Multihost experimental evolution of a plant RNA virus reveals local adaptation and host-specific mutations

For multihost pathogens, adaptation to multiple hosts has important implications for both applied and basic research. At the applied level, it is one of the main factors determining the probability and the severity of emerging disease outbreaks. At the basic level, it is thought to be a key mechanism for the maintenance of genetic diversity both in host and pathogen species. Using Tobacco etch potyvirus (TEV) and four natural hosts, we have designed an evolution experiment whose strength and novelty are the use of complex multicellular host organism as hosts and a high level of replication of different evolutionary histories and lineages. A pattern of local adaptation, characterized by a higher infectivity and virulence on host(s) encountered during the experimental evolution was found. Local adaptation only had a cost in terms of performance on other hosts in some cases. We could not verify the existence of a cost for generalists, as expected to arise from antagonistic pleiotropy and other genetic mechanisms generating a fitness trade-off between hosts. This observation confirms that this classical theoretical prediction lacks empirical support. We discuss the reasons for this discrepancy between theory and experiment in the light of our results. The analysis of full genome consensus sequences of the evolved lineages established that all mutations shared between lineages were host specific. A low degree of parallel evolution was observed, possibly reflecting the various adaptive pathways available for TEV in each host. Altogether, these results reveal a strong adaptive potential of TEV to new hosts without severe evolutionary constraints.     

Evolutionary genetics of aging in Daphnia

Evolutionary theory of aging stipulates that aging is inevitable consequence of low effectiveness of natural selection acting on traits expressed late in the life span of the organisms. Two main hypotheses exist: the neutralist mutation-accumulation theory and selectionist antagonistic pleiotropy theory. Both theories predict the increase of genetic variance with age; the antagonistic pleiotropy theory also predicts negative genetic correlation between fitness related traits in the beginning and in the end of the life span. In order to test these predictions we measured life expectancy and age specific mortality in cohorts of 26 c lones of Daphnia magna extracted from a single cyclic parthenogen population. Simultaneously, fecundity and age to maturity were measured in representatives of the same clones. Log mortality increased linearly with age, with little evidence for leveling off, although some replicate cohorts did show a significant leveling off of mortality. Genetic variance of log mortality was significantly higher in the last quarter of the life span than in earlier time intervals. There was a significant positive genetic correlation between early fecundity and early mortality, but not between early fecundity and late mortality. This indicates that, although there is a trade-off between fecundity and survival, this trade-off is not based on pleiotropy across ages and therefore the data does not support the prediction of antagonistic pleiotropy theory.     

Sex and the evolution of intrahost competition in RNA virus phi6.

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 phi6 when multiple bacteriophages coinfect the same host cell. Here, we tested the model''s predictions by determining whether sex favors more rapid adaptation of phi6 to a bacterial host, Pseudomonas phaseolicola. Replicate populations of phi6 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 phi6, and suggest that genetic conflicts can evolve in RNA viruses.     

The effect of purging on sexually selected traits through antagonistic pleiotropy with survival

Sexually selected traits are expected to evolve to a point where their positive effect on reproductive success is counterbalanced by their negative effect on survival. At the genetic level, such a trade-off implies antagonistic pleiotropy between survival and the expression of sexually selected traits. Yet, the consequences of such a genetic architecture have been largely overlooked in studies examining how inbreeding influences sexually selected traits. These studies have solely interpreted their results as an effect of increased homozygosity. An alternative, however, is that purging of recessive alleles deleterious for survival when inbreeding increases can negatively affect the expression of sexually selected traits through antagonistic pleiotropy. We tested this hypothesis by analyzing the effects of inbreeding on several male ornaments and life-history traits across 20 captive populations of guppies (Poecilia reticulata) with varying levels of inbreeding. Only one ornament, orange area, decreased in its expression with an increasing level of inbreeding. This was most likely due to purging because we found no within-population relationship between orange area and the inbreeding coefficient. We further tested this hypothesis by crossing unrelated individuals from the four most inbred populations, creating a group of individuals with purged genomes but restored heterozygosity. Restoration of heterozygosity only slightly increased orange area, confirming that the decrease in orange area in the inbred populations most likely resulted from purging. These results support previous studies suggesting the existence of antagonistic pleiotropy between ornament expression and survival.     

