Historical Contingency Drives Compensatory Evolution and Rare Reversal of Phage Resistance.

Bacteria and lytic viruses (phages) engage in highly dynamic coevolutionary interactions over time, yet we have little idea of how transient selection by phages might shape the future evolutionary trajectories of their host populations. To explore this question, we generated genetically diverse phage-resistant mutants of the bacterium Pseudomonas syringae. We subjected the panel of mutants to prolonged experimental evolution in the absence of phages. Some populations re-evolved phage sensitivity, whereas others acquired compensatory mutations that reduced the costs of resistance without altering resistance levels. To ask whether these outcomes were driven by the initial genetic mechanisms of resistance, we next evolved independent replicates of each individual mutant in the absence of phages. We found a strong signature of historical contingency: some mutations were highly reversible across replicate populations, whereas others were highly entrenched. Through whole-genome sequencing of bacteria over time, we also found that populations with the same resistance gene acquired more parallel sets of mutations than populations with different resistance genes, suggesting that compensatory adaptation is also contingent on how resistance initially evolved. Our study identifies an evolutionary ratchet in bacteria-phage coevolution and may explain previous observations that resistance persists over time in some bacterial populations but is lost in others. We add to a growing body of work describing the key role of phages in the ecological and evolutionary dynamics of their host communities. Beyond this specific trait, our study provides a new insight into the genetic architecture of historical contingency, a crucial component of interpreting and predicting evolution.

Genomic and phenotypic signatures of bacteriophage coevolution with the phytopathogen Pseudomonas syringae.

The rate and trajectory of evolution in an obligate parasite is critically dependent on those of its host(s). Adaptation to a genetically homogeneous host population should theoretically result in specialization, while adaptation to an evolving host population (i.e., coevolution) can result in various outcomes including diversification, range expansion, and/or local adaptation. For viruses of bacteria (bacteriophages, or phages), our understanding of how evolutionary history of the bacterial host(s) impacts viral genotypic and phenotypic evolution is currently limited. In this study, we used whole genome sequencing and two different metrics of phage impacts to compare the genotypes and phenotypes of lytic phages that had either coevolved with or were repeatedly passaged on an unchanging (ancestral) strain of the phytopathogen Pseudomonas syringae. Genomes of coevolved phages had more mutations than those of phages passaged on a constant host, and most mutations were in genes encoding phage tail-associated proteins. Phages from both passaging treatments shared some phenotypic outcomes, including range expansion and divergence across replicate populations, but coevolved phages were more efficient at reducing population growth (particularly of sympatric coevolved hosts). Genotypic similarity correlated with infectivity profile similarity in coevolved phages, but not in phages passaged on the ancestral host. Overall, while adaptation to either host type (coevolving or ancestral) led to divergence in phage tail proteins and infectivity patterns, coevolution led to more rapid molecular changes that increased bacterial killing efficiency and had more predictable effects on infectivity range. Together, these results underscore the important role of hosts in driving viral evolution and in shaping the genotype-phenotype relationship.

High-throughput mapping of the phage resistance landscape in E. coli.

Bacteriophages (phages) are critical players in the dynamics and function of microbial communities and drive processes as diverse as global biogeochemical cycles and human health. Phages tend to be predators finely tuned to attack specific hosts, even down to the strain level, which in turn defend themselves using an array of mechanisms. However, to date, efforts to rapidly and comprehensively identify bacterial host factors important in phage infection and resistance have yet to be fully realized. Here, we globally map the host genetic determinants involved in resistance to 14 phylogenetically diverse double-stranded DNA phages using two model Escherichia coli strains (K-12 and BL21) with known sequence divergence to demonstrate strain-specific differences. Using genome-wide loss-of-function and gain-of-function genetic technologies, we are able to confirm previously described phage receptors as well as uncover a number of previously unknown host factors that confer resistance to one or more of these phages. We uncover differences in resistance factors that strongly align with the susceptibility of K-12 and BL21 to specific phage. We also identify both phage-specific mechanisms, such as the unexpected role of cyclic-di-GMP in host sensitivity to phage N4, and more generic defenses, such as the overproduction of colanic acid capsular polysaccharide that defends against a wide array of phages. Our results indicate that host responses to phages can occur via diverse cellular mechanisms. Our systematic and high-throughput genetic workflow to characterize phage-host interaction determinants can be extended to diverse bacteria to generate datasets that allow predictive models of how phage-mediated selection will shape bacterial phenotype and evolution. The results of this study and future efforts to map the phage resistance landscape will lead to new insights into the coevolution of hosts and their phage, which can ultimately be used to design better phage therapeutic treatments and tools for precision microbiome engineering.

