Inter-individual variation is a fact of life and under-standing its ecological and evolutionary implications is one of the central subjects of biology. In populations of plants and animals, individual genotypic and phenotypic variation is recognized as having a substantial effect on population function, making individuality an impor-tant component of the structure–function relationship in ecology. However, in the case of bacteria and archaea, we have only started to learn how to interpret diversity among closely related genotypes in the context of the ecological and evolutionary processes that affect popu-lations. In fact, theoretical and empirical models of the population structure of environmental microorganisms have only recently emerged1–5, and it is these models that enable us to take a fresh look at the extent, causes and consequences of inter-individual variation among bacteria and archaea.
Populations are typically defined as coexisting indi-viduals of the same species, which means that they share a common gene pool. However, for bacteria and archaea — similarly to many eukaryotes — there is no broadly applicable species concept1,4. Despite this prob-lem, ecological populations of microorganisms can be operationally defined and are recognized as groups of coexisting individuals that are highly clustered on the genotypic and phenotypic levels, meaning that the variance between populations is much greater than the variance within populations6. Although popula-tions can be defined solely on the basis of gene flow and coexistence, several observations have shown that, when bacterial and archaeal genotypes are mapped with high
resolution onto physiochemical gradients or environ-mental resource patches, there is a strong correlation between population structure and ecological differentia-tion. For example, in the free-living, unicellular cyano-bacteria Prochlorococcus spp., ecological populations are differentiated according to light and temperature gra-dients in the ocean7, whereas genotypic clusters of the heterotrophic Vibrionaceae differentially associate with small organic particles and/or zooplankton and phyto-plankton in the same seawater samples8. Several other studies have mapped such fine-scale genotypic diversity with similar results9–13, providing evidence that genotypic clusters can represent ecologically cohesive popula-tions. Moreover, population genomics has shown that such ecologically associated clusters represent gene-flow units, although sufficient ecological and genomic data are currently available for only a handful of examples. In marineVibrio spp.14, acidiphilic bacteria15 and thermo-philic archaea16, genome-wide rates of homologous recombination are much higher within than between clusters, which means that these groups closely adhere to classically defined populations (BOX 1).
Although clustering and population differentiation can emerge via neutral processes, such as geographic isolation17 and population bottlenecks, theoretical analy-ses18,19 and empirical observations14 support the idea that natural selection is a trigger for the evolution of clus-ters in sympatry — which is the more likely scenario for bacteria and archaea — owing to their high dispersal potential. Such sympatric speciation can affect geno-typic diversity within populations in the following way.
PopulationsGroups or clusters of closely related organisms that occupy the same environment and exhibit population-specific gene flow. Although similar to the ‘ecotype’ concept, populations need only be separated by gene-flow barriers, whereas ecotypes are assumed to contain groups of individuals that have been optimized by selection to occupy a similar niche.
Explaining microbial genomic diversity in light of evolutionary ecologyOtto X. Cordero1 and Martin F. Polz2
Abstract | Comparisons of closely related microorganisms have shown that individual genomes can be highly diverse in terms of gene content. In this Review, we discuss several studies showing that much of this variation is associated with social and ecological interactions, which have an important role in the population biology of wild populations of bacteria and archaea. These interactions create frequency-dependent selective pressures that can either stabilize gene frequencies at intermediate levels in populations or promote fast gene turnover, which presents as low gene frequencies in genome surveys. Thus, interpretation of gene-content diversity requires the delineation of populations according to cohesive gene flow and ecology, as micro-evolutionary changes arise in response to local selection pressures and population dynamics.
1Department of Environmental Systems Science, Swiss Federal Institute of Technology Zurich (ETH-Zürich), CH-8092 Zürich, Switzerland.2Department of Civil and Environmental Engineering, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts 02139-4307, USA.e-mails: [email protected]; [email protected]:10.1038/nrmicro3218Published online 3 March 2014
Population bottlenecksDrastic reductions in the gene pool of a population caused by selective sweeps or demographic fluctuations (for example, few individuals being transmitted during infections).
Whole genomes can ‘hitchhike’ with adaptive loci, lead-ing to — at least initially — a highly clonal population structure19 (BOX 2). However, such clonal sweeps have not yet been observed among environmental bacteria and archaea. Instead, adaptive genomic regions have been shown to sweep independently of the rest of the genome in a population-specific manner, such that much of the genetic diversity is preserved by speciation events14,16. Prevention of genome-wide selective sweeps is possibly caused by frequency-dependent selection, resulting from ecological interactions and/or metabolic trade-offs20,
which, together with high rates of recombination, lead to a population structure that is characterized by the coexistence of multiple, distinct genotypic backgrounds with low genetic linkage (BOX 2).
Indeed, one initially puzzling observation for micro-biologists, who are traditionally used to thinking of bacterial populations as clones growing in a culture tube or at an infection site, is the high genotypic diversity of wild populations. Genomic analyses show high levels of heterogeneity both at the level of sequence similarity among shared genes and at the level of gene content21–24. Point mutations and homologous recombination are primarily responsible for the introduction of single-nucleotide polymorphisms, but homologous and non-homologous recombination can also lead to the rapid loss and gain of heterologous DNA in specific regions of the genome. Hence, these regions are characterized by extensive gene-content variation and form what is known as the flexible genome14,21,23, as opposed to the core genome, which contains conserved loci that diverge only by the accumulation of nucleotide polymorphisms. The functional implications of this genomic variation in the environment are mostly unknown.
