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Selected PublicationsTurner, P.E., V. Souza, and R.E. Lenski. 1996. Tests of ecological mechanisms promoting the stable coexistence of two bacterial genotypes. Ecology 77:2119-2129. Abstract:A series of competition experiments with two genotypes of Escherichia coli showed that each genotype was favored when it was the minority, allowing their stable coexistence. In these experiments, glucose was the sole source of carbon provided and its concentration was limiting to population density. Thus, the stable polymorphism does not conform to a simple model of competitive exclusion. We considered two hypotheses that might explain the observed coexistence: (1) a strictly demographic tradeoff, such that one genotype is competitively superior when glucose is abundant whereas the other genotype is the better competitor for sparse glucose; and (2) a cross-feeding interaction, whereby the superior competitor for glucose excretes a metabolite that acts as a second resource for which the other genotype is the better competitor. Although there was a demographic tradeoff, the advantage to the superior competitor at high glucose concentrations was too large (given the initial concentration of glucose used in these experiments) to allow the second genotype to invade when rare at the observed rate. Therefore, the second genotype must have some other advantage that allows it to readily invade a population of the superior competitor for glucose. Indeed, the second genotype could increase in abundance after glucose was depleted, but only in the presence of the superior competitor for glucose, thus implicating a cross-feeding interaction. These results confirm earlier studies showing that populations of E. coli can maintain ecologically relevant genetic diversity even in a simple environment. Souza, V., P.E. Turner, and R.E. Lenski. 1997. Long-term experimental evolution in Escherichia coli. V. effects of recombination with immigrant genotypes on the rate of bacterial evolution. Journal of Evolutionary Biology 10:743-769. Abstract:This study builds upon an earlier experiment that examined the dynamics of mean fitness in evolving populations of Escherichia coli in which mutations were the sole source of genetic variation. During thousands of generations in a constant environment, the rate of improvement in mean fitness of these asexual populations slowed considerably from an initially rapid pace. In this study, we sought to determine whether sexual recombination with novel genotypes would reaccelerate the rate of adaption in these populations. To that end, treatment populations were propagated for an additional 1000 generations in the same environment as their ancestors, but they were periodically allowed to mate with an immigrant pool of genetically distinct Hfr (high frequency recombination) donors. These donors could transfer genes to the resident populations by conjugation, but the donors themselves could not grow in the experimental environment. Control populations were propagated under identical conditions, but in the absence of sexual recombination with the donors. All twelve control populations retained the ancestral alleles at every locus that was scored. In contrast, the sexual recombination treatment yielded dramatic increases in genetic variation. Thus, there was a profound effect of recombination on the rate of genetic change. However, the increased genetic variation in the treatment populations had no significant effect on the rate of adaptive evolution, as measured by changes in mean fitness relative to a common competitor. We then considered three hypotheses that might reconcile these two outcomes: recombination pressure, hitchhiking of recombinant genotypes in association with beneficial mutations, and complex selection dynamics whereby certain genotypes may have a selective advantage only within a particular milieu of competitors. The estimated recombination rate was too low to explain the observed rate of genetic change, either alone or in combination with hitchhiking effects. However, we documented complex ecological interactions among some recombinant genotypes, suggesting that our method for estimating fitness relative to a common competitor might have underestimated the rate of adaptive evolution in the treatment populations. Turner, P.E., V.S. Cooper, and R.E. Lenski. 1998. Tradeoff between horizontal and vertical modes of transmission in bacterial plasmids. Evolution 52:315-329. Abstract:It is widely hypothesized that there is a fundamental conflict between horizontal (infectious) and vertical (intergenerational) modes of parasite transmission. Activities of a parasite that increase its rate of infectious transmission are presumed to reduce its host's fitness. This reduction in host fitness impedes vertical transmission of the parasite and thereby causes a tradeoff between horizontal and vertical transmission. Under these circumstances, and assuming no multiple infections (no within-host competition among parasites), the density of uninfected hosts in the environment should determine the optimum balance between modes of parasite transmission. When susceptible hosts are abundant, horizontal transmission is more important and simple models predict that selection should favor increased rates of horizontal transfer, even at the expense of reduced vertical transmission. Conversely, when hosts are scarce, vertical transmission is more important, which should favor increased vertical transmission even at the expense of reduced horizontal transmission. We tested the tradeoff hypothesis and key model predictions using conjugative plasmids and the bacteria that they infect. Plasmids evolved for 500 generations in environments with different densities of susceptible bacterial hosts. We observed that the plasmid’s conjugation rate increased evolutionarily at the expense of host fitness, demonstrating a systematic tradeoff between horizontal and vertical modes of plasmid transmission. Also, evolutionary reductions in conjugation rate repeatedly coincided with the loss of a particular plasmid-encoded antibiotic resistance gene. However, contrary to model predictions, susceptible host density had no significant effect on the evolution of horizontal versus