\u3ci\u3eEx Uno Plures\u3c/i\u3e: Clonal Reinforcement Drives Evolution of a Simple Microbial Community

A major goal of genetics is to define the relationship between phenotype and genotype, while a major goal of ecology is to identify the rules that govern community assembly. Achieving these goals by analyzing natural systems can be difficult, as selective pressures create dynamic fitness landscapes that vary in both space and time. Laboratory experimental evolution offers the benefit of controlling variables that shape fitness landscapes, helping to achieve both goals. We previously showed that a clonal population of E. coli experimentally evolved under continuous glucose limitation gives rise to a genetically diverse community consisting of one clone, CV103, that best scavenges but incompletely utilizes the limiting resource, and others, CV101 and CV116, that consume its overflow metabolites. Because this community can be disassembled and reassembled, and involves cooperative interactions that are stable over time, its genetic diversity is sustained by clonal reinforcement rather than by clonal interference. To understand the genetic factors that produce this outcome, and to illuminate the community’s underlying physiology, we sequenced the genomes of ancestral and evolved clones. We identified ancestral mutations in intermediary metabolism that may have predisposed the evolution of metabolic interdependence. Phylogenetic reconstruction indicates that the lineages that gave rise to this community diverged early, as CV103 shares only one Single Nucleotide Polymorphism with the other evolved clones. Underlying CV103’s phenotype we identified a set of mutations that likely enhance glucose scavenging and maintain redox balance, but may do so at the expense of carbon excreted in overflow metabolites. Because these overflow metabolites serve as growth substrates that are differentially accessible to the other community members, and because the scavenging lineage shares only one SNP with these other clones, we conclude that this lineage likely served as an ‘‘engine’’ generating diversity by creating new metabolic niches, but not the occupants themselves

Starvation-Associated Genome Restructuring Can Lead to Reproductive Isolation in Yeast

Knowledge of the mechanisms that lead to reproductive isolation is essential for understanding population structure and speciation. While several models have been advanced to explain post-mating reproductive isolation, experimental data supporting most are indirect. Laboratory investigations of this phenomenon are typically carried out under benign conditions, which result in low rates of genetic change unlikely to initiate reproductive isolation. Previously, we described an experimental system using the yeast Saccharomyces cerevisiae where starvation served as a proxy to any stress that decreases reproduction and/or survivorship. We showed that novel lineages with restructured genomes quickly emerged in starved populations, and that these survivors were more fit than their ancestors when re-starved. Here we show that certain yeast lineages that survive starvation have become reproductively isolated from their ancestor. We further demonstrate that reproductive isolation arises from genomic rearrangements, whose frequency in starving yeast is several orders of magnitude greater than an unstarved control. By contrast, the frequency of point mutations is less than 2-fold greater. In a particular case, we observe that a starved lineage becomes reproductively isolated as a direct result of the stress-related accumulation of a single chromosome. We recapitulate this result by demonstrating that introducing an extra copy of one or several chromosomes into naïve, i.e. unstarved, yeast significantly diminishes their fertility. This type of reproductive barrier, whether arising spontaneously or via genetic manipulation, can be removed by making a lineage euploid for the altered chromosomes. Our model provides direct genetic evidence that reproductive isolation can arise frequently in stressed populations via genome restructuring without the precondition of geographic isolation

Uncoupling reproduction from metabolism extends chronological lifespan in yeast

Studies of replicative and chronological lifespan in Saccharomyces cerevisiae have advanced understanding of longevity in all eukaryotes. Chronological lifespan in this species is defined as the agedependent viability of nondividing cells. To date this parameter has only been estimated under calorie restriction, mimicked by starvation. Because postmitotic cells in higher eukaryotes often do not starve, we developed a model yeast system to study cells as they age in the absence of calorie restriction. Yeast cells were encapsulated in a matrix consisting of calcium alginate to form ~3 mm beads that were packed into bioreactors and fed ad libitum. Under these conditions cells ceased to divide, became heat shock and zymolyase resistant, yet retained high fermentative capacity. Over the course of 17 d, immobilized yeast cells maintained 10%. Immobilized cells exhibited a stable pattern of gene expression that differed markedly from growing or starving planktonic cells, highly expressing genes in glycolysis, cell wall remodeling, and stress resistance, but decreasing transcription of genes in the tricarboxylic acid cycle, and genes that regulate the cell cycle, including master cyclins CDC28 and CLN1. Stress resistance transcription factor MSN4 and its upstream effector RIM15 are conspicuously up-regulated in the immobilized state, and an immobilized rim15 knockout strain fails to exhibit the long-lived, growth-Arrested phenotype, suggesting that altered regulation of the Rim15-mediated nutrient-sensing pathway plays an important role in extending yeast chronological lifespan under calorie-unrestricted conditions