The definition of bacterial and archaeal populations as ecologically distinct gene-flow units is an important step towards understanding microbial genotypic varia-tion in the context of evolutionary ecology. Within these units, changes in allele frequencies define evolutionary dynamics; however, owing to the high levels of gene gain and loss in microorganisms, gene frequencies (not only allele frequencies) need to be considered to quan-tify evolutionary change. In fact, division into core and flexible gene content is based on the analysis of gene frequencies, but population structure is usually poorly defined in these genomic comparisons, precluding the interpretation of these frequencies in terms of selective pressures. In other words, core and flexible genome partitions have typically been defined by comparing close relatives but mostly ignore ecological data and/or population structure.
In this Review, we summarize evidence suggesting that within the boundaries of ecological populations, gene-content variation reveals evolutionary dynamics and selective pressures that emerge from local ecological interactions. We will begin by discussing how geno typic diversity in these populations can relate to ecological function, primarily relying on well-studied animal and plant systems. This comparison enables questions relat-ing to bacterial and archaeal genotypic diversity to be framed in the broader context of evolutionary ecology. We also discuss the issue of genome diversity with a focus on intrapopulation gene content variation, which is more specific to bacteria and archaea than to eukary-otes, and we propose that the flexible genome should be re-defined using a framework of different evolutionary forces that cause certain types of genes to occur at low and medium frequency within the boundaries of eco-logical populations. This is illustrated by a discussion of recent work in which social and ecological interactions have been evaluated in wild microbial populations and shown to be associated with variable gene content. We
Box 1 | Bacterial and archaeal population structure
The extent to which bacteria and archaea can form genotypically diverse but ecologically cohesive populations (akin to plant and animal populations) has been challenging to address. Bacterial and archaeal genotypic variation is different from that of eukaryotes in that it is dominated by changes in gene content, in addition to sequence variation in alleles22–24. This observation has led to the categorization of gene complements that are derived from closely related bacteria and archaea into core and flexible genomes. The flexible genome varies on very short timescales and can introduce ecologically adaptive genes. These are acquired from a vast, external gene pool that originates from a range of closely and distantly related organisms and hence might, in combination with the high degree of dispersal potential, erode ecological cohesion among closely related genotypes87.
Despite such theoretical concerns, recent evidence suggests that bacteria and archaea are organized into ecologically cohesive genotypic clusters8,19. This view emerges most clearly for the core genome, from a comparison of protein-coding genes from a large number of genomes and metagenomic data sets3. Theory and empirical evidence suggest that these clusters require selection or gene-flow boundaries for their formation and that they have different ecological associations, which means that they can be used to address hypotheses regarding population-level processes4,88.
Recent data from environmental bacteria and archaea show the fine-grained ecological and genomic structure of these populations8,89, which suggests that they can be adapted to specific microenvironments (such as different types of suspended organic particles in ocean water). These populations also experience high rates of recombination but are separated by strong gene-flow boundaries5,16, enabling adaptive genes to spread horizontally within populations10,14,16. Individuals within these populations are also adapted for cell-to-cell interactions that occur within population boundaries37,80 (see intrapopulation versus interpopulation interactions). Such fine-scale structure is often observed within the bounds of taxonomic species; traditionally described species are subdivided into multiple ecological populations, which suggests that such patterns are the outcome of micro-evolutionary processes.
Vibrio spp. from coastal marine environments8, Leptospirillum spp. from acid mine drainage sites15, Sulpholobus icelandicus from hotsprings16 and streptococci from clinical environments90 are some of the key examples for which population structure and genomic diversity have been mapped with high resolution. These studies show that, in many environmental populations, clonal sweeps, in which a fitter genotype replaces an entire population of genotypes that share the same niche, are rare. Thus, genotypic diversity is maintained by speciation events, in which populations become adapted to a specific niche and develop gene-flow boundaries14,16. The fact that genotypic clusters cannot be traced back to a single founding ancestor differs from the mode of clonal expansion that is traditionally assumed for bacteria, and although clonal expansions (for example, of pathogenic clones) may happen, they are typically short-lived on evolutionary timescales.
Although much progress has been made in linking genotypic clusters to ecology, the role of the flexible genome has remained difficult to explain. Even isolates that are nearly identical in sequence across the core genome can have hundreds of unique genes, leading to the suggestion that much of the flexible gene content might be fitness neutral21,91. However, annotation of flexible genes hints at interactions with the local biotic environment36,48,50,60, which suggests that gene-content variation might be an evolutionary response to local, rapidly varying biotic interactions, such as competition and phage predation. Overall, understanding the ecological and selective forces that drive genome flexibility remains one of the central challenges in microbial population biology.
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SympatryThe coexistence of populations in the same geographic area, such that any gene flow barriers are not caused by geographic isolation but by genetic or behavioural mechanisms.
Clonal sweepsReductions of genome-wide variation in clonal populations owing to an increase in the frequency of one genotype that carries an adaptive mutation.
Frequency-dependent selection A type of selection in which the fitness of a phenotype depends on its frequency in the population.
Genetic linkageThe probability that genes are inherited together. When selection changes the frequency of one gene, it also changes the frequency of the linked genes in a process called hitchhiking.
conclude by outlining some of the open questions and future research directions.
