William C. Ratcliff

Experimental evolution of multicellularity

Multicellularity was one of the most significant innovations in the history of life, but its initial evolution remains poorly understood. Using experimental evolution, we show that key steps in this transition could have occurred quickly. We subjected the unicellular yeast Saccharomyces cerevisiae to an environment in which we expected multicellularity to be adaptive. We observed the rapid evolution of clustering genotypes that display a novel multicellular life history characterized by reproduction via multicellular propagules, a juvenile phase, and determinate growth. The multicellular clusters are uniclonal, minimizing within-cluster genetic conflicts of interest. Simple among-cell division of labor rapidly evolved. Early multicellular strains were composed of physiologically similar cells, but these subsequently evolved higher rates of programmed cell death (apoptosis), an adaptation that increases propagule production. These results show that key aspects of multicellular complexity, a subject of central importance to biology, can readily evolve from unicellular eukaryotes.

Poly-3-hydroxybutyrate (PHB) supports survival and reproduction in starving rhizobia.

The carbon that rhizobia in root nodules receive from their host powers both N(2) fixation, which mainly benefits the host, and rhizobium reproduction. Rhizobia also store energy in the lipid poly-3-hydroxybutyrate (PHB), which may enhance rhizobium survival when they are carbon limited, either in nodules or in the soil between hosts. There can be a conflict of interest between rhizobia and legumes over the rate of PHB accumulation, due to a metabolic tradeoff between N(2) fixation and PHB accumulation. To quantify the benefits of PHB to carbon-limited rhizobia, populations of genetically uniform rhizobia with high vs. low PHB (confirmed by flow cytometry) were generated by fractionating Sinorhizobium meliloti via density gradient centrifugation, and also by harvesting cells at early vs. late stationary phase. These rhizobia were starved for 165 days. PHB use during starvation was highly predictive of both initial reproduction and long-term population maintenance. Cultured S. meliloti accumulated enough PHB to triple their initial population size when starved, and to persist for c. 150 days before the population fell below its initial value. During the first 21 days of nodule growth, undifferentiated S. meliloti within alfalfa nodules accumulated enough PHB to support significant increases in reproduction and survival during starvation.

Experimental evolution of an alternating uni- and multicellular life cycle in Chlamydomonas reinhardtii

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.

Individual-level bet hedging in the bacterium Sinorhizobium meliloti.

The expression of phenotypic variability can enhance geometric mean fitness and act as a bet-hedging strategy in unpredictable environments. Metazoan bet hedging usually involves phenotypic diversification among an individuals offspring, such as differences in seed dormancy. Virtually all known microbial bet-hedging strategies, in contrast, rely on low-probability stochastic switching of a heritable phenotype by individual cells in a clonal group. This is less effective at generating within-group diversity when group size is small. Here we describe a novel microbial bet-hedging behavior that resembles individual-level metazoan bet hedging. Sinorhizobium meliloti stores carbon and energy in poly-3-hydroxybutyrate (PHB) as a contingency against carbon scarcity. We show that, when starved, dividing S. meliloti bet hedge by forming two daughter cells with different phenotypes. These have high and low PHB levels and are suited to long- and short-term starvation, respectively. The low-PHB cells have greater competitiveness for resources, whereas the high-PHB cells can survive for over a year without food, perhaps until a legume host is next available.

Alternative Actions for Antibiotics

Compounds recognized as having antibiotic functions may have other possible roles in microbial interactions. Microbes generate signals, which coordinate mutually beneficial activities (1). They also produce antibiotics that kill prey, suppress competitors, or deter predators (2). Recent observations have led to the view that antibiotics often act as mutually beneficial signals (3–6). Exposure to sublethal concentrations of antibiotics can indeed alter microbial metabolism and even change behavior in beneficial ways, triggering reactions such as fleeing or hiding within the protective environment of a microbial aggregate (biofilm). But the weapon-signal dichotomy of functions for these compounds is a false one—there may be other possible information-related actions of naturally produced antibiotics: cues and manipulation.

Tempo and mode of multicellular adaptation in experimentally evolved Saccharomyces cerevisiae.

Multicellular complexity is a central topic in biology, but the evolutionary processes underlying its origin are difficult to study and remain poorly understood. Here we use experimental evolution to investigate the tempo and mode of multicellular adaptation during a de novo evolutionary transition to multicellularity. Multicelled “snowflake” yeast evolved from a unicellular ancestor after 7 days of selection for faster settling through liquid media. Over the next 220 days, snowflake yeast evolved to settle 44% more quickly. Throughout the experiment the clusters evolved faster settling by three distinct modes. The number of cells per cluster increased from a mean of 42 cells after 7 days of selection to 114 cells after 227 days. Between days 28 and 65, larger clusters evolved via a twofold increase in the mass of individual cells. By day 227, snowflake yeast evolved to form more hydrodynamic clusters that settle more quickly for their size than ancestral strains. The timing and nature of adaptation in our experiment suggests that costs associated with large cluster size favor novel multicellular adaptations, increasing organismal complexity.