vertical modes of plasmid transmission. Turner, P.E., and L. Chao. 1998. Sex and the evolution of intrahost competition in RNA virus Φ6. Genetics 150:523-532. Abstract:Sex allows beneficial mutations that occur in separate lineages to be fixed in the same genome. For this reason, the Fisher-Muller model predicts that adaptation to the environment is more rapid in a large sexual population than in an equally large asexual population. Sexual reproduction occurs in populations of the RNA virus Φ6 when multiple bacteriophages coinfect the same host cell. Here, we tested the model's predictions by determining whether sex favors more rapid adaptation of Φ6 to a bacterial host, Pseudomonas phaseolicola. Replicate populations of Φ6 were allowed to evolve in either the presence or absence of sex for 250 generations. All experimental populations showed a significant increase in fitness relative to the ancestor, but sex did not increase the rate of adaptation. Rather, we found that the sexual and asexual treatments also differ because intense intrahost competition between viruses occurs during coinfection. Results showed that the derived sexual viruses were selectively favored only when coinfection is common, indicating that within-host competition detracts from the ability of viruses to exploit the host. Thus, sex was not advantageous because the cost created by intrahost competition was too strong. Our findings indicate that high levels of coinfection exceed an optimum where sex may be beneficial to populations of Φ6, and suggest that genetic conflicts can evolve in RNA viruses. Turner, P.E., C. Burch, K. Hanley, and L. Chao. 1999. Hybrid frequencies confirm limit to coinfection in the RNA bacteriophage Φ6. Journal of Virology 73:2420-2424. AbstractCoinfection of the same host cell by multiple viruses may lead to increased competition for limited cellular resources, thus reducing the fitness of an individual virus. Selection should favor viruses that can limit or prevent coinfection, and it is not surprising that many viruses have evolved mechanisms to do so. Here we explore whether coinfection is limited in the RNA bacteriophage Φ6 that infects Pseudomonas phaseolicola. We estimated the limit to coinfection in Φ6 by comparing the frequency of hybrids produced by two marked phage strains to that predicted by a mathematical model based on differing limits to coinfection. Our results provide an alternative method for estimating the limit to coinfection and confirm a previous estimate between two to three phages per host cell. In addition, our data reveal that the rate of coinfection at low phage densities may exceed that expected through random Poisson sampling. We discuss whether phage Φ6 has evolved an optimal limit that balances the costly and beneficial fitness effects associated with multiple infections. Turner, P.E., and L. Chao. 1999. Prisoner's dilemma in an RNA virus. Nature 398:441-443. Abstract:The evolution of competitive interactions among viruses was studied in the RNA phage Φ6 at high and low multiplicities of infection (that is, at high and low ratios of infecting phage to host cells). At high multiplicities, many phage infect and reproduce in the same host cell, whereas at low multiplicities the viruses reproduce mainly as clones. An unexpected result of this study was that phage grown at high rates of co-infection increased in fitness initially, but then evolved lowered fitness. Here we show that the fitness of the high-multiplicity phage relative to their ancestors generates a pay-off matrix conforming to the prisoner's dilemma strategy of game theory. In this strategy, defection (selfishness) evolves, despite the greater fitness pay-off that would result if all players were to cooperate. Viral cooperation and defection can be defined as, respectively, the manufacturing and sequestering of diffusible (shared) intracellular products. Because the low-multiplicity phage did not evolve lowered fitness, we attribute the evolution of selfishness to the lack of clonal structure and the mixing of unrelated genotypes at high multiplicity. Turner, P.E., and S.F. Elena. 2000. Cost of host radiation in an RNA virus. Genetics 156:1465-1470. Abstract:Although host radiation allows a parasite to expand its ecological niche, traits governing the infection of multiple host types can decrease fitness in the original or alternate host environments. Reasons for this reduction in fitness include slower replication due to added genetic material or modifications, fitness trade-offs across host environments, and weaker selection resulting from simultaneous adaptation to multiple habitats. We examined the consequences of host radiation using vesicular stomatitis virus (VSV) and mammalian host cells in tissue culture. Replicate populations of VSV were allowed to evolve for 100 generations on the original host (BHK cells), on either of two novel hosts (HeLa and MDCK cells), or in environments where the availability of novel hosts fluctuated in a predictable or random way. As expected, each experimental population showed a substantial fitness gain in its own environment, but those evolved on new hosts (constant or fluctuating) suffered reduced competitiveness on the original host, However, whereas evolution on one novel host negatively correlated with performance on the unselected novel host, adaptation in fluctuating environments led to fitness improvements in both novel habitats. Chao L, Hanley KA, Burch CL, et al. Kin selection and parasite evolution: Higher and lower virulence with hard and soft selection Quarterly Review of Biology 75 (3): 261-275 Sep 2000 Abstract:Conventional models predict that low genetic relatedness among parasites that coinfect the sa,ne host leads to the evolution of high parasite virulence. Such models assume adaptive, responses to hard selection only. We show that if soft selection is allowed to operate, low relatedness leads instead to the evolution of low virulence. With both hard and soft selection, low relatedness increases the conflict among coinfecting parasites. Although parasites can only respond to hard selection by evolving higher virulence and overexploiting their host, they can respond to soft selection ip evolving other adaptations, such as interference, that prevent overexploitation. Be cause interference can entail a cost, the host may actually De underexploited, and virulence will decrease as a result of soft selection. Our analysis also shows that responses to soft selection can have a much stronger effect than, responses to hard selection. After hard selection has raised virulence to a level that is an evolutionarily stable strategy, the population, as expected, cannot De invaded by more virulent phenotypes that respond only to hard selection. The population remains susceptible to invasion by a less virulent phenotype that responds to soft selection, however. Elena, S.F., A.V. Bordería, R. Sanjuán, and P.E. Turner. 