Genotypic diversity and ecological interactionsIn recent years, several studies of natural plant, insect and fish populations have been fundamental in docu-menting the ecological effects of intrapopulation geno-typic diversity and in predicting its global consequences for ecosystems25–31. Together, these studies show that niche complementation, which involves a reduction in competition between conspecifics, is common in ani-mal and plant populations. Niche complementation increases the productivity of populations and can trigger changes in the structure of communities. For example, increased genotypic diversity within populations of the perennial herb Solidago altissima resulted in increased primary productivity but also influenced the community structure of associated arthropod species28. These effects were the result of an increase in the diversity of ecologi-cal interactions between plant genotypes and resources as well as between plant genotypes and the arthro-pod community27,32–34. The increase in productivity in S. altissima illustrates how genotypic diversity can increase niche breadth and reduce competition, even though the differences between genotypes are not strong enough to
disrupt population cohesion. This ultimately improves resource utilization and productivity in polyclonal (as opposed to monoclonal) populations34,35. Furthermore, ecological interactions between members of a polyclonal population can lead to synergistic interactions that boost ecological success or affect the diversity of co-occurring species28,32. Indeed, synergism as a result of chemical interactions can be highly relevant in the case of bac-teria and archaea, as genotypic diversity is expected to increase the range of potential interactions owing to the tendency of secondary metabolites to be encoded in the flexible genome36–38.
Besides these immediate effects, genotypic diversity can also influence the long-term ecological success of populations by buffering against extreme changes in the environment. Several studies with plant and animal populations have shown that low-abundance genotypes can rescue a population after a perturbation of their habitat29,33,39. This has been shown in salmon populations that were tracked over a period of 50 years; temporal variability in population size was estimated to be at least twofold lower than it would have been if populations had been clonal40. In addition, genotypic diversity can also increase the evolvability of populations. Maintaining high genotypic diversity represents an evolutionary strategy to increase the chances of finding and fixing an adaptive genotype, and evolution can pre-emptively generate this diversity either by increasing the mutation rate41,42 or by facilitating the incorporation of novel DNA. The incor-poration of novel DNA has been observed in antibiotic-resistant Enterococcus faecalis strains43, in which the loss of defence mechanisms against mobile genetic elements enabled the rapid acquisition of antibiotic resistance genes.
Gene frequencies and population structureCompared to animals and plants, delineating the func-tional consequences of bacterial and archaeal genotypic diversity (which manifests, to a large extent, as varia-tion in gene content21,22) has been hampered by a lack of understanding of what constitutes a population in a natural environment (BOX 1). Although some insights into how diversity might increase productivity or reduce invasibility have been obtained by assembling artifi-cial communities of closely related microorganisms in microcosms44, few studies have considered the actual structure of natural populations. This is because most of the closely related strains that are available in isolate col-lections have been obtained from disparate geographi-cal locations and/or environmental conditions. That is, genotypes have typically been selected on the basis of their relatedness at the level of clades or taxonomy, irre-spective of whether they actually coexist and encounter each other in the environment. Such coarse-grained sampling overlooks the differential fixation of genes in distinct environments and their ecological relevance. For example, the cyanobacteria Prochlorococcus spp. maintain closely related populations in the Atlantic and Pacific oceans, which are differentiated from each other by nutrient-acquisition genes that have risen to near fixation in response to local nutrient supplies. In
Box 2 | Evolution of genotypic clusters
The ecotype theory remains the prominent explanation for the evolution of genotypic clusters and posits that the spread of adaptive genes within populations triggers genome-wide selective sweeps19. Theoretically, this process is highly plausible as measured rates of homologous recombination are orders of magnitude lower than even moderate rates of selection, so gene flow should not be high enough to unlink a gene that is under selection from the rest of the genome92,93. Consequently, as the gene that is under selection increases in frequency within the population, so should the genome it resides in, until it has outcompeted other genomes that share the same niche. Subsequent to a sweep, the successful genome is free to (neutrally) diversify until similar patterns of clustering are apparent at all loci except for occasional discordant alleles that are introduced by homologous recombination from other populations94.
Although the ecotype theory is consistent with the widespread clustering that is observed among microorganisms, it is at odds with observations of reduced diversity at single loci amidst high genome-wide polymorphism11,95, which suggests that genes can sweep independently of entire genomes. Recent comparative genomics of nascent populations of Vibrio cyclitrophicus14 have confirmed that adaptive genes can sweep in a population-specific manner, whereas the remainder of the genomes show signs of pervasive recombination. Similarly, in hotspring archaea, no evidence for genome-wide selective sweeps was found in the process of sympatric cluster formation16. In both cases, speciation is thought to be a gradual process that is initiated by differential ecological adaptation that leads to a reduction in interpopulation gene flow, owing to the separation of microhabitats5. Over time, population-specific mutations and recombination further differentiate populations until they become permanently distinct clusters.
It is currently unclear why theory predicts that genome-wide clonal sweeps are possible, but current data from environmental populations seem to reject their occurrence. At least two factors might contribute to this conflict. First, the rates of recombination that are reported in previous studies, which are primarily based on multilocus sequence analysis96, are probably much too low. This is because recombination can only be confidently inferred when segments of DNA with a high density of polymorphisms are detected, but such segments mostly stem from distant genotypes. Hence, these rates are more a measure of the degree of genetic isolation of a population than a measure of the rates of intrapopulation homologous recombination. Second, models of genome-wide selective sweeps assume the ecological equivalence of genotypes within a population; however, if niche complementarity has a role in these populations, selection would have to be much stronger to overcome such forces.
