By nature of their small size, dense growth and frequent need for extracellular metabolism, microbes face persistent public goods dilemmas. Genetic assortment is the only general solution stabilizing cooperation, but all known mechanisms structuring microbial populations depend on the availability of free space, an often unrealistic constraint. Here we describe a class of self-organization that operates within densely packed bacterial populations. Through mathematical modelling and experiments with Vibrio cholerae, we show how killing adjacent competitors via the Type VI secretion system (T6SS) precipitates phase separation via the ‘Model A' universality class of order-disorder transition mediated by killing. We mathematically demonstrate that T6SS-mediated killing should favour the evolution of public goods cooperation, and empirically support this prediction using a phylogenetic comparative analysis. This work illustrates the twin role played by the T6SS, dealing death to local competitors while simultaneously creating conditions potentially favouring the evolution of cooperation with kin.

The transition to multicellularity enabled the evolution of large, complex organisms, but early steps in this transition remain poorly understood. Here we show that multicellular complexity, including development from a single cell, can evolve rapidly in a unicellular organism that has never had a multicellular ancestor. We subject the alga Chlamydomonas reinhardtii to conditions that favour multicellularity, resulting in the evolution of a multicellular life cycle in which clusters reproduce via motile unicellular propagules. While a single-cell genetic bottleneck during ontogeny is widely regarded as an adaptation to limit among-cell conflict, its appearance very early in this transition suggests that it did not evolve for this purpose. Instead, we find that unicellular propagules are adaptive even in the absence of intercellular conflict, maximizing cluster-level fecundity. These results demonstrate that the unicellular bottleneck, a trait essential for evolving multicellular complexity, can arise rapidly via co-option of the ancestral unicellular form.

The evolution of multicellularity set the stage for sustained increases in organismal complexity [1][2][3][4][5] . However, a fundamental aspect of this transition remains largely unknown: how do simple clusters of cells evolve increased size when confronted by forces capable of breaking intracellular bonds? Here we show that multicellular snowflake yeast clusters 6-8 fracture due to crowding-induced mechanical stress. Over seven weeks (~291 generations) of daily selection for large size, snowflake clusters evolve to increase their radius 1.7-fold by reducing the accumulation of internal stress. During this period, cells within the clusters evolve to be more elongated, concomitant with a decrease in the cellular volume fraction of the clusters. The associated increase in free space reduces the internal stress caused by cellular growth, thus delaying fracture and increasing cluster size. This work demonstrates how readily natural selection finds simple, physical solutions to spatial constraints that limit the evolution of group size-a fundamental step in the evolution of multicellularity.The first step in the transition to multicellularity-prior to the origin of cellular division of labour, genetically regulated development and complex multicellular forms-was the evolution of simple multicellular clusters [1][2][3][4][5] . Long before simple clusters of cells can evolve traits characteristic of complex multicellularity, they must contend with physical forces-both internal and external-that are capable of breaking cell-cell bonds and thus limit cluster size. This physical challenge is critical for several reasons. First, large size is a likely prerequisite to the evolution of complex multicellularity 2,3 . Second, these forces act on long length scales that were probably irrelevant to a single-cell ancestor, and are thus evolutionarily novel. Finally, it is unclear how simple multicellular clusters that do not yet possess genetically regulated developmental systems can evolve novel multicellular morphology.Direct experimental investigation of the early steps in the transition to multicellularity has been challenging, largely because these transitions occurred long ago, and the evolutionary path to multicellularity has been obscured by extinction in most extant lineages 9,10 . Recently, however, this constraint has been circumvented through experimental evolution of novel multicellular organisms [6][7][8]11 , genetic reconstruction of early events 12 and experiments comparing extant multicellular taxa with their unicellular relatives 13,14 . To examine the biophysical basis of the evolution of increased size in a nascent multicellular organism, we employed the tractable 'snowflake' yeast model system [6][7][8] . Multicellular snowflake clusters evolved from the unicellular baker's yeast Saccharomyces cerevisiae under daily selection for rapid settling speed in liquid media 6 . The resulting snowflake growth form is the consequence of a single mutation in the ACE2 gene . This mutation prevents cell separation after ...

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