2001. Transmission bottlenecks and the evolution of fitness in rapidly evolving RNA viruses. Infection, Genetics and Evolution 1:41-48. Abstract:We explored the evolutionary importance of two factors in the adaptation of RNA viruses to their cellular hosts, size of viral inoculum used to initiate a new infection, and mode of transmission (horizontal versus vertical). Transmission bottlenecks should occur in natural populations of viruses and their profound effects on viral adaptation have been previously documented. However, the role of transmission mode has not received the same attention. Here we used a factorial experimental design to test the combined effects of inoculum (bottleneck) size and mode of transmission in evolution of vesicular stomatitis virus (VSV) in tissue culture, and compared our results to the predictions of a recent theoretical model. Our data were in accord with basic genetic principles concerning the balance between mutation, selection and genetic drift. In particular, attenuation of vertically transmitted viruses was a consequence of the random accumulation of deleterious mutations, whereas horizontally transmitted viruses experiencing similar bottlenecks did not suffer the same fitness losses because effective bottleneck size was actually determined by the number of host individuals. In addition, high levels of viral fitness in horizontally transmitted populations were explained by competition among viral variants. V. Souza, M. Travisano, P.E. Turner, and L.E. Eguiarte. 2002. Does experimental evolution reflect patterns in natural populations? E. coli strains from long-term studies compared with wild isolates. Antonie von Leeuwenhoek Journal 81:143-153. AbstractOur results show that experimental evolution mimics evolution in nature. In particular, only 1000 generations of periodic recombination with immigrant genotypes is enough for linkage disequilibrium values in experimental populations to change from a maximum linkage value to a value similar to the one observed in wild strains of E. coli. Our analysis suggests an analogy between the recombination experiment and the evolutionary history of E. coli; the E. coli genome is a patchwork of genes laterally inserted in a common backbone, and the experimental E. coli chromosome is a patchwork where some sites are highly prone to recombination and others are very clonal. In addition, we propose a population model for wild E. coli where gene flow (recombination and migration) are an important source of genetic variation, and where certain hosts act as selective sieves; i.e., the host digestive system allows only certain strains to adhere and prosper as resident strains generating a particular microbiota in each host. Therefore we suggest that the strains from a wide range of wild hosts from different regions of the world may present an ecotypic structure where adaptation to the host may play an important role in the population structure. Turner, P.E., and L. Chao. 2003. Escape from prisoner’s dilemma in RNA phage Φ6. American Naturalist 161(3):497-505. Abstract: We previously examined competitive interactions among viruses by allowing the RNA phage Φ6 to evolve at high and low multiplicities of infection (ratio of infecting viruses to bacterial cells). Derived high-multiplicity phages were competitively advantaged relative to their ancestors during coinfection, but their fixation caused population fitness to decline. These data conform to the evolution of lowered fitness in a population of defectors, as expected from the Prisoner's Dilemma of game theory. However, the generality of this result is unknown; the evolution of viruses at other multiplicities may alter the fitness payoffs associated with conflicting strategies of cooperation and defection. Here we examine the change in matrix variables by propagating the ancestor under strictly clonal conditions, allowing cooperation the chance to evolve. In competitions involving derived cooperators and their selfish counterparts, our data reveal a new outcome where the two strategies are predicted to coexist in a mixed polymorphism. Thus, we demonstrate that the payoff matrix is not a constant in Φ6. Rather, clonal selection allows viruses the opportunity to escape the Prisoner's Dilemma. We discuss mechanisms that may afford selfish genotypes an advantage during intrahost competition and the relevance in our system for alternative ecological interactions among viruses. Turner, P.E. 2003. Searching for the advantages of virus sex. Origins of Life and Evolution of the Biosphere 33(1):95-108 Abstract:Sex (genetic exchange) is a nearly universal phenomenon in biological populations. But this is surprising given the costs associated with sex. For example, sex tends to break apart co-adapted genes, and sex causes a female to inefficiently contribute only half the genes to her offspring. Why then did sex evolve? One famous model poses that sex evolved to combat Muller’s ratchet, the mutational load that accrues when harmful mutations drift to high frequencies in populations of small size. In contrast, the Fisher-Muller Hypothesis predicts that sex evolved to promote genetic variation that speeds adaptation in novel environments. Sexual mechanisms occur in viruses, which feature high rates of deleterious mutation and frequent exposure to novel or changing environments. Thus, confirmation of one or both hypotheses would shed light on the selective advantages of virus sex. Experimental evolution has been used to test these classic models in the RNA bacteriophage Φ6, a virus that experiences sex via reassortment of its chromosomal segments. Empirical data suggest that sex might have originated in Φ6 to assist in purging deleterious mutations from the genome. However, results do not support the idea that sex evolved because it provides beneficial variation in novel environments. Rather, experiments show that too much sex can be bad for Φ6; promiscuity allows selfish viruses to evolve and spread their inferior genes to subsequent generations. Here I discuss various explanations for the evolution of segmentation in RNA viruses, and the added cost of sex when large numbers of viruses co-infect the same cell. Burch, C.L., Turner, P.E., and K. Hanley. 2003. Patterns of epistasis