Successive passaging of a plant-associated microbiome reveals robust habitat and host genotype-dependent selection.

There is increasing interest in the plant microbiome as it relates to both plant health and agricultural sustainability. One key unanswered question is whether we can select for a plant microbiome that is robust after colonization of target hosts. We used a successive passaging experiment to address this question by selecting upon the tomato phyllosphere microbiome. Beginning with a diverse microbial community generated from field-grown tomato plants, we inoculated replicate plants across 5 plant genotypes for 4 45-d passages, sequencing the microbial community at each passage. We observed consistent shifts in both the bacterial (16S amplicon sequencing) and fungal (internal transcribed spacer region amplicon sequencing) communities across replicate lines over time, as well as a general loss of diversity over the course of the experiment, suggesting that much of the naturally observed microbial community in the phyllosphere is likely transient or poorly adapted within the experimental setting. We found that both host genotype and environment shape microbial composition, but the relative importance of genotype declines through time. Furthermore, using a community coalescence experiment, we found that the bacterial community from the end of the experiment was robust to invasion by the starting bacterial community. These results highlight that selecting for a stable microbiome that is well adapted to a particular host environment is indeed possible, emphasizing the great potential of this approach in agriculture and beyond. In light of the consistent response of the microbiome to selection in the absence of reciprocal host evolution (coevolution) described here, future studies should address how such adaptation influences host health.

Multiyear Time-Shift Study of Bacteria and Phage Dynamics in the Phyllosphere.

AbstractCoevolution shapes diversity within and among populations but is difficult to study directly. Time-shift experiments, where individuals from one point in time are experimentally challenged against individuals from past, contemporary, and/or future time points, are a powerful tool to measure coevolution. This approach has proven useful both in directly measuring coevolutionary change and in distinguishing among coevolutionary models. However, these data are only as informative as the time window over which they were collected, and inference from shorter coevolutionary windows might conflict with those from longer time periods. Previous time-shift experiments from natural microbial communities of horse chestnut tree leaves uncovered an apparent asymmetry, whereby bacterial hosts were more resistant to bacteriophages from all earlier points in the growing season, while phages were most infective to hosts from only the recent past. Here, we extend the time window over which these infectivity and resistance ranges are observed across years and confirm that the previously observed asymmetry holds over longer timescales. These data suggest that existing coevolutionary theory should be revised to include the possibility of differing models for hosts and their parasites and examined for how such asymmetries might reshape the predicted outcomes of coevolution.

Temporally Selective Modification of the Tomato Rhizosphere and Root Microbiome by Volcanic Ash Fertilizer Containing Micronutrients.

