Giant Starship Elements Mobilize Accessory Genes in Fungal Genomes.

Accessory genes are variably present among members of a species and are a reservoir of adaptive functions. In bacteria, differences in gene distributions among individuals largely result from mobile elements that acquire and disperse accessory genes as cargo. In contrast, the impact of cargo-carrying elements on eukaryotic evolution remains largely unknown. Here, we show that variation in genome content within multiple fungal species is facilitated by Starships, a newly discovered group of massive mobile elements that are 110 kb long on average, share conserved components, and carry diverse arrays of accessory genes. We identified hundreds of Starship-like regions across every major class of filamentous Ascomycetes, including 28 distinct Starships that range from 27 to 393 kb and last shared a common ancestor ca. 400 Ma. Using new long-read assemblies of the plant pathogen Macrophomina phaseolina, we characterize four additional Starships whose activities contribute to standing variation in genome structure and content. One of these elements, Voyager, inserts into 5S rDNA and contains a candidate virulence factor whose increasing copy number has contrasting associations with pathogenic and saprophytic growth, suggesting Voyager's activity underlies an ecological trade-off. We propose that Starships are eukaryotic analogs of bacterial integrative and conjugative elements based on parallels between their conserved components and may therefore represent the first dedicated agents of active gene transfer in eukaryotes. Our results suggest that Starships have shaped the content and structure of fungal genomes for millions of years and reveal a new concerted route for evolution throughout an entire eukaryotic phylum.

A generalist nematode destabilises plant competition: no evidence for direct effects, but strong evidence for indirect effects on rhizobium abundance.

Difficulties quantifying pathogen load and mutualist abundance limit our ability to connect disease dynamics to host community ecology. For example, specific predictions about how differential pathogen load is hypothesised to drive host competitive outcomes are rarely tested. Additionally, although infection is known to affect mutualists, we rarely measure the magnitude of pathogen effects on mutualist abundance across host competitive contexts. We tested for both mechanisms in a plant-rhizobia-nematode system. We paired the legume Medicago lupulina with intraspecific and interspecific plant competitors, with and without a generalist nematode parasite Meloidogyne sp. Relative change in plant biomass was used to determine how nematode inoculation affected plant competitive outcomes. We counted nematode galls to test for direct effects of parasitism on plant competition and rhizobia nodules to test for indirect effects of nematode presence on rhizobium abundance. Parasites were destabilising despite similar nematode load across competition treatments. During interspecific compared with intraspecific competition, nematode inoculation decreased nodulation on M. lupulina, increased nodulation on Trifolium repens and had no effect on nodulation on Chamaecrista fasciculata. We found no support for hypothesised direct effects of nematode load on competitive outcomes and strong but idiosyncratic indirect effects of nematode inoculation on rhizobium abundance.

The Evolutionary Forces Shaping Cis- and Trans-Regulation of Gene Expression within a Population of Outcrossing Plants.

Understanding the persistence of genetic variation within populations has long been a goal of evolutionary biology. One promising route toward achieving this goal is using population genetic approaches to describe how selection acts on the loci associated with trait variation. Gene expression provides a model trait for addressing the challenge of the maintenance of variation because it can be measured genome-wide without information about how gene expression affects traits. Previous work has shown that loci affecting the expression of nearby genes (local or cis-eQTLs) are under negative selection, but we lack a clear understanding of the selective forces acting on variants that affect the expression of genes in trans. Here, we identify loci that affect gene expression in trans using genomic and transcriptomic data from one population of the obligately outcrossing plant, Capsella grandiflora. The allele frequencies of trans-eQTLs are consistent with stronger negative selection acting on trans-eQTLs than cis-eQTLs, and stronger negative selection acting on trans-eQTLs associated with the expression of multiple genes. However, despite this general pattern, we still observe the presence of a trans-eQTL at intermediate frequency that affects the expression of a large number of genes in the same coexpression module. Overall, our work highlights the different selective pressures shaping variation in cis- and trans-regulation.

Constraints Imposed by a Natural Landscape Override Offspring Fitness Effects to Shape Oviposition Decisions in Wild Forked Fungus Beetles.

Oviposition site decisions often maximize offspring fitness, but costs constraining choice can cause females to oviposit in poor developmental environments. It is unclear whether these constraints cumulatively outweigh offspring fitness to determine oviposition decisions in wild populations. Understanding how constraints shape oviposition in natural landscapes is a critical step toward revealing how maternal behavior influences fundamental phenomena like the evolution of specialization and the use of sink environments. Here, we used a genetic capture-recapture technique to reconstruct the oviposition decisions of individual females in a natural metapopulation of a beetle (Bolitotherus cornutus) that oviposits on three fungus species. We measured larval fitness-related traits (mass, development time, survival) on each fungus and compared the oviposition preferences of females in laboratory versus field tests. Larval fitness differed substantially among fungi, and females preferred a high-quality (high larval fitness) fungus in laboratory trials. However, females frequently laid eggs on the lowest-quality fungus in the wild. They preferred high-quality fungi when moving between oviposition sites, but this preference disappeared as the distance between sites increased and was inconsistent between study plots. Our results suggest that constraints on oviposition preferences in natural landscapes are sufficiently large to drive oviposition in poor developmental environments even when offspring fitness consequences are severe.