in RNA viruses: a review of the evidence from vaccine design. Journal
of Evolutionary Biology 16:1223-1235. Abstract:Epistasis results when the fitness effects of a mutation change depending on the presence or absence of other mutations in the genome. The predictions of many influential evolutionary hypotheses are determined by the existence and form of epistasis. One rich source of data on the interactions among deleterious utations that has gone untapped by evolutionary biologists is the literature on the design of live, attenuated vaccine viruses. Rational vaccine design depends upon the measurement of individual and combined effects of deleterious mutations. In the current study we have reviewed data from 29 vaccine-oriented studies using 14 different RNA viruses. Our analyses indicate: (1) that no consistent tendency toward a particular form of epistasis exists across RNA viruses and (2) that significant interactions among groups of mutations within individual viruses occur but are not common. RNA viruses are significant pathogens of human disease, as well as tractable model systems for evolutionary studies - we discuss the relevance of our findings in both contexts. Turner, P.E. 2004. Phenotypic plasticity in bacterial plasmids.
Genetics 167:9-20. Abstract:Co-infection of a single host by multiple virus genotypes or species is common in nature, facilitating studies of ecological interactions between viruses at the cellular level. When two or more viruses co-infect the same host cell, this can have profound consequences for the fitness (growth performance) of an individual virus. Co-infection may be advantageous to an individual virus due to increased pathogenesis, enhanced transmission, or the opportunity for genetic exchange (sex) which produces the raw material for natural selection. In contrast, co-infection may be disadvantageous to an individual virus because it increases the likelihood of competition for proteins and other resource products available within the cell. One intriguing cost of co-infection is the recent evidence that intra-cellular interactions between viruses can be antagonistic, where a virus genotype evolves to specialize in parasitizing other co-infecting viruses. Here I review laboratory experiments involving the RNA bacteriophage Φ6, which demonstrate the evolution of parasitism when viruses are propagated in environments where co-infection is common. Frequency-dependent selection is shown to govern the fitness of these parasitic (cheater) genotypes, because their benefit of cheating depends on the relative abundance of ordinary and cheater genotypes encountered within the host cell. I relate why the evolution of parasitic viruses may be relevant for observed limits to the absolute number of phages that can simultaneously infect a single cell. The evolution of parasitic interactions in viruses infecting animal and plant hosts is briefly discussed. I suggest directions for future research on the evolutionary ecology of virus co-infection, especially the need to study phage interactions under natural (non laboratory) conditions. Froissart, R., C. Wilke, R. Montville, S. Remold, L. Chao, and
P.E. Turner. 2004. Co-infection weakens selection against epistatic
mutations in RNA viruses. Genetics 168:9-19. Abstract:Co-infection by multiple viruses affords opportunities for the evolution of cheating strategies to use intracellular resources. Cheating may be costly, however, when viruses infect cells alone. We previously allowed the RNA bacteriophage Φ6 to evolve for 250 generations in replicated environments allowing coinfection of Pseudomonas phaseolicola bacteria. Derived genotypes showed great capacity to compete during co-infection, but suffered reduced performance in solo infections. Thus, the evolved viruses appear to be cheaters that sacrifice between-host fitness for within-host fitness. It is unknown, however, which stage of the lytic growth cycle is linked to the cost of cheating. Here, we examine the cost through burst assays, where lytic infection can be separated into three discrete phases (analogous to phage life history): dispersal stage, latent period (juvenile stage), and burst (adult stage). We compared growth of a representative cheater and its ancestor in environments where the cost occurs. The cost of cheating was shown to be reduced fecundity, because cheaters feature a significantly smaller burst size (progeny produced per infected cell) when infecting on their own. Interestingly, latent period (average burst time) of the evolved virus was much longer than that of the ancestor, indicating the cost does not follow a life history trade-off between timing of reproduction and lifetime fecundity. Our data suggest that interference competition allows high fitness of derived cheaters in mixed infections, and we discuss preferential encapsidation as one possible mechanism. Dennehy, J.J. and P.E. Turner. 