Gene content The collection of genes in a genome. At the population level, each gene can be multiallelic.
Flexible genomeThe set of genes that are present in only a fraction of the members of a clade.
Core genomeThe set of genes that are present in all members of a clade or population.
Niche complementation The reduction in intraspecies competition among members of a population owing to differences in resource use and interspecies interactions or to ecological facilitation.
ConspecificsMembers of the same species or population.
Niche breadthThe range of environments and resources to which a population is adapted.
EvolvabilityThe evolutionary potential of a population; that is, the ability of the population to generate novel adaptive mutants.
MicrocosmsExperimental assemblages of organisms that are designed to represent simplified models of biological communities.
Negative frequency-dependent selectionA type of frequency-dependent selection that favours rare phenotypes in a population.
other words, these genes form part of the core genome of the local population45. However, if the Atlantic and Pacific genotypes were to be treated as one closely related clade (or species), these genes would appear as part of the flexible genome. Because of this problem, care must be taken to only compare genomes that are connected by contemporary, unrestricted gene flow so that the effects of environmental selection on the frequency of geno-types can be evaluated in terms of ecological processes.
By operationally defining populations as genotypes that are genetically clustered, connected by gene flow and that possess cohesive ecology, the spectrum of gene frequencies can be used to generate hypotheses about environmental selection pressures and ecological inter-actions. In contrast to animals and plants, for which full genomes are difficult to obtain, hundreds or even thou-sands of microbial genomes from the same ecological population can now be sequenced with relative ease and at low cost. This gives microbiologists the unique possi-bility of using gene-frequency data from across the entire genome to understand population ecology.
Categorization by gene frequencies. Although genes that belong to close relatives are traditionally categorized in a binary fashion into core and flexible genomes (that is, genes that are present in all genomes or that are only present in some genomes, respectively), we propose that a division into high-, medium- and low-frequency genes is more informative and accurate, as these categories can be linked to different evolutionary processes, including both selection and neutral gene-content variation (FIG. 1). For example, within the boundary of an ecological popu-lation, high-frequency genes mostly evolve by vertical inheritance and homologous recombination. Such genes most probably encode essential metabolic and house-keeping functions that are under purifying selection. By contrast, low-frequency genes are gained and lost at such high rates that they are often found as singletons, which suggests that they are derived from large gene pools that transcend population boundaries46–48. Although a large percentage of these genes have no known func-tion and could be selectively neutral and transient in genomes21,49, many low-frequency genes have been sug-gested to be under strong negative frequency-dependent selection (meaning that they confer an advantage only when they are rare in a population) often because they encode potential targets that are recognized by preda-tors (such as phage) and the immune system50. Between these two extremes are medium-frequency genes, which can be lost and gained in a population-specific manner by homologous recombination. As detailed later in this Review, many of these medium-frequency genes differ from low-frequency genes in that they can experience other types of frequency-dependent selective pressures, such as those that result from interactions between geno-types both within and between populations.
Given the above considerations, the study of ecologi-cal interactions within and between natural populations should lead to a better understanding of the origins of genotypic diversity in natural populations of microor-ganisms as well as its functional consequences (BOX 3).
Studying interactions between strains that have known population structures can reveal the potential roles of niche complementation and ecological dynamics, which are unpredictable when single genotypes are examined. At the same time, linking ecology to variation in gene frequencies enables us to generate hypotheses about how different types of ecological processes influence geno-typic diversity and to gain some insight into the micro-evolutionary mechanisms that underpin the observed patterns of interactions. In the following sections, we discuss low-frequency and medium-frequency genes and their relationship to biological interactions as well as the underlying evolutionary mechanisms.
Low-frequency genes and genetic linkageSome phenotypes only confer an advantage when they are present at low frequency in a population51–53. For example, rare patterns or colouration in some animals facilitates predator evasion, which, in turn, selects for high variation in these traits53,54. In bacterial and archaeal populations there are similar trait variations, and they seem to be driven primarily by changes in gene con-tent48,50,55. Although surface structures, such as lipid A and O antigen, are widespread in Gram-negative bac-teria, genes that encode different structural variants of these antigens are typically found at very low frequen-cies in microbial populations56. Maintaining these vari-ants at low frequency facilitates evasion from predators and host immune responses57,58, and it is thought that
▶Figure 1 | Gene frequencies and their interpretation in terms of evolutionary and ecological processes. Populations are recognized as genotypic clusters separated by gene flow boundaries and can have distinct habitats. a | High-frequency genes (green and orange arrows; also represented by short black lines in the gene flow map) are primarily maintained by vertical inheritance and homologous recombination. These genes are observed across multiple ecological populations and typically encode core metabolic and housekeeping functions that are independent of the different environments. b | High-frequency genes (High*) can also segregate ecological populations. After being gained or lost in a population-specific manner, these genes could follow similar patterns of gene flow as other core genes. They are potentially involved in habitat-specific functions (for example, the adaptation to use either the orange or green substrates as a nutrient source). c | Medium-frequency genes flow by vertical inheritance, homologous recombination and gene loss. As illustrated in the figure, without considering population structure (in other words, that the green and orange genes are derived from two distinct populations), the frequency of these genes would be indistinguishable from that of the High* genes (50%). Recent studies suggest that some of these genes might be involved in local biological interactions (such as those that are mediated by public goods), which create frequency-dependent selection. d | Low-frequency genes reflect extremely high rates of gene turnover, which represents an evolutionary strategy to diversify, often precipitated by negative frequency-dependent selection emerging from interactions with predators (such as phage) or with the immune system.