ANTAGONISTIC PLEIOTROPIC EFFECT OF SECOND-CHROMOSOME INVERSIONS ON BODY SIZE AND EARLY LIFE-HISTORY TRAITS IN DROSOPHILA BUZZATII

A simple way to think of evolutionary trade-offs is to suppose genetic effects of opposed direction that give rise to antagonistic pleiotropy. Maintenance of additive genetic variability for fitness related characters, in association with negative correlations between these characters, may result. In the cactophilic species Drosophila buzzatii, there is evidence that second-chromosome polymorphic inversions affect size-related traits. Because a trade-off between body size and larval developmental time has been reported in Drosophila, we study here whether or not these inversions also affect larva-adult viability and developmental time. In particular, we expect that polymorphic inversions make a statistically significant contribution to the genetic correlation between body size (as measured by thorax length) and larval developmental time. This contribution is expected to be in the direction predicted by the trade-off, namely, those flies whose karyotypes cause them to be genetically larger should also have a longer developmental time than flies with other karyotypes. Using two different experimental approaches, a statistically significant contribution of the second-chromosome inversions to the phenotypic variances of body size and developmental time in D. buzzatii was found. Further, these inversions make a positive contribution to the total genetic correlation between the traits, as expected by the suggested trade-off. The data do not provide evidence as to whether the genetic correlation is due to antagonistic pleiotropic gene action or to gametic disequilibrium of linked genes that affect one or both traits. The results do suggest, however, a possible explanation for the maintenance of inversion polymorphism in this species.     

The effect of antagonistic pleiotropy on the estimation of the average coefficient of dominance of deleterious mutations

We investigate the impact of antagonistic pleiotropy on the most widely used methods of estimation of the average coefficient of dominance of deleterious mutations from segregating populations. A proportion of the deleterious mutations affecting a given studied fitness component are assumed to have an advantageous effect on another one, generating overdominance on global fitness. Using diffusion approximations and transition matrix methods, we obtain the distribution of gene frequencies for nonpleiotropic and pleiotropic mutations in populations at the mutation-selection-drift balance. From these distributions we build homozygous and heterozygous chromosomes and assess the behavior of the estimators of dominance. A very small number of deleterious mutations with antagonistic pleiotropy produces substantial increases on the estimate of the average degree of dominance of mutations affecting the fitness component under study. For example, estimates are increased three- to fivefold when 2% of segregating loci are over-dominant for fitness. In contrast, strengthening pleiotropy, where pleiotropic effects are assumed to be also deleterious, has little effect on the estimates of the average degree of dominance, supporting previous results. The antagonistic pleiotropy model considered, applied under mutational parameters described in the literature, produces patterns for the distribution of chromosomal viabilities, levels of genetic variance, and homozygous mutation load generally consistent with those observed empirically for viability in Drosophila melanogaster.     

A search for trade-offs among life history traits inDrosophila melanogaster

Summary Are there underlying developmental and physiological properties of organisms that can be used to build a general theory of life history evolution? Much of the theoretical work on the evolution of life histories is based on the premise of negative developmental and genetic correlations among life history traits. If negative correlations do not exist as a general rule then no general theory taking them into account is possible. Negative genetic correlations among life history traits can come about by antagonistic pleiotropy. One cause of antagonistic pleiotropy is cost allocation trade-offs. Since cost allocation trade-offs are due to underlying physiological constraints they are expected to be common to closely related groups. A second form of antagonistic pleiotropy is specialization of genotypes to different niches. This type of antagonistic pleiotropy is expected to be specific to each population. We looked for trade-offs in life history traits of longevity and fecundity inDrosophila melanogaster. We used a half-sib mating design and raised the offspring at two temperatures, 19°C and 25°C. Correlations between longevity and fecundity showed some evidence of antagonistic pleiotropy at high temperature with no evidence of any trade-offs at low temperature. Correlations of early and late fecundity traits did show evidence of cost allocation trade-offs at both temperatures. Antagonistic pleiotropy was also found for cross-environmental correlations of fecundity traits. We conclude that, although life history trade-offs can not be generally assumed, they are frequently found among functionally related traits. Thus, we provide guidelines for the development of general theories of life history evolution.     