2004. Reduced fecundity is the cost of cheating in RNA virus Φ6. Proceedings of the Royal Society: Biological Sciences 271:2275-2282. Abstract:Co-infection by multiple viruses affords opportunities for the evolution of cheating strategies to use intracellular resources. Cheating may be costly, however, when viruses infect cells alone. We previously allowed the RNA bacteriophage Φ6 to evolve for 250 generations in replicated environments allowing coinfection of Pseudomonas phaseolicola bacteria. Derived genotypes showed great capacity to compete during co-infection, but suffered reduced performance in solo infections. Thus, the evolved viruses appear to be cheaters that sacrifice between-host fitness for within-host fitness. It is unknown, however, which stage of the lytic growth cycle is linked to the cost of cheating. Here, we examine the cost through burst assays, where lytic infection can be separated into three discrete phases (analogous to phage life history): dispersal stage, latent period (juvenile stage), and burst (adult stage). We compared growth of a representative cheater and its ancestor in environments where the cost occurs. The cost of cheating was shown to be reduced fecundity, because cheaters feature a significantly smaller burst size (progeny produced per infected cell) when infecting on their own. Interestingly, latent period (average burst time) of the evolved virus was much longer than that of the ancestor, indicating the cost does not follow a life history trade-off between timing of reproduction and lifetime fecundity. Our data suggest that interference competition allows high fitness of derived cheaters in mixed infections, and we discuss preferential encapsidation as one possible mechanism. Turner, P.E. 2005. Parasitism between co-infecting bacteriophages. Pp. 309-332 in R. Desharnais (ed.) Population Dynamics and Laboratory Ecology, vol. 37 of Advances in Ecological Research. Elsevier Press. Abstract:Co-infection of a single host by multiple virus genotypes or species is common in nature, facilitating studies of ecological interactions between viruses at the cellular level. When two or more viruses co-infect the same host cell, this can have profound consequences for the fitness (growth performance) of an individual virus. Co-infection may be advantageous to an individual virus due to increased pathogenesis, enhanced transmission, or the opportunity for genetic exchange (sex) which produces the raw material for natural selection. In contrast, co-infection may be disadvantageous to an individual virus because it increases the likelihood of competition for proteins and other resource products available within the cell. One intriguing cost of co-infection is the recent evidence that intra-cellular interactions between viruses can be antagonistic, where a virus genotype evolves to specialize in parasitizing other co-infecting viruses. Here I review laboratory experiments involving the RNA bacteriophage Φ6, which demonstrate the evolution of parasitism when viruses are propagated in environments where co-infection is common. Frequency-dependent selection is shown to govern the fitness of these parasitic (cheater) genotypes, because their benefit of cheating depends on the relative abundance of ordinary and cheater genotypes encountered within the host cell. I relate why the evolution of parasitic viruses may be relevant for observed limits to the absolute number of phages that can simultaneously infect a single cell. The evolution of parasitic interactions in viruses infecting animal and plant hosts is briefly discussed. I suggest directions for future research on the evolutionary ecology of virus co-infection, especially the need to study phage interactions under natural (non laboratory) conditions. Turner, P.E. 2005. Cheating viruses and game theory. American Scientist 93:428-435. Abstract:The theory of games can explain how viruses evolve when they compete against one another in a test of evolutionary fitness. Game theory has been applied to the experimental evolution of viruses in the laboratory. This is a field that is relatively new, but is proving to be powerful for testing fundamental questions in evolutionary biology. Silander, O., D. Weinreich, K. Wright, K. O'Keefe, C. Rang, P.E. Turner, and L. Chao. 2005. Widespread genetic exchange among terrestrial bacteriophages Proceedings of the National Academy of Sciences USA 102(52):19009-19014. Abstract:Bacteriophages are the most numerous entities in the biosphere. Despite this numerical dominance, the genetic structure of bacteriophage populations is poorly understood. Here we present a biogeography study involving 25 novel bacteriophages from the Cystoviridae clade, a group characterized by a double stranded RNA genome divided into three segments. Previous laboratory manipulation has found that when multiple Cystoviruses infect a single host cell, they undergo (i) rare intrasegment recombination events, and (ii) frequent genetic reassortment between segments. Analyzing linkage disequilibrium within segments, we find no evidence of intrasegment recombination in wild populations, consistent with (i). An extensive analysis of linkage disequilibrium between segments supports frequent reassortment, on a time scale similar to the genomic mutation rate. The absence of linkage disequilibrium within this group of phage is consistent with expectations for a completely sexual population, despite that some segments have more than 50% nucleotide divergence at four-fold degenerate sites. This extraordinary rate of genetic exchange between highly unrelated individuals is unprecedented in any taxa. We discuss our results in light of the biological species concept applied to viruses. Montville, R., R. Froissart, S.K. Remold, O. Tenaillon, and P.E. Turner. 2005. Evolution of mutational robustness in RNA viruses. PLoS Biology 3(11):1939-1945. Abstract:Mutational (genetic) robustness is phenotypic constancy in the face of mutational changes to the genome. Robustness is critical to the understanding of evolution because phenotypically expressed genetic variation is the fuel of natural selection. Nonetheless, the evidence for adaptive evolution of mutational robustness in biological populations is controversial. Robustness should be selectively favored when mutation rates are high, a common feature of RNA viruses. However, selection for robustness may be relaxed under virus co-infection because complementation between virus genotypes can buffer mutational effects. We therefore hypothesized that selection for genetic robustness in viruses will be weakened with increasing frequency of co-infection. To test this idea, we used populations of RNA phage Φ6 that were experimentally evolved at low and high levels of co-infection and subjected lineages of these viruses to mutation accumulation through population bottlenecking. The data demonstrate that viruses evolved under high co-infection show greater mean magnitude and variance in the fitness changes generated by addition of random mutations, confirming our hypothesis that they experience weakened selection for robustness. Our study further suggests that co-infection of host cells may be advantageous to RNA viruses only in the short term. In addition, we observed higher mutation rates in the more robust viruses, indicating that evolution of robustness might foster less-accurate genome replication in RNA viruses. O'Keefe, K.J., N.M. Morales, H. Ernstberger, G. Benoit, and P.E. Turner. 