Research Evolution of multicellularity, bet hedging and microbial cooperation Multicellularity We evolve simple multicellular organisms in the lab The evolution of multicellular organisms from unicellular ancestors was critical to the evolution of large, complex organisms. While multicellularity has evolved more than 25 times independently during the last 3.5 billion years on Earth, the first steps in this transition remain poorly understood. By evolving novel multicellularity in the lab, we find that the transition to multicellularity may be less constrained than previously thought. We exposed the unicellular yeast Saccharomyces cerevisiae to selection for rapid settling through liquid media, favoring strains that evolved greater size. In all 10 replicate populations, ‘snowflake’ yeast clusters (see left picture) evolved. Unlike flocculating yeast, snowflake yeast adhere to one another only through mother-daughter cell adhesion, resulting in genetic uniformity among the cells in the cluster. Snowflake yeast display a novel multicellular life history: they have determinate growth, reproduce by making multicellular propagules, and these propagules are functionally juvenile (they have to grow to their parent’s size before they can reproduce themselves). We found that once snowflake clusters evolve, whole clusters become the unit of selection (they either make it to the bottom of the tube and survive, or fail to do so and perish). As a result, we see the evolution of multicellular traits, like changes in cluster morphology that speed settling and growth, and even a simple form of division of labor through programmed cell death (PCD). Our large-cluster forming snowflake yeast strains evolve higher rates of PCD. Dead cells act as break-points within the cluster, allowing snowflake yeast to regulate the number and size of offspring they produce. We demonstrated that this can be adaptive in these two modeling papers (one and two). See some criticisms of our 2012 PNAS paper and my responses here. In our 2013 Evolution paper, we showed that cluster-level adaptation takes place in several discrete phases. First, clusters evolve to settle faster simply by increasing the number of cells per group. Next, we see an increase in the size of individual cells (increasing cluster size), and the evolution of elevated programmed cell death. Finally, we see the evolution of more hydrodynamic clusters that settle more efficiently. We believe that this pattern reflects a key constraint: large clusters grow slowly (diffusion limitation), favoring traits that increase settling speed without reducing growth rates. So how hard is it really to get a snowflake yeast cluster? It turns out to be remarkably easy! In our 2015 Nature Communications paper,  we show that knocking out a single gene (ACE2) is sufficient to transform a unicellular yeast into a snowflake. ACE2 regulates daughter-cell separation after mitosis, so this mutation causes daughter cells to remain attached to mother cells after budding. This simple microevolutionary change turns out to have profound macroevolutionary consequences. Snowflake yeast clusters are geometrically elegant. In fact, the distribution of cells within a cluster follows predictions from Pascal’s triangle (figure to the right). This simple growth form has a few important properties. First, it results in clonal development, because snowflake yeast clusters undergo unicellular genetic bottlenecks at reproduction, and the only way that cells can join a cluster is to be born into them. This limits the potential for genetic conflict to erode multicellular complexity. Second, it increases the heritability of multicellular traits, exposing the cluster-level effects of every de novo mutation to selection. Thus, knocking out a single gene both results in the formation of clusters and also provides the basis upon which the ability to evolve as a multicellular individual is founded. Indeed, preliminary experiments suggest that snowflake yeast have a substantial advantage over yeast that form chimeric clusters through aggregation. One previously unexpected consequence of multicellular adaptation is what Eric Libby and I have termed the ‘ratcheting’ hypothesis. As we describe in our perspective in Science, traits evolved by cells early in the transition to multicellularity that are adaptive in clusters, but maladaptive to single cells, may entrench the lineage in a multicellular way of life. Cutting off the unicellular escape route may facilitate the evolution of increased multicellular complexity by maintaining strong selection on multicellular traits. One example of ratcheting in snowflake yeast may be elevated rates of programmed cell death (left), which can increase the fitness of cells in snowflake clusters, but should be costly to single cells not living in clusters. In our 2016 Phil. Trans. Biol. follow-up, we examine how ratcheting can stabilize the transition to multicellularity in more detail. We show that ratcheting can evolve in two ways: cells can evolve traits in a multicellular context that are costly to isolated cells (like PCD), or cells in groups can evolve traits that directly limit the rate at which they revert to unicellularity. One of the neatest aspects of the paper is that we