The experimental evolution of herbicide resistance in Chlamydomonas reinhardtii results in a positive correlation between fitness in the presence and absence of herbicides

Pleiotropic fitness trade-offs will be key determinants of the evolutionary dynamics of selection for pesticide resistance. However, for herbicide resistance, empirical support for a fitness cost of resistance is mixed, and it is therefore also questionable what further ecological trade-offs can be assumed to apply to herbicide resistance. Here, we test the existence of trade-offs by experimentally evolving herbicide resistance in Chlamydomonas reinhardtii. Although fitness costs are detected for all herbicides, we find that, counterintuitively, the most resistant populations also have the lowest fitness costs as measured by growth rate in the ancestral environment. Furthermore, after controlling for differences in the evolutionary dynamics of resistance to different herbicides, we also detect significant positive correlations between resistance, fitness in the ancestral environment and cross-resistance to other herbicides. We attribute this to the highest levels of nontarget-site resistance being achieved by fixing mutations that more broadly affect cellular physiology, which results in both more cross-resistance and less overall antagonistic pleiotropy on maximum growth rate. Consequently, the lack of classical ecological trade-offs could present a major challenge for herbicide resistance management.     

Sexual antagonism for resistance and tolerance to infection in Drosophila melanogaster

A critical task in evolutionary genetics is to explain the persistence of heritable variation in fitness-related traits such as immunity. Ecological factors can maintain genetic variation in immunity, but less is known about the role of other factors, such as antagonistic pleiotropy, on immunity. Sexually dimorphic immunity—with females often being more immune-competent—may maintain variation in immunity in dioecious populations. Most eco-immunological studies assess host resistance to parasites rather than the host''s ability to maintain fitness during infection (tolerance). Distinguishing between resistance and tolerance is important as they are thought to have markedly different evolutionary and epidemiological outcomes. Few studies have investigated tolerance in animals, and the extent of sexual dimorphism in tolerance is unknown. Using males and females from 50 Drosophila melanogaster genotypes, we investigated possible sources of genetic variation for immunity by assessing both resistance and tolerance to the common bacterial pathogen Pseudomonas aeruginosa. We found evidence of sexual dimorphism and sexual antagonism for resistance and tolerance, and a trade-off between the two traits. Our findings suggest that antagonistic pleiotropy may be a major contributor to variation in immunity, with implications for host–parasite coevolution.     

A TRADE-OFF FOR HOST PLANT UTILIZATION IN THE BLACK BEAN APHID,APHIS FABAE

Many studies on insect herbivores have sought to find trade-offs between utilization of alternate host plants, both to understand the prevalence of specialization and to appreciate the likelihood of sympatric speciation due to disruptive selection. To date, few studies have found trade-offs. Seventy-seven clones of the black bean aphid, Aphis fabae, were collected from field sites in East Anglia, U.K., over an area of about 10,000 km². These clones exhibit a trade-off in fitness between two alternative hosts, broad bean and nasturtium. This pattern is maintained in the F2 generation. The predominance of broad bean in the area, the fact that clones were only sampled from one of these two hosts, and the absence of “master-of-all-trades” genotypes after recombination all point to the importance of antagonistic pleiotropy rather than linkage disequilibrium in maintaining this trade-off. It is concluded that this population presents strong evidence for a fundamental trade-off for host utilization.     

High frequency of mutations that expand the host range of an RNA virus

The ability of a virus population to colonize a novel host is predicted to depend on the equilibrium frequency of potential colonists (i.e., genotypes capable of infecting the novel host) in the source population. In this study, we investigated the determinants of the equilibrium frequency of potential colonists in the RNA bacteriophage 6. We isolated 40 spontaneous mutants capable of infecting a novel Pseudomonas syringae host and sequenced their host attachment genes to identify the responsible mutations. We observed 16 different mutations in the host attachment gene and used a new statistical approach to estimate that 39 additional mutations were missed by our screen. Phenotypic and fitness assays confirmed that the proximate mechanism underlying host range expansion was an increase in the ability to attach to the novel host and that acquisition of this ability most often imposed a cost for growth rate on two standard hosts. Considered in a population genetic framework, our data suggest that host range mutations should exist in phage populations at an equilibrium frequency (3 x 10(-4)) that exceeds the phage mutation rate by more than two orders of magnitude. Thus, colonization of novel hosts is unlikely to be limited by an inability to produce appropriate mutations.     