2006. Laboratory-dependent bacterial ecology: a cautionary tale. Applied and Environmental Microbiology 72:3032-3035. Abstract:Laboratory lore cautions that the behavior of cultured organisms can depend on subtle environmental factors and may sometimes even differ across laboratories. This is despite considerable efforts to employ identical diets and other prescribed environmental conditions at every location. Here we aim to demonstrate that laboratory dependence is real and quantifiable and that it raises interesting and valuable questions of general biological importance. We show that a simple bacterial community exhibits strikingly different behavior between two laboratories, in spite of standardized laboratory growth protocols. The community is composed of two Escherichia coli genotypes in a serial culture (seasonal) habitat. Previously, the two strains exhibited negatively frequency-dependent fitness which promoted their ecological coexistence at near equal frequencies. We show that when the pair of strains is moved to another laboratory they retain frequency-dependent fitness, but a new equilibrium is established in favor of one competitor. We attribute the altered competitive behavior to an unexpected difference in the culture medium between labs, most likely in the composition of deionized water. We show theoretically that the altered equilibrium could result from changes in each of two important growth parameters, Vmax (maximal growth rate) and Ks (resource concentration where growth is half-maximal). However, our empirical measurements rule out a large contribution by the parameter Ks. Duffy, S., P.E. Turner, and C. L. Burch. 2006. Pleiotropic costs of niche expansion in the RNA bacteriophage Φ6. Genetics 172:751-757. Abstract:Natural and experimental systems have failed to universally demonstrate a trade-off between generalism and specialism. When a trade-off does occur it is difficult to attribute its cause to antagonistic pleiotropy without dissecting the genetic basis of adaptation, and few previous experiments provide these genetic data. Here we investigate the evolution of expanded host range (generalism) in the RNA virus Φ6, an experimental model system allowing adaptive mutations to be readily identified. We isolated 10 spontaneous host range mutants on each of three novel Pseudomonas hosts and determined whether these mutations imposed fitness costs on the standard laboratory host. Sequencing revealed that each mutant had one of nine nonsynonymous initiations in the Φ6 gene P3, important in host attachment. Seven of these nine imitations were costly on the original host, confirming the existence of antagonistic pleiotropy. In addition to this genetically imposed cost, we identified an epigenetic cost of generalism that occurs when phage transition between host types. Our results confirm the existence in Φ6 of two costs of generalism, genetic and environmental, but they also indicate that the cost is not always large. The possibility for cost-free niche expansion implies that varied ecological conditions may favor host shifts in RNA viruses. Draghi, J., and P.E. Turner. 2006. DNA secretion and its implications for bacterial evolution. Microbiology 152:2683-2688. Abstract:Natural genetic transformation can facilitate gene transfer in many genera of bacteria and requires the presence of extracellular DNA. Although cell lysis can contribute to this extracellular DNA pool, several studies have suggested that the secretion of DNA from living bacteria may also provide genetic material for transformation. This paper reviews the evidence for specific secretion of DNA from intact bacteria into the extracellular environment and examines this behaviour from a population-genetics perspective. A mathematical model demonstrates that the joint action of DNA secretion and transformation creates a novel type of gene-level natural selection. This model demonstrates that gene-level selection could explain the existence of DNA secretion mechanisms that provide no benefit to individual cells or populations of bacteria. Additionally, the model predicts that any trait affecting DNA secretion will experience selection at the gene level in a transforming population. This analysis confirms that the secretion of DNA from intact bacterial cells is fully explicable with evolutionary theory, and reveals a novel mechanism for bacterial evolution. Dennehy, J.J., N.A. Friedenberg, Y.W. Yang, and P.E. Turner. 2006. Bacteriophage migration via nematode vectors: host-parasite-consumer interactions in laboratory microcosms. Applied and Environmental Microbiology 72:1974-1979. Abstract:Pathogens vectored by nematodes pose serious agricultural, economic, and health threats; however, little is known of the ecological and evolutionary aspects of pathogen transmission by nematodes. Here we describe a novel model system with two trophic levels, bacteriophages and nematodes, each of which competes for bacteria. We demonstrate for the first time that nematodes are capable of transmitting phages between spatially distinct patches of bacteria. This model system has considerable advantages, including the ease of maintenance and manipulation at the laboratory bench, the ability to observe many generations in short periods, and the capacity to freeze evolved strains for later comparison to their ancestors. More generally, experimental studies of complex multispecies interactions, host-pathogen coevolution, disease dynamics, and the evolution of virulence may benefit from this model system because current models (e.g., chickens, mosquitoes, and malaria parasites) are costly to maintain, are difficult to manipulate, and require considerable space. Our initial explorations centered on independently assessing the impacts of nematode, bacterium, and phage population densities on virus migration between host patches. Our results indicated that virus transmission increases with worm density and host bacterial abundance; however, transmission decreases with initial phage abundance, perhaps because viruses eliminate available hosts before migration can occur. We discuss the microbial growth dynamics that underlie these results, suggest mechanistic explanations for nematode transmission of phages, and propose intriguing possibilities for future research. Dennehy, J.J., N.A. Friedenberg, R.D. Holt, and P.E. Turner. 