Food crops are grown with fertilizers containing nitrogen, phosphorus, and potassium (macronutrients) along with magnesium, calcium, boron, and zinc (micronutrients) at different ratios during their cultivation. Soil and plant-associated microbes have been implicated to promote plant growth, stress tolerance, and productivity. However, the high degree of variability across agricultural environments makes it difficult to assess the possible influences of nutrient fertilizers on these microbial communities. Uncovering the underlying mechanisms could lead us to achieve consistently improved food quality and productivity with minimal environmental impacts. For this purpose, we tested a commercially available fertilizer (surface-mined volcanic ash deposit Azomite) applied as a supplement to the normal fertilizer program of greenhouse-grown tomato plants. Because this treatment showed a significant increase in fruit production at measured intervals, we examined its impact on the composition of below-ground microbial communities, focusing on members identified as "core taxa" that were enriched in the rhizosphere and root endosphere compared to bulk soil and appeared above their predicted neutral distribution levels in control and treated samples. This analysis revealed that Azomite had little effect on microbial composition overall, but it had a significant, temporally selective influence on the core taxa. Changes in the composition of the core taxa were correlated with computationally inferred changes in functional pathway enrichment associated with carbohydrate metabolism, suggesting a shift in available microbial nutrients within the roots. This finding exemplifies how the nutrient environment can specifically alter the functional capacity of root-associated bacterial taxa, with the potential to improve crop productivity. IMPORTANCE Various types of soil fertilizers are used routinely to increase crop yields globally. The effects of these treatments are assessed mainly by the benefits they provide in increased crop productivity. There exists a gap in our understanding of how soil fertilizers act on the plant-associated microbial communities. The underlying mechanisms of nutrient uptake are widely complex and, thus, difficult to evaluate fully but have critical influences on both soil and plant health. Here, we presented a systematic approach to analyzing the effects of fertilizer on core microbial communities in soil and plants, leading to predictable outcomes that can be empirically tested and used to develop simple and affordable field tests. The methods described here can be used for any fertilizer and crop system. Continued effort in advancing our understanding of how fertilizers affect plant and microbe relations is needed to advance scientific understanding and help growers make better-informed decisions.

Water stress and disruption of mycorrhizas induce parallel shifts in phyllosphere microbiome composition.

Water and nutrient acquisition are key drivers of plant health and ecosystem function. These factors impact plant physiology directly as well as indirectly through soil- and root-associated microbial responses, but how they in turn affect aboveground plant-microbe interactions are not known. Through experimental manipulations in the field and growth chamber, we examine the interacting effects of water stress, soil fertility, and arbuscular mycorrhizal fungi on bacterial and fungal communities of the tomato (Solanum lycopersicum) phyllosphere. Both water stress and mycorrhizal disruption reduced leaf bacterial richness, homogenized bacterial community composition among plants, and reduced the relative abundance of dominant fungal taxa. We observed striking parallelism in the individual microbial taxa in the phyllosphere affected by irrigation and mycorrhizal associations. Our results show that soil conditions and belowground interactions can shape aboveground microbial communities, with important potential implications for plant health and sustainable agriculture.

Resistance gained, resistance lost: An explanation for host-parasite coexistence.

Host populations are under continual selection by parasites due to reduced fitness of infected individuals relative to uninfected individuals. This should select for host resistance against parasites, and ample evidence from the laboratory and natural populations demonstrates that hosts can respond rapidly to parasitism by evolving resistance. Why then do parasites still exist? In part, this is due to ongoing arms races as parasites evolve counteradaptations to overcome resistance and to the presence of spatial structure and refuges. However, host-parasite coexistence can also be explained through loss of resistance over time due either to selection against costly resistance mechanisms or constant loss of resistance via reversion mutations.

Application of ecological and evolutionary theory to microbiome community dynamics across systems.

A fundamental aim of microbiome research is to understand the factors that influence the assembly and stability of host-associated microbiomes, and their impact on host phenotype, ecology and evolution. However, ecological and evolutionary theories applied to predict microbiome community dynamics are largely based on macroorganisms and lack microbiome-centric hypotheses that account for unique features of the microbiome. This special feature sets out to drive advancements in the application of eco-evolutionary theory to microbiome community dynamics through the development of microbiome-specific theoretical and conceptual frameworks across plant, human and non-human animal systems. The feature comprises 11 research and review articles that address: (i) the effects of the microbiome on host phenotype, ecology and evolution; (ii) the application and development of ecological and evolutionary theories to investigate microbiome assembly, diversity and stability across broad taxonomic scales; and (iii) general principles that underlie microbiome diversity and dynamics. This cross-disciplinary synthesis of theoretical, conceptual, methodological and analytical approaches to characterizing host-microbiome ecology and evolution across systems addresses key research gaps in the field of microbiome research and highlights future research priorities.

The impact of bacteriophages on phyllosphere bacterial abundance and composition.