Frequency and Fitness Consequences of Bacteriophage Φ6 Host Range Mutations

Viruses readily mutate and gain the ability to infect novel hosts, but few data are available regarding the number of possible host range-expanding mutations allowing infection of any given novel host, and the fitness consequences of these mutations on original and novel hosts. To gain insight into the process of host range expansion, we isolated and sequenced 69 independent mutants of the dsRNA bacteriophage Φ6 able to infect the novel host, Pseudomonas pseudoalcaligenes. In total, we found at least 17 unique suites of mutations among these 69 mutants. We assayed fitness for 13 of 17 mutant genotypes on P. pseudoalcaligenes and the standard laboratory host, P. phaseolicola. Mutants exhibited significantly lower fitnesses on P. pseudoalcaligenes compared to P. phaseolicola. Furthermore, 12 of the 13 assayed mutants showed reduced fitness on P. phaseolicola compared to wildtype Φ6, confirming the prevalence of antagonistic pleiotropy during host range expansion. Further experiments revealed that the mechanistic basis of these fitness differences was likely variation in host attachment ability. In addition, using computational protein modeling, we show that host-range expanding mutations occurred in hotspots on the surface of the phage''s host attachment protein opposite a putative hydrophobic anchoring domain.     

The basis of antagonistic pleiotropy in hfq mutations that have opposite effects on fitness at slow and fast growth rates

Mutations beneficial in one environment may cause costs in different environments, resulting in antagonistic pleiotropy. Here, we describe a novel form of antagonistic pleiotropy that operates even within the same environment, where benefits and deleterious effects exhibit themselves at different growth rates. The fitness of hfq mutations in Escherichia coli affecting the RNA chaperone involved in small-RNA regulation is remarkably sensitive to growth rate. E. coli populations evolving in chemostats under nutrient limitation acquired beneficial mutations in hfq during slow growth (0.1 h⁻¹) but not in populations growing sixfold faster. Four identified hfq alleles from parallel populations were beneficial at 0.1 h⁻¹ and deleterious at 0.6 h⁻¹. The hfq mutations were beneficial, deleterious or neutral at an intermediate growth rate (0.5 h⁻¹) and one changed from beneficial to deleterious within a 36 min difference in doubling time. The benefit of hfq mutations was due to the greater transport of limiting nutrient, which diminished at higher growth rates. The deleterious effects of hfq mutations at 0.6 h⁻¹ were less clear, with decreased viability a contributing factor. The results demonstrate distinct pleiotropy characteristics in the alleles of the same gene, probably because the altered residues in Hfq affected the regulation of expression of different genes in distinct ways. In addition, these results point to a source of variation in experimental measurement of the selective advantage of a mutation; estimates of fitness need to consider variation in growth rate impacting on the magnitude of the benefit of mutations and on their fitness distributions.     

Antagonistic and reinforcing pleiotropy: a study of differences in development time in wing dimorphic insects

Morphological dimorphisms are found in many different taxa. Wing dimorphism in insects, in which some individuals possess wings and associated flight muscles and are thus volant while others lack a functional flight apparatus and are thus flightless, is a typical example of such types of dimorphisms. It has been extensively studied and such studies have demonstrated that the volant form, although possessing the advantage of flight capability, suffers a fitness cost in a delay in the onset of reproduction after emergence into the adult form and a reduced fecundity. Previous comparative analyses have suggested that there is no consistent trend for development time (hatching to adult) to differ between the two morphs. The present study analyses the phenotypic and genetic correlations between development time and wing morph in the cricket Gryllus firmus. It is shown that the macropterous (volant) morph develops faster than the micropterous (flightless morph). This trade-off is manifested at both thephenotypic and genetic level. Further, a comparative analysis shows that the same phenotypic trade-off is generally found in other Orthopteran species so far studied, but in other orders the micropterous morph develops faster. Provided that the phenotypic trade-off is genetically based, in the Orthoptera the fitness advantage of the earlier onset of reproduction in micropterous females is offset by the extended development time (antagonistic pleiotropy). However, in other orders there is reinforcing pleiotropy in that the micropterous females develop faster and reproduce sooner than the macropterous morph. These results highlight the complexity of fitness interactions and the need to study a phenomenon across several taxa.     