show that these two traits can interact synergistically- the presence of either kind of ratcheting trait selects for the other. This has a pretty intuitive explanation: if cells in multicellular groups evolve traits that are costly in a single-cell context (type 1 ratcheting), then evolving reversion-limiting traits (type 2 ratcheting) is good. And in the opposite case, if reversion is rare, then it frees up the lineage to evolve traits in a multicellular context that are very costly to isolated cells. The take-away message from this paper is that it's pretty easy, at least in theory world, for a lineage that switches between uni and multicellular states to evolve traits that lock them into a multicellular lifestyle- an important step for evolving complex multicellular phenotypes. In collaboration with physicist Peter Yunker, I’ve been exploring the physical basis of multicellular adaptation in snowflake yeast. In our 2017 Nature Physics paper, we show snowflake yeast figure out physics of cellular packing in just a few months which took humans hundreds of years to work out, allowing them to evolve larger size. In broad terms, we're examining a fundamental (but neglected) phase in the evolution of multicellularity, in which incipient groups of cells evolve tougher bodies that can withstand forces acting on long (multicellular) length scales. To them, this is a totally novel evolutionary challenge. To evolve tougher bodies, snowflake yeast end up evolving more elongate cells, which reduces the intensity of packing among cells in the cluster interior, reducing cell-cell stress and resulting in larger clusters. I really like this figure (right), in which we compress individual clusters with an AFM and see how much energy it takes before the cluster fragments. For the ancestor with more round cells (blue), larger clusters rapidly become more brittle, while the elongated cell strain can tolerate a lot more energy input before fragmenting. Interestingly, the inferred spontaneous size at fragmentation (open black circles) is within 5% of the mean cluster size at stationary phase for each strain! We have some 3D modeling in here that neatly complements these results, too. Fragmentation does something else that's neat: it creates a life cycle. Life cycles are super important for early multicellular critters- reproduction is an absolutely essential part of the Darwinian algorithm. In a recent paper in a great issue of Philosophical Transactions of the Royal Society, we (Matthew Herron, Eric Libby, and Peter Conlin) show how early life cycles can shepherd the fragile beginnings of a major transition. We also use modeling to examine how different early life cycles affect the rate of fixation of different kinds of beneficial mutations. The life cycle which arises in snowflake yeast for 'free' (that is, as a consequence of physics, see above!), is nearly ideal for spurring a major transition: regular genetic bottlenecks limit the potential for within-group evolution, their canalized growth form helps with the emergence of heritable multicellular traits from mutations that only directly affect the properties of an individual cell, and their life cycle, which lacks a persistent unicellular phase, favors the fixation of 'ratcheting' mutations that limit evolutionary reversion to the pre-ETI state. The early evolution of multicellularity is essentially an applied physics problem: to evolve a body, a group of cells must evolve new biophysics. Fortunately, physical constraints play a key role in generating a multicellular life cycle that allows groups of cells to be effective Darwinian Individuals, thus allowing them to evolve tougher bodies. Cool, eh? In collaboration with Matthew Herron, Frank Rosenzweig and Michael Travisano, we have been pursuing similar experiments in the unicellular green alga Chlamydomonas reinhardii. This alga is closely related to the multicellular volvocine algae, allowing us to compare experimentally- evolved traits to those that occur in nature. In a pilot experiment (Ratcliff et al, 2014), we observe the evolution of a novel multicellular life cycle. Cells form clusters by producing a viscous extracellular matrix. After transfer to fresh medium, motile cells burst from the cluster, then settle down and form new clusters. This demonstrates that a unicellular genetic bottleneck, a trait that strongly facilitates multicellular-level adaptation, can arise early in this evolutionary transition and in the absence of selection for among-cell conflict mediation. We are following up on this work using predators (i.e., Paramecium and rotifers) to select for increased size, a more ecologically-realistic selective agent than centrifugation. Next steps Now that we have a model system spanning the transition from single-celled to simple multicellular organisms, we’re uniquely suited to research several aspects of this evolutionary process. For example, we’re studying how cells evolve into parts of a novel multicellular individual, and how multicellular development can evolve from scratch. We are also working to determine the genetic basis of emergent multicellular traits, and how the function, physically, to give rise to novel adaptations. Snowflake with red dead cells Snowflake yeast reproduction