Interactions between bacteria and bacteriophage viruses (phages) are known to influence pathogen growth and virulence, microbial diversity and even biogeochemical cycling. Lytic phages in particular infect and lyse their host cells, and can therefore have significant effects on cell densities as well as competitive dynamics within microbial communities. Despite the known impacts of lytic phages on the ecology and evolution of bacteria in free-living communities, little is known about the role of lytic phages in host-associated microbiomes. We set out to characterize the impact of phages in the tomato phyllosphere, that is the bacteria associated with above-ground plant tissues, by transferring microbial communities from field-grown tomato plants to juvenile plants grown under mostly sterile conditions in either the presence or absence of their associated phage community. In three separate experiments, we found that the presence of phages affects overall bacterial abundance during colonization of new host plants. Furthermore, bacterial community analysis using 16S rRNA amplicon sequencing shows that phages significantly alter the relative abundance of dominant community members and can influence both within- and among-host diversity. These results underscore the importance of lytic phages in host-associated microbiomes and are relevant to microbiome transplantation approaches, as they suggest transferring nonbacterial components of the microbiome among hosts is likely to have a strong impact on growth of both the resident and colonizing microbiota.

Host-parasite fluctuating selection in the absence of specificity.

Fluctuating selection driven by coevolution between hosts and parasites is important for the generation of host and parasite diversity across space and time. Theory has focused primarily on infection genetics, with highly specific 'matching-allele' frameworks more likely to generate fluctuating selection dynamics (FSD) than 'gene-for-gene' (generalist-specialist) frameworks. However, the environment, ecological feedbacks and life-history characteristics may all play a role in determining when FSD occurs. Here, we develop eco-evolutionary models with explicit ecological dynamics to explore the ecological, epidemiological and host life-history drivers of FSD. Our key result is to demonstrate for the first time, to our knowledge, that specificity between hosts and parasites is not required to generate FSD. Furthermore, highly specific host-parasite interactions produce unstable, less robust stochastic fluctuations in contrast to interactions that lack specificity altogether or those that vary from generalist to specialist, which produce predictable limit cycles. Given the ubiquity of ecological feedbacks and the variation in the nature of specificity in host-parasite interactions, our work emphasizes the underestimated potential for host-parasite coevolution to generate fluctuating selection.

A signature of tree health? Shifts in the microbiome and the ecological drivers of horse chestnut bleeding canker disease.

Host susceptibility to pathogens can be shaped by genetic, ecological, and evolutionary factors. The ability to predict the spread of disease therefore requires an integrated understanding of these factors, including effects of pests on pathogen growth and competition between pathogens and commensal microbiota for host resources. We examined interactions between the leaf-mining moth Cameraria ohridella, the bacterial causal agent of bleeding canker disease Pseudomonas syringae pv aesculi, and the bark-associated microbiota of horse chestnut (Aesculus hippocastanum) trees. Through surveys of > 900 trees from 60 sites in the UK, we tested for ecological or life history predictors of leaf miner infestation, bleeding canker, or coinfection. Using culture-independent sequencing, we then compared the bark microbiomes from 46 trees to measure the association between microbiome composition and key ecological variables, including the severity of disease. Both pest and pathogen were found to respond to tree characteristics, but neither explained damage inflicted by the other. However, we found a clear loss of microbial diversity and associated shift in microbiome composition of trees as a function of disease. These results show a link between bark-associated microbiota and tree health that introduces the intriguing possibility that tree microbiota play key roles in the spread of disease.

Adaptation of the pathogen, Pseudomonas syringae, during experimental evolution on a native vs. alternative host plant.