Different Selective Pressures Lead to Different Genomic Outcomes as Newly-Formed Hybrid Yeasts Evolve

Background: Interspecific hybridization occurs in every eukaryotic kingdom. While hybrid progeny are frequently at a selective disadvantage, in some instances their increased genome size and complexity may result in greater stress resistance than their ancestors, which can be adaptively advantageous at the edges of their ancestors\u27 ranges. While this phenomenon has been repeatedly documented in the field, the response of hybrid populations to long-term selection has not often been explored in the lab. To fill this knowledge gap we crossed the two most distantly related members of the Saccharomyces sensu stricto group, S. cerevisiae and S. uvarum, and established a mixed population of homoploid and aneuploid hybrids to study how different types of selection impact hybrid genome structure. Results: As temperature was raised incrementally from 31 degrees C to 46.5 degrees C over 500 generations of continuous culture, selection favored loss of the S. uvarum genome, although the kinetics of genome loss differed among independent replicates. Temperature-selected isolates exhibited greater inherent and induced thermal tolerance than parental species and founding hybrids, and also exhibited ethanol resistance. In contrast, as exogenous ethanol was increased from 0% to 14% over 500 generations of continuous culture, selection favored euploid S. cerevisiae x S. uvarum hybrids. Ethanol-selected isolates were more ethanol tolerant than S. uvarum and one of the founding hybrids, but did not exhibit resistance to temperature stress. Relative to parental and founding hybrids, temperature-selected strains showed heritable differences in cell wall structure in the forms of increased resistance to zymolyase digestion and Micafungin, which targets cell wall biosynthesis. Conclusions: This is the first study to show experimentally that the genomic fate of newly-formed interspecific hybrids depends on the type of selection they encounter during the course of evolution, underscoring the importance of the ecological theatre in determining the outcome of the evolutionary play

Recurrent Rearrangement During Adaptive Evolution in an Interspecific Yeast Hybrid Suggests a Model for Rapid Introgression

Genome rearrangements are associated with eukaryotic evolutionary processes ranging from tumorigenesis to speciation. Rearrangements are especially common following interspecific hybridization, and some of these could be expected to have strong selective value. To test this expectation we created de novo interspecific yeast hybrids between two diverged but largely syntenic Saccharomyces species, S. cerevisiae and S. uvarum, then experimentally evolved them under continuous ammonium limitation. We discovered that a characteristic interspecific genome rearrangement arose multiple times in independently evolved populations. We uncovered nine different breakpoints, all occurring in a narrow similar to 1-kb region of chromosome 14, and all producing an interspecific fusion junction within the MEP2 gene coding sequence, such that the 59 portion derives from S. cerevisiae and the 39 portion derives from S. uvarum. In most cases the rearrangements altered both chromosomes, resulting in what can be considered to be an introgression of a several-kb region of S. uvarum into an otherwise intact S. cerevisiae chromosome 14, while the homeologous S. uvarum chromosome 14 experienced an interspecific reciprocal translocation at the same breakpoint within MEP2, yielding a chimaeric chromosome; these events result in the presence in the cell of two MEP2 fusion genes having identical breakpoints. Given that MEP2 encodes for a high-affinity ammonium permease, that MEP2 fusion genes arise repeatedly under ammonium-limitation, and that three independent evolved isolates carrying MEP2 fusion genes are each more fit than their common ancestor, the novel MEP2 fusion genes are very likely adaptive under ammonium limitation. Our results suggest that, when homoploid hybrids form, the admixture of two genomes enables swift and otherwise unavailable evolutionary innovations. Furthermore, the architecture of the MEP2 rearrangement suggests a model for rapid introgression, a phenomenon seen in numerous eukaryotic phyla, that does not require repeated backcrossing to one of the parental species

