Improved use of a public good selects for the evolution of undifferentiated multicellularity

RESEARCH ARTICLE elife.elifesciences.org Improved use of a public good selects for the evolution of undifferentiated multicellularity John H Koschwanez1*, Kevin R Foster2, Andrew W Murray1 FAS Center for Systems Biology and Department of Molecular and Cellular Biology, Harvard University, Cambridge, United States; 2Department of Zoology, University of Oxford, Oxford, United Kingdom 1 Abstract We do not know how or why multicellularity evolved. We used the budding yeast, Saccharomyces cerevisiae, to ask whether nutrients that must be digested extracellularly select for the evolution of undifferentiated multicellularity. Because yeast use invertase to hydrolyze sucrose extracellularly and import the resulting monosaccharides, single cells cannot grow at low cell and sucrose concentrations. Three engineered strategies overcame this problem: forming multicellular clumps, importing sucrose before hydrolysis, and increasing invertase expression. We evolved populations in low sucrose to ask which strategy they would adopt. Of 12 successful clones, 11 formed multicellular clumps through incomplete cell separation, 10 increased invertase expression, none imported sucrose, and 11 increased hexose transporter expression, a strategy we had not engineered. Identifying causal mutations revealed genes and pathways, which frequently contributed to the evolved phenotype. Our study shows that combining rational design with experimental evolution can help evaluate hypotheses about evolutionary strategies. DOI: 10.7554/eLife.00367.001 *For correspondence: Competing interests: The authors declare that no competing interests exist. Funding: See page 24 Received: 01 November 2012 Accepted: 14 February 2013 Published: 02 April 2013 Reviewing editor: Diethard Tautz, Max Planck Institute for Evolutionary Biology, Germany Copyright Koschwanez et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. Introduction Multicellular organisms have evolved from a unicellular ancestor at least 25 times (Grosberg and Strathmann, 2007), but we know little about what selected for the simplest form of multicellularity: an undifferentiated clump of cells produced by the repeated division of a single cell. Two driving forces have been proposed, protection from a variety of factors (including predation [Kessin et al., 1996], environmental stress [Smukalla et al., 2008], and phagocytosis [Boraas et al., 1998]) and more efficient nutrient usage (Dworkin, 1972; Pfeiffer and Bonhoeffer, 2003; Koschwanez et al., 2011; Alegado et al., 2012). In earlier work, we showed that sharing public goods favors clumps over isolated cells and proposed that sharing could have selected for simple multicellularity (Koschwanez et al., 2011). The budding yeast, Saccharomyces cerevisiae, utilizes sucrose by secreting invertase (Dodyk and Rothstein, 1964; Carlson et al., 1981). Over 95% of this enzyme remains in the cell wall (Esmon et al., 1987; Tammi et al., 1987), where it hydrolyzes sucrose into glucose and fructose, which are imported into the cell by a variety of hexose transporters (Meijer et al., 1996; Reifenberger et al., 1997). Lab yeast strains cannot grow from low density in low concentrations of sucrose because of diffusion: each cell captures only a small fraction of the sugars that sucrose hydrolysis releases, and the molecules released by other, distant cells are at very low concentration. As a result, cells cannot capture enough glucose and fructose to grow. Forming multicellular clumps overcomes this failure; cells in a clump can capture glucose and fructose diffusing from their neighbors and grow in concentrations of sucrose where low concentrations of individual cells cannot. Koschwanez et al. eLife 2013;2:e00367. DOI: 10.7554/eLife.00367 1 of 27 Research article Genomics and evolutionary biology eLife digest Life first appeared on Earth more than 3 billion years ago in the form of single-celled microorganisms. The diverse array of complex life forms that we see today evolved from these humble beginnings, but it is not clear what triggered the evolution of multicellular organisms from single cells. One of the simplest multicellular eukaryotes is the yeast, Saccharomyces cerevisiae—a fungus that has been used for centuries in baking and brewing and, more recently, as a model organism in molecular biology. Yeast cells feed on sugar (sucrose), but are unable to absorb it directly from their surroundings. Instead they secrete an enzyme called invertase, which breaks down the sucrose into simpler components that cells can take up with the help of sugar transporters. However, single yeast cells living in a low-sucrose environment face a problem: most of the simple sugars that they produce diffuse out of reach. To overcome this difficulty, the cells could form multicellular clumps, which would enable each cell to consume the sugars that drift away from its neighbours. Alternatively, the cells could increase their production of invertase, or they could begin to take up sucrose directly. Using genetic engineering, Koschwanez et al. produced three strains of yeast, each with one of these traits, and confirmed that all three strategies do indeed help fungi to grow in low sucrose. But could any of these traits evolve spontaneously? To test this possibility, Koschwanez et al. introduced wild-type yeast cells into a low-sucrose environment and studied any populations of cells that managed to survive. Of 12 that did, 11 had acquired the ability to form multicellular clumps, while 10 had increased their expression of invertase. Surprisingly, none had evolved the ability to import sucrose. However, 11 of the populations that survived also displayed an adaptation that the researchers had not predicted beforehand: they all expressed higher levels of the sugar transporters that take up sucrose breakdown products. The work of Koschwanez et al. suggests that the benefits of being able to share invertase and, therefore, simple sugars, may have driven the evolution of multicellularity in ancient organisms. Moreover, their use of rational design (engineered mutations) combined with experimental evolution (allowing colonies to grow under selection pressure and studying the strategies that they adopt) offers a new approach to studying evolution in the lab. DOI: 10.7554/eLife.00367.002 Speculating on evolution based on experiments with engineered strains is problematic. How well is the ease of engineering a strategy correlated with its evolutionary accessibility? Are multiple mutations required? Do these mutations reduce fitness in other environmental conditions? Are other strategies more accessible? Do certain combinations of strategies outcompete single strategies? And finally, how many different strategies does a set of parallel cultures adopt? Experimentally evolving populations, ch (...truncated)