The specialization and distribution of pathogens among species has substantial impact on disease spread, especially when reservoir hosts can maintain high pathogen densities or select for increased pathogen virulence. Theory predicts that optimal within-host growth rate will vary among host genotypes/species and therefore that pathogens infecting multiple hosts should experience different selection pressures depending on the host environment in which they are found. This should be true for pathogens with broad host ranges, but also those experiencing opportunistic infections on novel hosts or that spill over among host populations. There is very little empirical data, however, regarding how adaptation to one host might directly influence infectivity and growth on another. We took an experimental evolution approach to examine short-term adaptation of the plant pathogen, Pseudomonas syringae pathovar tomato, to its native tomato host compared with an alternative host, Arabidopsis, in either the presence or absence of bacteriophages. After four serial passages (20 days of selection in planta), we measured bacterial growth of selected lines in leaves of either the focal or alternative host. We found that passage through Arabidopsis led to greater within-host bacterial densities in both hosts than did passage through tomato. Whole genome resequencing of evolved isolates identified numerous single nucleotide polymorphisms based on our novel draft assembly for strain PT23. However, there was no clear pattern of clustering among plant selection lines at the genetic level despite the phenotypic differences observed. Together, the results emphasize that previous host associations can influence the within-host growth rate of pathogens.

Bacteria-phage interactions across time and space: merging local adaptation and time-shift experiments to understand phage evolution.

The study of parasite local adaptation to host populations offers important insight into the spatial scale of host-parasite interactions. For parasites adapting to local hosts, the process is continually driven by change in the host population, in response to either the parasite or alternative selection pressures. In the case of reciprocal coevolutionary change, this adaptation should generate a pattern whereby parasites are most fit against hosts from the recent past (which have not yet responded to parasite-mediated selection) and least fit against future host populations (with increased resistance). I argue that combining data on local adaptation across space with data on evolutionary responses over time can offer novel insight into the process of adaptation. Using bacteriophages from horse chestnut trees, I compare infectivity on bacterial hosts isolated from either the same tree or different trees over multiple months of the growing season and find that phage adaptation to local hosts is most pronounced on bacterial hosts from the recent past. These results confirm that phages are well adapted to bacterial populations living within eukaryotes and more broadly suggest that local adaptation studies may underestimate the magnitude of parasite evolution, as host and parasite adaptation are confounded within contemporary time points.

Bacteria-phage interactions in natural environments.

Phages are considered the most abundant and diverse biological entities on Earth and are notable not only for their sheer abundance, but also for their influence on bacterial hosts. In nature, bacteria-phage relationships are complex and have far-reaching consequences beyond particular pairwise interactions, influencing everything from bacterial virulence to eukaryotic fitness to the carbon cycle. In this review, we examine bacteria and phage distributions in nature first by highlighting biogeographic patterns and nonhost environmental influences on phage distribution, then by considering the ways in which phages and bacteria interact, emphasizing phage life cycles, bacterial responses to phage infection, and the complex patterns of phage host specificity. Finally, we discuss phage impacts on bacterial abundance, genetics, and physiology, and further aim to clarify distinctions between current theoretical models and point out areas in need of future research.

Phage biology: The ins and outs of prophages in bacterial populations.

Bacterial genomes often harbor integrated viruses (prophages), which provide novel functions but also lyse cells under stressful conditions. A new paper combines mathematical models with experimental evolution to determine how prophages are maintained in bacterial populations despite their fitness costs.

A link between evolution and society fostering the UN sustainable development goals.

Given the multitude of challenges Earth is facing, sustainability science is of key importance to our continued existence. Evolution is the fundamental biological process underlying the origin of all biodiversity. This phylogenetic diversity fosters the resilience of ecosystems to environmental change, and provides numerous resources to society, and options for the future. Genetic diversity within species is also key to the ability of populations to evolve and adapt to environmental change. Yet, the value of evolutionary processes and the consequences of their impairment have not generally been considered in sustainability research. We argue that biological evolution is important for sustainability and that the concepts, theory, data, and methodological approaches used in evolutionary biology can, in crucial ways, contribute to achieving the UN Sustainable Development Goals (SDGs). We discuss how evolutionary principles are relevant to understanding, maintaining, and improving Nature Contributions to People (NCP) and how they contribute to the SDGs. We highlight specific applications of evolution, evolutionary theory, and evolutionary biology's diverse toolbox, grouped into four major routes through which evolution and evolutionary insights can impact sustainability. We argue that information on both within-species evolutionary potential and among-species phylogenetic diversity is necessary to predict population, community, and ecosystem responses to global change and to make informed decisions on sustainable production, health, and well-being. We provide examples of how evolutionary insights and the tools developed by evolutionary biology can not only inspire and enhance progress on the trajectory to sustainability, but also highlight some obstacles that hitherto seem to have impeded an efficient uptake of evolutionary insights in sustainability research and actions to sustain SDGs. We call for enhanced collaboration between sustainability science and evolutionary biology to understand how integrating these disciplines can help achieve the sustainable future envisioned by the UN SDGs.