Geometry Shapes Evolution of Early Multicellularity

Organisms have increased in complexity through a series of major evolutionary transitions, in which formerly autonomous entities become parts of a novel higher-level entity. One intriguing feature of the higher-level entity after some major transitions is a division of reproductive labor among its lower-level units in which reproduction is the sole responsibility of a subset of units. Although it can have clear benefits once established, it is unknown how such reproductive division of labor originates. We consider a recent evolution experiment on the yeast Saccharomyces cerevisiae as a unique platform to address the issue of reproductive differentiation during an evolutionary transition in individuality. In the experiment, independent yeast lineages evolved a multicellular “snowflake-like” cluster formed in response to gravity selection. Shortly after the evolution of clusters, the yeast evolved higher rates of cell death. While cell death enables clusters to split apart and form new groups, it also reduces their performance in the face of gravity selection. To understand the selective value of increased cell death, we create a mathematical model of the cellular arrangement within snowflake yeast clusters. The model reveals that the mechanism of cell death and the geometry of the snowflake interact in complex, evolutionarily important ways. We find that the organization of snowflake yeast imposes powerful limitations on the available space for new cell growth. By dying more frequently, cells in clusters avoid encountering space limitations, and, paradoxically, reach higher numbers. In addition, selection for particular group sizes can explain the increased rate of apoptosis both in terms of total cell number and total numbers of collectives. Thus, by considering the geometry of a primitive multicellular organism we can gain insight into the initial emergence of reproductive division of labor during an evolutionary transition in individuality.

An oscillating tragedy of the commons in replicator dynamics with game-environment feedback

Significance Classical game theory addresses how individuals make decisions given suitable incentives, for example, whether to use a commons rapaciously or with restraint. However, classical game theory does not typically address the consequences of individual actions that reshape the environment over the long term. Here, we propose a unified approach to analyze and understand the coupled evolution of strategies and the environment. We revisit the originating tragedy of the commons example and evaluate how overuse of a commons resource changes incentives for future action. In doing so, we identify an oscillatory tragedy of the commons in which the system cycles between deplete and replete environments and cooperation and defection behavior, highlighting new challenges for control and influence of feedback-evolving games. A tragedy of the commons occurs when individuals take actions to maximize their payoffs even as their combined payoff is less than the global maximum had the players coordinated. The originating example is that of overgrazing of common pasture lands. In game-theoretic treatments of this example, there is rarely consideration of how individual behavior subsequently modifies the commons and associated payoffs. Here, we generalize evolutionary game theory by proposing a class of replicator dynamics with feedback-evolving games in which environment-dependent payoffs and strategies coevolve. We initially apply our formulation to a system in which the payoffs favor unilateral defection and cooperation, given replete and depleted environments, respectively. Using this approach, we identify and characterize a class of dynamics: an oscillatory tragedy of the commons in which the system cycles between deplete and replete environmental states and cooperation and defection behavior states. We generalize the approach to consider outcomes given all possible rational choices of individual behavior in the depleted state when defection is favored in the replete state. In so doing, we find that incentivizing cooperation when others defect in the depleted state is necessary to avert the tragedy of the commons. In closing, we propose directions for the study of control and influence in games in which individual actions exert a substantive effect on the environmental state.

Bacterial persistence and bet hedging in Sinorhizobium meliloti.

We recently described a novel bet-hedging mechanism in which the bacterium Sinorhizobium meliloti responds to starvation by forming two discrete cell types via cell division. The old-pole daughter cell retains most of the resource, polyhydroxybutyrate (PHB) and is capable of surviving long-term starvation, while the low-PHB, new-pole daughter cell is capable of quickly resuming growth when starvation ends. Here we present additional data showing that the high-PHB, old-pole cells are similar to bacterial persisters, characterized by metabolic dormancy and antibiotic tolerance. Using two independent methods, we generated clonal populations of S. meliloti that varied in the frequency of the high- and low-PHB phenotypes, and then challenged these populations with ampicillin. Populations containing more high-PHB cells were significantly more antibiotic-tolerant. In a separate experiment, we used GFP fluorescence as a marker of overall metabolic activity. After 24 hours of starvation, new-pole cells were 64% brighter than their old-pole sister cells, demonstrating that the divergence in metabolic rate is rapid.

Ratcheting the evolution of multicellularity

Traits that entrench cells in a group lifestyle may pave the way for complexity Multicellularity is one of the major transitions that allowed the evolution of large, complex organisms, fundamentally reshaping Earths ecology (1). Early steps in this process remain poorly resolved, because known transitions occurred hundreds of millions of years ago (2) and few transitional forms persist. It is generally accepted that the first steps toward multicellularity were the formation of cellular clusters, followed by the success or failure of those clusters depending on their traits. As clusters of cells adapted, cells lost their evolutionary autonomy, becoming mutually reliant parts in a new higher-level whole (1, 3). This transition may be facilitated by a “ratcheting” process in which cells adopt traits that entrench them in a group lifestyle, stabilizing the group and paving the way for the evolution of multicellular complexity.