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Genomic islandsLocalized regions in a genome at which horizontal gene transfer and gene loss occur at high rates, resulting in high gene-content diversity at these loci between close relatives.
selection favours high gene turnover at these loci, resulting in the observed low gene-frequency pattern.
There are two mutational mechanisms that ensure genes are maintained at a low frequency: high rates of gene turnover by homologous and non-homologous recombination, and gene loss. Low-frequency genes tend to be encoded in genomic islands55 or plasmids, which typically encode several transposases and integrases that function to increase the rate of DNA turnover. Thus, low frequency genes are associated with highly unstable gene content in localized regions of the genome and so are not associated with robust differentiation between geno-types. Such rapid turnover can result in a high number of unique genes in individual genotypes, as shown in a recent study of Vibrio cyclotrophicus populations from the marine environment; each strain carried up to 200 unique genes out of a total of ~4,000 genes, despite
displaying >99% nucleotide similarity in their shared genes14. Previous analyses of hypervariable gene content have shown that frequent, habitat-specific horizontal gene transfer might be a mechanism of adaptation to local environments59. Accordingly, recently transferred genes in fully sequenced genomes36,60 are enriched for those that encode defence mechanisms, efflux pumps, transporters and one-component regulatory genes, which combine sensor and DNA-binding domains in one protein61. A common feature of these classes of genes is that they all encode functions that enable organisms to respond to transient selective pressures in their local environment.
Connecting with genetic linkage. The fact that varia-tion in gene content occurs on fast ecological timescales in genomic islands47,48, whereas the rest of the genome evolves at much slower rates, implies that there is low genetic linkage between low-frequency genes and the rest of the genome. Genes with high linkage tend to hitchhike with each other, making it possible for selec-tion on one gene to affect the frequency of the linked genes62,63. However, when genetic linkage is low, hitch-hiking is minimal and genes can change in frequency in the population without interfering with each other. For this reason, low-frequency genes that have high rates of recombination and loss are predicted to have little influ-ence on the frequency of other genes in the population.
Understanding genetic linkage is crucial for pre-dicting how ecological interactions, such as predation by phage, affect the diversity of microbial populations. According to the ‘kill-the-winner’ model64, predation exerts top-down control on populations by selectively removing individuals that rise to high frequency owing to higher fitness. This means that those hosts that are more successful than potential competitors, in terms of resource exploitation and growth rate, are subject to increased predation pressure by highly host-specific phage. Although this idea was originally developed to explain species coexistence, it has more recently been proposed as an explanation for genotypic diversity within populations as part of the ‘constant diversity hypoth-esis’ (REF. 50). The fact that proteins that are recognized by phage are unique to different genotypes within popu-lations seems to suggest that phage control genotypes in a highly specific manner. However, this is where the concept of genetic linkage becomes important. Low gene frequency in microbial populations is indicative of high gene turnover, not of stable genotype differentiation, which brings into question whether the ‘constant diver-sity hypothesis’ is applicable to natural populations of bacteria with high recombination rates. Because of the low linkage between phage receptors and the rest of the genome, the negative frequency-dependent selection that affects these receptors could have little effect on the diversity of other genes in the genome.
An alternative way to understand the interaction between phage and bacteria is to consider it at the popula-tion level instead of at the level of single genotypes (FIG. 2). The negative frequency-dependent selection that emerges from predatory interactions favours diversity at those loci that encode determinants recognized by predators.
Public goodsSecreted products that can be used by coexisting bacteria, including cheaters, which do not incur the metabolic cost of production.
Classical experimental evolution studies have sug-gested that resistance to phage predators arises by point mutation in such loci65; however, comparative genomics has shown that genes that are targeted by phage are frequently segregated into genomic islands, where high rates of non-homologous recombination lead to rapid gene turnover, which results in low linkage to the rest of the genome50,66. Owing to the high rates of diversifi-cation that these co-evolutionary dynamics generate67, snapshots of the genetic composition of the population at any point in time reveal a high number of extremely low-frequency genes. However, because of their low genetic linkage with the rest of the genome, there should be little correlation between the frequency of adaptive loci in the core genome and predation intensity (that is, predation intensity does not increase with core genotype abundance), which would occur if the core genotype and phage receptors were linked. Instead, predation should translate into a density-independent mortality rate that represents the probability of carrying the ‘wrong’ recep-tor (that is, one recognized by phage). This probability depends on the available pool of genes that encode phage receptors, not on individual genotype density (FIG. 2). To the extent that gene pools are population specific, total population size can still be regulated by top-down con-trol, whereas the genotypic composition of the popu-lation can change independently of phage predation. This scenario makes the kill-the-winner-type control by phage a rational explanation for the coexistence of ecologically similar populations if they are separated by gene-flow boundaries, but an unlikely explanation for preventing clonal expansion within prokaryotic populations.
In summary, the fitness effect of low-frequency genes is rooted in their extreme variability, which results from the selective advantage that fast diversification provides in the face of changing ecological interactions. Fast gene turnover can provide tremendous ecological advantages to populations, such as the ability to evade predators or the immune system. As the mechanism to ensure variability relies on frequent DNA acquisition and loss, low-frequency genes tend be associated with mobile elements that are not associated with specific geno-types. Their intrinsic low level of genetic linkage implies that core genomes can evolve nearly independently of low-frequency genes.