A window into the transcriptomic basis of genotype-by-genotype interactions in the legume-rhizobia mutualism.

The maintenance of genetic variation in the benefits provided by mutualists is an evolutionary puzzle (Heath & Stinchcombe, ). Over time, natural selection should favour the benefit strategy that confers the highest fitness, eroding genetic variation in partner quality. Yet abundant genetic variation in partner quality exists in many systems (Heath & Stinchcombe, ). One possible resolution to this puzzle is that the genetic identity of both a host and its partner affects the benefits each mutualist provides to the other, a pattern known as a genotype-by-genotype interaction (Figure ). Mounting evidence suggests that genotype-by-genotype interactions between partners are pervasive at the phenotypic level (Barrett, Zee, Bever, Miller, & Thrall, ; Heath, ; Hoeksema & Thompson, ). Ultimately, however, to link these phenotypic patterns to the maintenance of genetic variation in mutualisms we need to answer two questions: How much variation in mutualism phenotypes is attributable to genotype-by-genotype interactions, and what mutualistic functions are influenced by each partner and by the interaction between their genomes? In this issue of Molecular Ecology, Burghardt et al. (2017) use transcriptomics to address both questions in the legume-rhizobia mutualism.

Evolutionary response when selection and genetic variation covary across environments.

Although models of evolution usually assume that the strength of selection on a trait and the expression of genetic variation in that trait are independent, whenever the same ecological factor impacts both parameters, a correlation between the two may arise that accelerates trait evolution in some environments and slows it in others. Here, we address the evolutionary consequences and ecological causes of a correlation between selection and expressed genetic variation. Using a simple analytical model, we show that the correlation has a modest effect on the mean evolutionary response and a large effect on its variance, increasing among-population or among-generation variation in the response when positive, and diminishing variation when negative. We performed a literature review to identify the ecological factors that influence selection and expressed genetic variation across traits. We found that some factors - temperature and competition - are unlikely to generate the correlation because they affected one parameter more than the other, and identified others - most notably, environmental novelty - that merit further investigation because little is known about their impact on one of the two parameters. We argue that the correlation between selection and genetic variation deserves attention alongside other factors that promote or constrain evolution in heterogeneous landscapes.

Herbivory modifies plant symbiont number and impact on host plant performance in the field.

Species interactions are a unifying theme in ecology and evolution. Both fields are currently moving beyond their historical focus on isolated pairwise relationships to understand how ecological communities affect focal interactions. Additional species can modify both the number of interactions and the fitness consequences of each interaction (i.e., selection). Although only selection affects the evolution of the focal interaction, the two are often conflated, limiting our understanding of the evolution of multispecies interactions. We manipulated aboveground herbivory on the legume Medicago lupulina in the field and quantified its effect on number of symbionts and the per-symbiont impact on plant performance in two belowground symbioses: mutualistic rhizobia bacteria (Ensifer meliloti) and parasitic root-knot nematodes (Meloidogyne hapla). We found that herbivores modified the number of rhizobia nodules, as well as the benefit per nodule. However, each effect was specific to a distinct herbivory regime: natural herbivory affected nodule number, whereas leafhoppers (Cicadellidae) weakened the per nodule benefit. We did not detect any effect of herbivory on nematode gall number or the cost of infection. Our data demonstrate that distinguishing between symbiont number from the fitness consequences of symbiosis is crucial to accurately infer how pairwise interactions will evolve in a community.

The soil microbiome increases plant survival and modifies interactions with root endosymbionts in the field.

Evidence is accumulating that the soil microbiome-the community of microorganisms living in soils-has a major effect on plant traits and fitness. However, most work to date has taken place under controlled laboratory conditions and has not experimentally disentangled the effect of the soil microbiome on plant performance from the effects of key endosymbiotic constituents. As a result, it is difficult to extrapolate from existing data to understand the role of the soil microbiome in natural plant populations. To address this gap, we performed a field experiment using the black medick Medicago lupulina to test how the soil microbiome influences plant performance and colonization by two root endosymbionts (the mutualistic nitrogen-fixing bacteria Ensifer spp. and the parasitic root-knot nematode Meloidogyne hapla) under natural conditions. We inoculated all plants with nitrogen-fixing bacteria and factorially manipulated the soil microbiome and nematode infection. We found that plants grown in microbe-depleted soil exhibit greater mortality, but that among the survivors, there was no effect of the soil microbiome on plant performance (shoot biomass, root biomass, or shoot-to-root ratio). The soil microbiome also impacted parasitic nematode infection and affected colonization by mutualistic nitrogen-fixing bacteria in a plant genotype-dependent manner, increasing colonization in some plant genotypes and decreasing it in others. Our results demonstrate the soil microbiome has complex effects on plant-endosymbiont interactions and may be critical for survival under natural conditions.