Selective Pressure Causes an RNA Virus to Trade Reproductive Fitness for Increased Structural and Thermal Stability of a Viral Enzyme

The modulation of fitness by single mutational substitutions during environmental change is the most fundamental consequence of natural selection. The antagonistic tradeoffs of pleiotropic mutations that can be selected under changing environments therefore lie at the foundation of evolutionary biology. However, the molecular basis of fitness tradeoffs is rarely determined in terms of how these pleiotropic mutations affect protein structure. Here we use an interdisciplinary approach to study how antagonistic pleiotropy and protein function dictate a fitness tradeoff. We challenged populations of an RNA virus, bacteriophage Φ6, to evolve in a novel temperature environment where heat shock imposed extreme virus mortality. A single amino acid substitution in the viral lysin protein P5 (V207F) favored improved stability, and hence survival of challenged viruses, despite a concomitant tradeoff that decreased viral reproduction. This mutation increased the thermostability of P5. Crystal structures of wild-type, mutant, and ligand-bound P5 reveal the molecular basis of this thermostabilization—the Phe207 side chain fills a hydrophobic cavity that is unoccupied in the wild-type—and identify P5 as a lytic transglycosylase. The mutation did not reduce the enzymatic activity of P5, suggesting that the reproduction tradeoff stems from other factors such as inefficient capsid assembly or disassembly. Our study demonstrates how combining experimental evolution, biochemistry, and structural biology can identify the mechanisms that drive the antagonistic pleiotropic phenotypes of an individual point mutation in the classic evolutionary tug-of-war between survival and reproduction.     

Intra-locus sexual conflict and sexually antagonistic genetic variation in hermaphroditic animals

Intra-locus sexual conflict results when sex-specific selection pressures for a given trait act against the intra-sexual genetic correlation for that trait. It has been found in a wide variety of taxa in both laboratory and natural populations, but the importance of intra-locus sexual conflict and sexually antagonistic genetic variation in hermaphroditic organisms has rarely been considered. This is not so surprising given the conceptual and theoretical association of intra-locus sexual conflict with sexual dimorphism, but there is no a priori reason why intra-locus sexual conflict cannot occur in hermaphroditic organisms as well. Here, I discuss the potential for intra-locus sexual conflict in hermaphroditic animals and review the available evidence for such conflict, and for the existence of sexually antagonistic genetic variation in hermaphrodites. I argue that mutations with asymmetric effects are particularly likely to be important in mediating sexual antagonism in hermaphroditic organisms. Moreover, sexually antagonistic genetic variation is likely to play an important role in inter-individual variation in sex allocation and in transitions to and from gonochorism (separate sexes) in simultaneous hermaphrodites. I also describe how sequential hermaphrodites may experience a unique form of intra-locus sexual conflict via antagonistic pleiotropy. Finally, I conclude with some suggestions for further research.     

Emergence of mammalian cell-adapted vesicular stomatitis virus from persistent infections of insect vector cells

Arboviruses (arthropod-borne viruses) represent quintessential generalists, with the ability to infect and perform well in multiple hosts. However, antagonistic pleiotropy imposed a cost during the adaptation to persistent replication of vesicular stomatitis virus in sand fly cells and resulted in strains that initially replicated poorly in hamster cells, even when the virus was allowed to replicate periodically in the latter. Once a debilitated strain started replicating continuously in mammalian cells, fitness increased significantly. Fitness recovery did not entail back mutations or compensatory mutations, but instead, we observed the replacement of persistence-adapted genomes by mammalian cell-adapted strains with a full set of new, unrelated sequence changes. These mammalian cell-adapted genomes were present at low frequencies in the populations with a history of persistence for up to a year and quickly became dominant during mammalian infection, but coexistence was not stable in the long term. Periodic acute replication in mammalian cells likely contributed to extending the survival of minority genomes, but these genomes were also found in strictly persistent populations.