Medium-frequency genes and interactionsAlthough some genes that are detected at medium fre-quencies might be in the process of being lost from the population, their distribution can also result from spe-cific types of frequency-dependent interactions that pre-vent these genes from rising to high frequency. However, in contrast to low frequency genes, they typically do not reside in hypervariable regions (such as genomic islands) and, as detailed below, are probably gained and replaced by homologous recombination. Key examples of the type of selective pressures that are operating include those that emerge from metabolic trade-offs or from social interactions, which can stabilize genes at medium fre-quencies owing to the partitioning of functional roles within the population. In this section, we discuss recent studies that show how social interactions can create medium gene-frequency patterns.
To illustrate how social interactions can drive genes to medium frequencies, consider the production of a secreted growth factor in a population. When the encoding gene (or genes) is at high frequency, there will be an excess of producers contributing to a common pool of the growth factor. This overabundance creates a short-term incentive for the emergence of cheater genotypes, which have either lost or suppressed the biosynthesis gene (or genes), but can nevertheless use the growth factor without incurring production costs. Producers and cheaters can coexist if producers have preferential access to the public goods, which typically occurs in spatially structured populations, such as bio-films68,69, resulting in medium frequencies of producer and cheater genotypes.
This conceptual model was recently tested in a study of siderophore production among >1,000 marine Vibrio spp. that represented several ecologically defined popula-tions38. This study revealed the link between social inter-actions and genotypic diversity in nature. Siderophores are iron-chelating molecules that are secreted by bacte-ria to scavenge poorly soluble iron70. Vibrio spp. encode several types of siderophores, such as aerobactin71 and vibrioferrin72, each of which is captured by specific receptors. Because iron–siderophore complexes are formed outside the cell, non-producing cells that express the appropriate receptors can access the iron without incurring the costs of siderophore production73,74. This makes siderophores one of the key examples of public goods produced by bacteria75.
Box 3 | Interactions and population structure in aquatic environments
Owing to the potentially high rates of mixing and dispersal that bacteria and archaea can experience in the environment, it is not intuitive to picture how these microorganisms engage in stable ecological interactions that have a substantial effect on their genetic composition. Aquatic environments, such as the ocean, function as an ideal platform to explore how, despite global mixing, ecological interactions between microorganisms occur and to illustrate why ecologically, genetically and socially cohesive populations exist6. Although seemingly homogeneous to the eye, ocean water is highly structured at small (that is, millimetre to micrometre) scales6,97,98. These spatial heterogeneities arise from a broad range of sources, such as phytoplankton exudates, chitinous particles and other forms of detrital material that tend to form nutrient-rich aggregates99 to which bacteria can adhere100. This microscale structure facilitates resource partitioning and spatial segregation despite the high rates of global mixing in the ocean. Resource partitioning occurs when populations preferentially consume certain types of nutrients, such as different Vibrio spp. populations whose environmental distributions indicate specialization for algae-derived detritus particles or chitin-rich copepods84. The spatial compartmentalization that is provided by particles also contributes to defining population structure. Owing to the high concentration of nutrients in these aggregates, particle-attached bacteria are highly metabolically active and can live at high cell densities in multispecies communities101,102. These conditions favour social and ecological interactions, as shown by recent work with Vibrio splendidus populations, in which the exploitation of secreted compounds was more prevalent on particles and had measurable effects on genetic diversity38. Collectively, resource specialization and spatial structure can create the conditions for conspecifics to encounter each other often and to assemble polyclonal populations. In these polyclonal populations, chemical communication, antagonism and the sharing of public-goods, among other traits, can lead to synergistic effects that can have a substantial effect on the ecological functions of natural populations.
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In the ecologically segregated Vibrio spp. populations, the genetic diversity of the siderophore operons could be accounted for by the frequency-dependent dynam-ics that are associated with public-goods interactions. Siderophore production is a medium-frequency trait, and only ~40% of individuals in a population are able to produce these compounds, whereas nearly all members of the population can reap their benefit. Moreover, tran-sitions between producer and non-producer phenotypes occurred via changes in gene content, such that non-producers lost the siderophore-biosynthesis machin-ery but retained the corresponding receptors, which is consistent with the model of a social cheater (FIG. 3).
Interestingly, switching between cheater and producer social roles seemed to be mediated by homologous recombination. The only difference in gene content of the siderophore operon between the cheaters and the producers is the deletion of the biosynthesis genes in the internal region of the operon in the cheater genotypes; thus, homologous recombination could replace operon variants and switch social roles. Such switching is prob-ably controlled by population-specific gene flow, as there is a sharp decline in homologous recombination rates between divergent sequences, which means that gene exchange between genotypes from different populations
is rare18,76. Moreover, as the siderophore operons are not encoded in genomic islands, a higher degree of linkage is expected between the siderophore genes and other core genes (compared with phage receptors). Whether the degree of linkage is enough to interfere with clonal sweeps and drive core-genome diversity remains unclear.
The idea that social roles are internally regulated (that is, within populations) contrasts with the idea of environmental bacteria existing as isolated creatures that undergo only short clonal bursts by exploiting transient opportunities77. The presence of cheaters at medium fre-quencies and the apparent switching of social roles by recombination would suggest that interactions between genotypes drive diversity within populations. In this regard, it is important to bear in mind that, given the non-clonality of these populations, multiple public goods and their associated genes are expected to be distributed across the genotypes of the population, which increases the chance that these types of interactions affect core-genome diversity even if genetic linkage is only modest.