<i>Ex Uno Plures</i>: Clonal Reinforcement Drives Evolution of a Simple Microbial Community

<div><p>A major goal of genetics is to define the relationship between phenotype and genotype, while a major goal of ecology is to identify the rules that govern community assembly. Achieving these goals by analyzing natural systems can be difficult, as selective pressures create dynamic fitness landscapes that vary in both space and time. Laboratory experimental evolution offers the benefit of controlling variables that shape fitness landscapes, helping to achieve both goals. We previously showed that a clonal population of <i>E. coli</i> experimentally evolved under continuous glucose limitation gives rise to a genetically diverse community consisting of one clone, CV103, that best scavenges but incompletely utilizes the limiting resource, and others, CV101 and CV116, that consume its overflow metabolites. Because this community can be disassembled and reassembled, and involves cooperative interactions that are stable over time, its genetic diversity is sustained by clonal reinforcement rather than by clonal interference. To understand the genetic factors that produce this outcome, and to illuminate the community's underlying physiology, we sequenced the genomes of ancestral and evolved clones. We identified ancestral mutations in intermediary metabolism that may have predisposed the evolution of metabolic interdependence. Phylogenetic reconstruction indicates that the lineages that gave rise to this community diverged early, as CV103 shares only one <u>S</u>ingle <u>N</u>ucleotide <u>P</u>olymorphism with the other evolved clones. Underlying CV103's phenotype we identified a set of mutations that likely enhance glucose scavenging and maintain redox balance, but may do so at the expense of carbon excreted in overflow metabolites. Because these overflow metabolites serve as growth substrates that are differentially accessible to the other community members, and because the scavenging lineage shares only one SNP with these other clones, we conclude that this lineage likely served as an “engine” generating diversity by creating new metabolic niches, but not the occupants themselves.</p></div

Gene expression and SNPs among loci that mediate glycolysis and fermentation.

<p>Mutations that may affect glycolysis and fermentation are restricted to the glucose-scavenger CV103. Green denotes lower transcription level in monoculture chemostats relative to the ancestor JA122. Red indicates higher transcript levels and grey denotes no transcript level change relative to JA122. Blue colored genes are those that show variable expression; normalized log₂ expression ratios of the evolved strain relative to JA122 are shown in the inset table (for details see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004430#s3" target="_blank">Materials and Methods</a>). Positive values indicate increased expression in the evolved isolates while negative values denote decreased expression. Stars and corresponding text denote the location and type of particular SNPs. Consistent with strain representations in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004430#pgen-1004430-g001" target="_blank">Figure 1</a>, light blue stars indicate ancestral mutations present in JA122, purple indicates SNPs present in CV101, yellow indicates SNPs in CV103, green indicates those in CV115/116, brown indicates SNPs shared by CV101/CV103/CV115/CV116 and pink denotes SNPs shared by CV101 and CV115/116.</p

Gene expression and SNPs among loci that mediate glycerol and acetate uptake/metabolism.

<p>As in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004430#pgen-1004430-g002" target="_blank">Figure 2</a>, a green color indicates that the gene has a lower transcription level in monoculture chemostats relative to the ancestor JA122. A red color denotes elevated monoculture transcript levels while grey denotes no change in transcript level. Expression levels of loci that vary significantly in a strain-specific manner are shown in blue; for these genes, the normalized log₂ expression ratios of the evolved strain relative to JA122 are shown in the inset table (for details see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004430#s3" target="_blank">Materials and Methods</a>). Positive values indicate increased expression in the evolved isolates while negative values denote decreased expression. Transcript ratios for these genes, relative to the ancestor JA122 grown under identical conditions, are presented in the inset table. Stars and corresponding text denote the location and type of particular SNPs. Consistent with strain representations in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004430#pgen-1004430-g001" target="_blank">Figure 1</a>, light blue stars indicate ancestral mutations present in JA122, purple indicates SNPs present in CV101, yellow indicates SNPs in CV103, green indicates those in CV115/116, brown indicates SNPs shared by CV101/CV103/CV115/CV116 and pink denotes SNPs shared by CV101 and CV115/116.</p