2006. Virus ecology and the maintenance of novel host use. American Naturalist 167:429-439. Abstract:Viruses can occasionally emerge by infecting new host species. However, the first phase of emergence hinges upon ecological sustainability of the virus population, which is a product of both within-host population growth and between-host transmission. Insufficient growth or transmission can force extinction of the virus population prior to the second phase of emergence, characterized by genetic adaptations that improve host use. We examined the primary phase of emergence by studying the population dynamics of RNA phages in replicated laboratory environments containing native and novel host bacteria. We developed a simple model based on in vitro data for phage growth rate over a range of initial population densities on both hosts, to predict the breadth of transmission rates allowing viral persistence on each species. Validation of these predictions using serial passage experiments revealed a range of transmission rates for which the native host was a source while the novel host was a sink. In this critical range of transmission rates, periodic exposure to the native host was sufficient for the maintenance of the viral population at high abundance on the novel host. We argue that this effect should facilitate adaptive evolution by the virus to utilize the novel host – a crucial second phase of emergence. Dennehy, J.J., N.A. Friedenberg, Y.W. Yang, and P.E. Turner. 2007. Virus population extinction via ecological traps. Ecology Letters 10: 230-240. Abstract:Populations are at risk of extinction when unsuitable or when sink habitat exceeds a threshold frequency in the environment. Sinks that present cues associated with high quality habitats, termed ecological traps, have especially detrimental effects on net population growth at metapopulation scales. Ecological traps for viruses arise naturally, or can be engineered, via the expression of viral-binding sites on cells that preclude viral reproduction. We present a model for virus population growth in a heterogeneous host community, parameterized with data from populations of the RNA bacteriophage phi-6 presented with mixtures of suitable host bacteria and either neutral or trap cells. We demonstrate that viruses can sustain high rates of population growth in the presence of neutral non-hosts as long as some host cells are present, whereas trap cells dramatically reduce viral fitness. In addition, we demonstrate that the efficacy of traps for viral elimination is frequency dependent in spatially structured environments such that population viability is a nonlinear function of habitat loss in dispersal-limited virus populations. We conclude that the ecological concepts applied to species conservation in altered landscapes can also contribute to the development of trap cell therapies for infectious human viruses. Dennehy, J.J., S.A. Abedon, and P.E. Turner. 2007. Host density impacts relative fitness of bacteriophage Φ6 genotypes in structured habitats. Evolution 61: 2516-2527 Abstract:Spatially structured environments may impact evolution by restricting population sizes, limiting opportunities for genetic mixis, or weakening selection against deleterious genotypes. When habitat structure impedes dispersal, low-productivity (less virulent) infectious parasites may benefit from their prudent exploitation of local hosts. Here we explored the combined ability for habitat structure and host density to dictate the relative reproductive success of differentially productive parasites. To do so, we allowed two RNA bacteriophage Φ6 genotypes to compete in structured and unstructured (semi-solid versus liquid) habitats while manipulating the density of Pseudomonas hosts. In the unstructured habitats, the more productive phage strain experienced a relatively constant fitness advantage regardless of starting host density. By contrast, in structured habitats, restricted phage dispersal may have magnified the importance of local productivity, thus allowing the relative fitness of the less productive virus to improve as host density increased. Further data suggested that latent period (duration of cellular infection) and especially burst size (viral progeny produced per cell) were the phage 'life history' traits most responsible for our results. We discuss the relevance of our findings for selection occurring in natural phage populations and for the general evolutionary epidemiology of infectious parasites. Duffy, S., C. L. Burch, and P.E. Turner. 2007. Evolution of host specificity drives reproductive isolation among RNA viruses. Evolution 61:2614-2622. Abstract:Ecological speciation hypotheses claim that assortative mating evolves as a consequence of divergent natural selection for ecologically important traits. Reproductive isolation is expected to be particularly likely to evolve by this mechanism in species such as phytophagous insects that mate in the habitats where they eat. We tested this expectation by monitoring the evolution of reproductive isolation in laboratory populations of an RNA virus that undergoes genetic exchange only when multiple virus genotypes co-infect the same host. We subjected four populations of the RNA bacteriophage 6 to 150 generations of natural selection on a novel host. Although there was no direct selection acting on host range in our experiment, three out of the four populations lost the ability to infect one or more alternative hosts. In the most extreme case, one of the populations evolved a host range that does not contain any of the hosts infectible by the wild type 6. Whole genome sequencing confirmed that the resulting reproductive isolation was due to a single nucleotide change, highlighting the ease with which an emerging RNA virus can decouple its evolutionary fate from that of its ancestor. Our results uniquely demonstrate the evolution of reproductive isolation in allopatric experimental populations. Furthermore, our data confirm the biological credibility of simple 'no-gene' mechanisms of assortative mating, in which this trait arises as a pleiotropic effect of genes responsible for ecological adaptation. Kysela, D.T., and P.E. Turner. 