Intrapopulation and interpopulation interactionsTo understand the potential for synergism in polyclonal populations, one of the basic questions we can ask is: are ecological or social interactions distributed in a manner that is consistent with population structure? For exam-ple, is chemical communication more readily achieved within populations than between them? How do differ-ences in the degree of interactions within and between populations influence the composition of populations and their ecological functions? The following examples show that asymmetries in interactions correlate with population structure and, in some cases, with the pos-session of specific genes, which can either be at high or medium frequency within the population (FIG. 4).
One study78 examined the interdependence between social interactions and population structure in the social bacterium Myxococcus xanthus. These soil bacteria have a complex life cycle, with cells aggregating under star-vation conditions to form fruiting bodies, in which a fraction of the cells become stress-resistant spores79. In polyclonal cultures (comprising 36 M. xanthus geno-types), impaired development and sporulation was more commonly observed among genotypes from geographi-cally distant locations than in those originating from the same square centimetre of soil (FIG. 4a). This suggests that ecological similarity and social conflict are inversely cor-related in M. xanthus. However, there was no correlation between social conflict and the phylogenetic distance of three sequenced housekeeping genes. Moreover, swarm-ing cells were able to discriminate against closely related genotypes (which were identical at the three housekeep-ing loci) to prevent inter-genotype mixing. This indi-cates that social structure in these organisms evolves at a faster rate than the rate of sequence diversification of slowly evolving protein-encoding genes, which are used for genotyping. For this reason, the genetic underpin-nings of such fine-grained social structure in M. xanthus could potentially be revealed only by whole-genome sequencing studies that capture highly variable regions of the genome.
Figure 2 | The role of low gene frequencies and low genetic linkage in bacteria–phage interactions. Phage–bacteria interactions in microbial populations are determined by the availability of mobile gene pools that are unlinked from the genome of the host. Populations of phage interact with their bacterial hosts by attaching to specific receptors on the cell surface, which leads to negative frequency-dependent selection103. The evolutionary response of bacteria is to diversify the repertoire of phage receptors expressed on their surface in order to escape predation. This is achieved by encoding phage receptors in hypervariable genomic regions (such as genomic islands), which increases the rate of gene turnover and thus enables the acquisition of new receptors from external gene pools. When the rate of gene flow is high, receptor genes are essentially unlinked from the rest of the genome and predation rates become independent of genotype frequency. In other words, only the receptor gene is subject to negative frequency-dependent selection, rather than the entire genotype, as postulated by the ‘kill the winner’ model64.
Interference competitionA form of competition that involves direct antagonistic interactions between individuals.
BacteriocinsProteinaceous toxins produced by bacteria that kill or inhibit the growth of close relatives.
A more recent study showed that quorum sensing signals are recognized more often by members of the same ecological population than by members of distant populations (FIG. 4b). Among different soil isolates of Bacillus spp., signalling was found to be effective among clustered genotypes, but it was often impaired between clusters80. Although these populations were identified by sequence similarity only, they are expected to represent ecological populations or ecotypes2 (BOX 2). The apparent signalling specificity that is observed in these clusters is mediated by genetic differences in the comQXPA locus, which encodes the signal-transduction system that con-trols quorum sensing in Bacillus spp. Previous studies of this system had found that genetic polymorphisms in the comQXPA locus are typically enriched in the genes that are dedicated to the biosynthesis of the signalling peptide as well as in the receptor for the peptide81. This could provide a mechanism for the rapid generation of novel ‘key–lock’ pairs that, as the study of wild Bacillus isolates showed, can be specific to ecological populations. These findings suggest that different genotypes within the same ecological population can function in a coordi-nated manner and exclude genotypes from other popula-tions even if they are closely related. Finally, asymmetry in interactions within and between populations has also been observed in oceanic Vibrio spp., as exemplified by antagonistic interactions between individual isolates from distinct ecological populations37. Owing to niche overlap among close relatives, interference competition as a result of
antibiotic production was expected to be most advanta-geous if directed against members of the same popula-tion, as was previously documented for bacteriocins82,83. By contrast, this study showed that antibiotics (mostly small molecules, such as peptides) produced by marine Vibrio spp. were primarily effective against members of other ecological populations and not against conspe-cifics in the same genotypic cluster (FIG. 4c). Although these populations are differentiated by their propensity to associate with different types of marine particles8, populations frequently mix in the intestines of inverte-brates84 and, possibly, those of vertebrates. This suggests that the observed high levels of interpopulation antago-nism re inforce gene-flow boundaries by preventing the mixing of antibiotic-producer genotypes with antibiotic-sensitive genotypes from distinct ecological populations.
This study also showed that antibiotic production by Vibrio spp. is a medium-frequency trait in the popula-tion. Nearly half of the strains within a cluster of close relatives were able to produce some type of inhibitory substance, although not all of these substances were the same and each had the ability to target a different set of individuals in other populations. By contrast, nearly all individuals within a population were resistant to substances that were produced by conspecifics (despite the diversity of products), which indicates that genes encoding resistance for each of the specific antibiot-ics produced were present at a high frequency within the same population. For example, one of the more
Figure 3 | Social cheating in natural populations and its role in generating medium-frequency genes. In oceanic Vibrio spp. populations, siderophore production is a medium-frequency trait that does not have any clear phylogenetic pattern, suggesting that frequent and rapid switching of social roles (such as production and cheating) occurs. This is exemplified by the siderophore vibrioferrin, for which the receptor and biosynthesis genes are encoded in a single operon. Vibrio spp. populations have evolved to have an operon variant that has lost the biosynthesis genes but that maintains the receptor genes. Cells that express this operon variant can access iron (in the form of Fe3+) but do not incur the costs of vibrioferrin production and are therefore known as cheaters. It is probable that the cheater variant was generated once and later spread through the population by homologous recombination, resulting in the siderophore biosynthesis genes appearing as medium-frequency genes in the population.