Drivers and consequences of bacteriophage host range.

Bacteriophages are obligate parasites of bacteria characterized by the breadth of hosts that they can infect. This "host range" depends on the genotypes and morphologies of the phage and the bacterial host, but also on the environment in which they are interacting. Understanding phage host range is critical to predicting the impacts of these parasites in their natural host communities and their utility as therapeutic agents, but is also key to predicting how phages evolve and in doing so drive evolutionary change in their host populations, including through movement of genes among unrelated bacterial genomes. Here, we explore the drivers of phage infection and host range from the molecular underpinnings of the phage-host interaction to the ecological context in which they occur. We further evaluate the importance of intrinsic, transient, and environmental drivers shaping phage infection and replication, and discuss how each influences host range over evolutionary time. The host range of phages has great consequences in phage-based application strategies, as well as natural community dynamics, and we therefore highlight both recent developments and key open questions in the field as phage-based therapeutics come back into focus.

Within-host adaptation alters priority effects within the tomato phyllosphere microbiome.

To predict the composition and function of ecological communities over time, it is essential to understand how in situ evolution alters priority effects between resident and invading species. Phyllosphere microbial communities are a useful model system to explore priority effects because the system is clearly spatially delineated and can be manipulated experimentally. We conducted an experimental evolution study with tomato plants and the early-colonizing bacterium species Pantoea dispersa, exploring priority effects when P. dispersa was introduced before, simultaneously with or after competitor species. P. dispersa rapidly evolved to invade a new niche within the plant tissue and altered its ecological interactions with other members of the plant microbiome and its effect on the host. Prevailing models have assumed that adaptation primarily improves the efficiency of resident species within their existing niches, yet in our study system, the resident species expanded its niche instead. This finding suggests potential limitations to the application of existing ecological theory to microbial communities.

Leaf side determines the relative importance of dispersal versus host filtering in the phyllosphere microbiome.

Leaves harbor distinct microbial communities that can have an important impact on plant health and microbial ecosystems worldwide. Nevertheless, the ecological processes that shape the composition of leaf microbial communities remain unclear, with previous studies reporting contradictory results regarding the importance of bacterial dispersal versus host selection. This discrepancy could be driven in part because leaf microbiome studies typically consider the upper and lower leaf surfaces as a single entity despite these habitats possessing considerable anatomical differences. We characterized the composition of bacterial phyllosphere communities from the upper and lower leaf surfaces across 24 plant species. Leaf surface pH and stomatal density were found to shape phyllosphere community composition, and the underside of leaves had lower richness and higher abundances of core community members than upper leaf surfaces. We found fewer endemic bacteria on the upper leaf surfaces, suggesting that dispersal is more important in shaping these communities, with host selection being a more important force in microbiome assembly on lower leaf surfaces. Our study illustrates how changing the scale in which we observe microbial communities can impact our ability to resolve and predict microbial community assembly patterns on leaf surfaces. IMPORTANCE Leaves can harbor hundreds of different bacterial species that form unique communities for every plant species. Bacterial communities on leaves are really important because they can, for example, protect their host against plant diseases. Usually, bacteria from the whole leaf are considered when trying to understand these communities; however, this study shows that the upper and lower sides of a leaf have a very different impact on how these communities are shaped. It seems that the bacteria on the lower leaf side are more closely associated with the plant host, and communities on the upper leaf side are more impacted by immigrating bacteria. This can be really important when we want to treat, for example, crops in the field with beneficial bacteria or when trying to understand host-microbe interactions on the leaves.