2007. Optimal bacteriophage mutation rates for phage therapy. Journal of Theoretical Biology 249:411-421. Abstract:The mutability of bacteriophages offers a particular advantage in the treatment of bacterial infections not afforded by other antimicrobial therapies. When phage-resistant bacteria emerge, mutation may generate phage capable of exploiting and thus limiting population expansion among these emergent types. However, while mutation potentially generates beneficial variants, it also contributes to a genetic load of deleterious mutations. Here we model the influence of varying phage mutation rate on the efficacy of phage therapy. All else being equal, phage types with historical mutation rates of approximately 0.1 deleterious mutations per genome per generation offer a reasonable balance between beneficial mutational diversity and deleterious mutational load. We determine that increasing phage inoculum density can undesirably increase the peak density of a mutant bacterial class by limiting the in situ production of mutant phage variants. Engineering increases in phage mutation rates may help to minimize mutational load and provide even greater protection against emergent bacterial types, but only with very weak selective coefficients for de novo deleterious mutations (below ~0.01). Increases to the mutation rate beyond the optimal value at mutation-selection balance may therefore prove generally undesirable. Remold, S., A. Rambaut, and P.E. Turner. 2008. Evolutionary genomics of host adaptation in VSV. Molecular Biology and Evolution 25(6):1138-1147. Abstract:Populations experiencing similar selection pressures can sometimes diverge in the genetic architectures underlying evolved complex traits. We used RNA virus populations of large size and high mutation rate to study the impact of historical environment on genome evolution, thus increasing our ability to detect repeatable patterns in the evolution of genetic architecture. Experimental vesicular stomatitis virus (VSV) populations were evolved on HeLa cells, on MDCK cells, or on alternating hosts. Turner and Elena (2000) previously showed that virus populations evolved in single-host environments achieved high fitness on their selected hosts but failed to increase in fitness relative to their ancestor on the unselected host, and that alternating-host-evolved populations had high fitness on both hosts. Here we determined the complete consensus sequence for each evolved population after 95 generations to gauge whether the parallel phenotypic changes were associated with parallel genomic changes. We also analyzed the patterns of allele substitutions to discern whether differences in fitness across hosts arose through true pleiotropy, or the presence of a mutation that is beneficial in both hosts but also one or more mutation(s) at other loci that are costly in the unselected environment (mutation accumulation). We found that ecological history may influence to what extent pleiotropy and mutation accumulation contribute to fitness asymmetries across environments. We discuss the degree to which current genetic architecture is expected to constrain future evolution of complex traits, such as host use by RNA viruses. McBride, R.C., C.B. Ogbunugafor, and P.E. Turner. 2008. Robustness promotes evolvability to thermotolerance in an RNA virus. BMC Evolutionary Biology. (In press). Abstract:The ability for an evolving population to adapt to a novel environment is achieved through a balance of robustness and evolvability. Robustness is the invariance of phenotype in the face of perturbation and evolvability is the capacity to adapt in response to selection. Genetic robustness has been posited, depending on the underlying mechanism, to either decrease the efficacy of selection, or increase the possibility of future adaptation. However, the true effect of genetic robustness on evolvability in biological systems remains uncertain. Here we demonstrate that genetic robustness increases evolvability in laboratory populations of the RNA virus phi-6. We observed that populations founded by robust clones evolved greater resistance to heat shock, relative to populations founded by brittle (less-robust) clones. Thus, we provide empirical evidence for the idea that robustness can promote evolvability, and further suggest that evolvability can arise indirectly via selection for robustness, rather than through direct selective action. Our data imply that greater tolerance of mutational change is associated with virus adaptability in a new niche, a finding generally relevant to evolutionary biology, and informative for elucidating how viruses might evolve to emerge in new habitats and/or overcome novel therapies. Abedon, S.A., S. Duffy, and P.E. Turner. 2008. Bacteriophage, Ecology, in The Encyclopaedia of Microbiology, 3rd Edition, M. Schaechter (ed.), Elsevier (In press). Abstract:Bacteriophages, also known as phages, are the viruses that infect bacteria. Phages are extremely abundant in aquatic and terrestrial environments, and are seemingly present wherever their host bacteria can thrive. Phage ecology is the study of the interactions between phages and biological populations and communities, including other phages, bacteria and eukaryotes. In addition, phage ecologists study phage interactions with, and impacts on, the abiotic components of habitats, often in the contexts of energy flow and nutrient cycling. Here we review phage ecology from several perspectives: phage environmental microbiology (phage prevalence, diversity, functioning, etc. within environments), phage population ecology (phage interactions with similar phages), phage community ecology (phage interactions with dissimilar phages and other organisms), and phage ecosystem ecology (phage impacts on the biotic and abiotic aspects of ecosystems). |