Population boundary
Siderophore non-producer
Siderophore producer
Vibrioferrin cheater
Vibrioferrin producer
Vibrioferrin Fe3+
PvuE
PvuD PvuB
PvuC PvuA
Transport genes
PsuA PvuE
PvuD PvuB
PvuC PvuA PsuAPvsA PvsC PvsE
PvsB PvsD
1 5,000 10,000 1 5,000 10,00015,000 17,108
Biosynthesis genes
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broad-range antibiotics (which is a non-ribosomal poly-ketide peptide) was produced by only five of 30 Vibrio ordalii genotypes, but all 30 genotypes encoded resist-ance to the antibiotic. Using a combination of genetic and genomic approaches it was established that this antibiotic is encoded by a gene cluster that was recently
acquired by horizontal gene transfer and is present only in the five producers. This mobile gene cluster did not contain resistance genes, which suggests that resistance mechanisms must have pre-existed in the population to enable integration of the gene cluster into the genome.
The most interesting aspects of these studies are their ecological implications. The fact that individuals within populations share similar resource preferences but show relatively low levels of social conflict sug-gests that these genotypes frequently coexist with each other in the environment85. The frequent re-assembly of populations has important consequences, as interactions that can increase niche complementarity between geno-types depend on close spatial proximity, which enables the sharing of public goods or the synergistic action of other secreted compounds. The case of antagonism in Vibrio spp. is a key example that illustrates the potential for synergism, as polyclonal populations can produce a larger repertoire of inhibitory substances, potentially outperforming monoclonal populations in their ability to deter competitors from distant species. Recurrent population assembly, driven by high resource similarity and low social conflict between conspecifics, also has important evolutionary consequences, as co-adaptation (for example, by complementary gene loss86) is more likely to occur if populations frequently reassemble.
ConclusionsWe have highlighted the importance of defining the flexible genome within the boundaries of ecologi-cal populations, in which gene frequencies can be analysed in terms of local selective pressures. Within these boundaries, high gene frequencies reflect stable selective pressures at the population level, whereas flexible gene content can be partitioned into medium and low gene frequencies to distinguish between dif-ferent forms of frequency-dependent selection and to generate hypotheses about ecological and evolutionary dynamics.
The collection of studies discussed in this Review connect flexible gene-content variation with social and ecological interactions within and between ecological populations. It is well recognized that bacteria are capable of multiple ecological interactions; however, such inter-actions are rarely studied in the context of wild popula-tions, which can be highly genotypically diverse despite being connected by gene flow. We suggest that interac-tions can explain part of that genetic diversity by gen-erating frequency-dependent selective pressures, which favour low and medium gene frequencies. An impor-tant consequence is that such frequency-dependent selection can prevent clonal sweeps14, especially if the genes it acts on are embedded in regions of the genome where linkage is relatively high (that is, within the core genome). The fact that environmental bacteria and archaea are not just simple assemblages of clones, but are ecological populations in which genotypes are connected by interactions and gene flow, has important implica-tions. It means that many of the ecological effects that are mediated by microbial activity, such as rates of nutrient turnover, virulence and community dynamics, might be
Figure 4 | Asymmetry in intrapopulation versus interpopulation interactions. a | The effect of polyclonality in Myxococcus xanthus fruiting body development. Polyclonal mixtures of strains (light blue and dark blue cells) produce fewer spores than monoclonal strains (light blue cells) when genotypes from relatively distant locations are experimentally combined. This negative response to polyclonality shows that individuals from different populations are less compatible than individuals from the same population. The genetic determinants of this incompatibility are probably encoded at hypervariable loci of the M. xanthus genome. b | The effect of population structure on the ability of Bacillus spp. to communicate by quorum sensing. As opposed to communication within the same genotypic cluster (light green cells), communication between genotypically distinct clusters (light and dark green cells) is often impaired owing to population-specific divergence in the comQXPA locus. c | Antagonistic interactions in Vibrio spp. populations in the ocean. Anti biotic-mediated antagonism was found to occur less often within (left panel) than between (right panel) populations. Conspecifics within populations were resistant to each other, even though each strain was able to produce a diverse range of different antibiotics.
emergent properties of population-level diversity. This can give rise to several possible effects, and future studies should investigate to what extent additional mechanisms described for animal and plant populations (for example,
niche complementation, with its potential cascade of effects on productivity and community structure) are at work in natural ecological populations of bacteria and archaea.
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AcknowledgementsThe authors wish to thank J. Friedman for valuable discus-sions. Funding for M.F.P was provided by US National Science Foundation grant DEB 0821391, US National Institute of Environmental Health Sciences grant P30-ES002109, the Moore Foundation and the Broad Institute’s Scientific Planning and Allocation of Resources Committee (SPARC) programme.
Competing interests statement The authors declare no competing interests.
