proteins myoglobin and myohemerythrin in snowflake yeast, a model system of simple, undifferentiated multicellularity. These proteins increased the depth and rate of oxygen diffusion, increasing the fitness of snowflake yeast growing aerobically. Experiments show that, paradoxically, oxygen-binding proteins confer a greater fitness benefit for larger organisms under high, not low, O2 conditions. We show via biophysical modeling that this is because facilitated diffusion is more efficient when oxygen is abundant, transporting a greater quantity of O2 which can be used for metabolism. By alleviating anatomical diffusion limitations to oxygen consumption, the evolution of O2-binding proteins in the oxygen-rich Neoproterozoic may have been a key breakthrough enabling the evolution of increasingly large, complex multicellular metazoan lineages.

A key step in the evolutionary transition to multicellularity is the origin of multicellular groups as biological individuals capable of adaptation. Comparative work, supported by theory, suggests clonal development should facilitate this transition, although this hypothesis has never been tested in a single model system. We evolved 20 replicate populations of otherwise isogenic clonally reproducing ‘snowflake’yeast (Δace2/∆ ace2) and aggregative ‘floc’yeast (GAL1p:: FLO1/GAL1p:: FLO1) with daily selection for rapid growth in liquid media, which favors faster cell division, followed by selection for rapid sedimentation, which favors larger multicellular groups. While both genotypes adapted to this regime, growing faster and having higher survival during the groupselection phase, there was a stark difference in evolutionary dynamics. Aggregative floc yeast obtained nearly all their increased fitness from faster growth, not improved group survival; indicating that selection acted primarily at the level of cells. In contrast, clonal snowflake yeast mainly benefited from higher group-dependent fitness, indicating a shift in the level of Darwinian individuality from cells to groups. Through genome sequencing and mathematical modeling, we show that the genetic bottlenecks in a clonal life cycle also drive much higher rates of genetic drift—a result with complex implications for this evolutionary transition. Our results highlight the central role that early multicellular life cycles play in the process of multicellular adaptation.

Simple multicellularity evolves readily in diverse unicellular species, but nascent multicellular groups are prone to reversion to unicellularity. Successful transitions to multicellularity therefore require subsequent mutations that promote the entrenchment of the higher-level unit, stabilizing it through time. Here we explore the causes of entrenchment using digital evolution. When faced with a trade-off between cellular metabolic productivity and information fidelity, digital "multicells" often evolve reproductive division of labor. Because digital "unicells" cannot circumvent this trade-off, unicellular revertants tend to exhibit low fitness relative to their differentiated multicellular ancestors. Thus, division of labor can drive the entrenchment of multicellularity. More generally, division of labor may play a crucial role in major transitions, enriching the complexity and functionality of higher-level units while enhancing their evolutionary stability.

Where is the line between a robot and an organism? Does such a line even exist? Definitions, even those that are fundamental, have long been problematic in biology. For example, we still argue about how to define species, 1 organisms, 2 Darwinian fitness, 3 or even life itself. 4 Don’t believe me? Go on Twitter and defend any definition of the mentioned concepts as the One True Definition! It should come as little surprise that pioneering work in biological robotics is as controversial as it is exciting. Take for example the article published in December2021 in the Proceedings of the National Academy of Sciences by Sam Kreigman and Douglas Blackiston at Tufts University and colleagues. 5 This article, entitled ‘‘Kinematic self-replication in reconfigurable organisms,’’5 is the third installment of the authors’‘‘xenobots’’series. In their prior study, this interdisciplinary team of roboticists, developmental biologists, and …

The initial transition from competing individuals to cooperative groups is difficult to overcome. Previous studies have looked into this changeover by examining populations of Pseudomonas aeruginosa. These bacterial species were able to aggregate in the presence of extracellular polymers and this provided the groups with an increased antibiotic tolerance. This behavior has not been shown in Vibrio cholerae. V. cholerae encodes a neighbor-killing mechanism called the Type VI Secretion System (T6SS) but it was still found to be able to aggregate just like P. aeruginosa. We hypothesize that despite containing the T6SS mechanism, V. cholerae is still able to form cooperative bacterial communities with T6SS-susceptible cells due to how aggregations limit the mobility of individual V. cholerae cells. To test this, we will grow T6SS cells with susceptible cells in a medium containing extracellular polymers. Then we will …