Priority effects alter the colonization success of a host-associated parasite and mutualist.

Priority effects shape the assembly of free-living communities and host-associated communities. However, the current literature does not fully incorporate two features of host-symbiont interactions, correlated host responses to multiple symbionts and ontogenetic changes in host responses to symbionts, leading to an incomplete picture of the role of priority effects in host-associated communities. We factorially manipulated the inoculation timing of two plant symbionts (mutualistic rhizobia bacteria and parasitic root-knot nematodes) and tested how host age at arrival, arrival order, and arrival synchrony affected symbiont colonization success in the model legume Medicago truncatula. We found that host age, arrival order, and arrival synchrony significantly affected colonization of one or both symbionts. Host age at arrival only affected nematodes but not rhizobia: younger plants were more heavily infected than older plants. By contrast, arrival order only affected rhizobia but not nematodes: plants formed more rhizobia nodules when rhizobia arrived before nematodes. Finally, synchronous arrival decreased colonization both symbionts, an effect that depended on host age. Our results demonstrate that priority effects compromise the host's ability to control colonization by two major symbionts and suggest that the role of correlated host responses and host ontogeny in the assembly of host-associated communities deserve further attention.

Using clear plastic CD cases as low-cost mini-rhizotrons to phenotype root traits.

PREMISE: We developed a novel low-cost method to visually phenotype belowground structures in the plant rhizosphere. We devised the method introduced here to address the difficulties encountered growing plants in seed germination pouches for long-term experiments and the high cost of other mini-rhizotron alternatives. METHODS AND RESULTS: The method described here took inspiration from homemade ant farms commonly used as an educational tool in elementary schools. Using compact disc (CD) cases, we developed mini-rhizotrons for use in the field and laboratory using the burclover Medicago lupulina. CONCLUSIONS: Our method combines the benefits of pots and germination pouches. In CD mini-rhizotrons, plants grew significantly larger than in germination pouches, and unlike pots, it is possible to measure roots without destructive sampling. Our protocol is a cheaper, widely available alternative to more destructive methods, which could facilitate the study of belowground phenotypes and processes by scientists with fewer resources.

Genetic conflict with a parasitic nematode disrupts the legume-rhizobia mutualism.

Genetic variation for partner quality in mutualisms is an evolutionary paradox. One possible resolution to this puzzle is that there is a tradeoff between partner quality and other fitness-related traits. Here, we tested whether susceptibility to parasitism is one such tradeoff in the mutualism between legumes and nitrogen-fixing bacteria (rhizobia). We performed two greenhouse experiments with the legume Medicago truncatula. In the first, we inoculated each plant with the rhizobia Ensifer meliloti and with one of 40 genotypes of the parasitic root-knot nematode Meloidogyne hapla. In the second experiment, we inoculated all plants with rhizobia and half of the plants with a genetically variable population of nematodes. Using the number of nematode galls as a proxy for infection severity, we found that plant genotypes differed in susceptibility to nematode infection, and nematode genotypes differed in infectivity. Second, we showed that there was a genetic correlation between the number of mutualistic structures formed by rhizobia (nodules) and the number of parasitic structures formed by nematodes (galls). Finally, we found that nematodes disrupt the rhizobia mutualism: nematode-infected plants formed fewer nodules and had less nodule biomass than uninfected plants. Our results demonstrate that there is genetic conflict between attracting rhizobia and repelling nematodes in Medicago. If genetic conflict with parasitism is a general feature of mutualism, it could account for the maintenance of genetic variation in partner quality and influence the evolutionary dynamics of positive species interactions.

Geographically structured genetic variation in the Medicago lupulina-Ensifer mutualism.

Gene flow between genetically differentiated populations can maintain variation in species interactions, especially when population structure is congruent between interacting species. However, large-scale empirical comparisons of the population structure of interacting species are rare, particularly in positive interspecific interactions (mutualisms). One agriculturally and ecologically important mutualism is the partnership between legume plants and rhizobia. Through characterizing and comparing the population genomic structure of the legume Medicago lupulina and two rhizobial species (Ensifer medicae and E. meliloti), we explored the spatial scale of population differentiation between interacting partners in their introduced range in North America. We found high proportions of E. meliloti in southeastern populations and high proportions of E. medicae in northwestern populations. Medicago lupulina and the Ensifer genus showed similar patterns of spatial genetic structure (isolation by distance). However, we detected no evidence of isolation by distance or population structure within either species of bacteria. Genome-wide nucleotide diversity within each of the two Ensifer species was low, suggesting limited introduction of strains, founder events, or severe bottlenecks. Our results suggest that there is potential for geographically structured coevolution between M. lupulina and the Ensifer genus, but not between M. lupulina and either Ensifer species.

No evidence for adaptation to local rhizobial mutualists in the legume Medicago lupulina.

Local adaptation is a common but not ubiquitous feature of species interactions, and understanding the circumstances under which it evolves illuminates the factors that influence adaptive population divergence. Antagonistic species interactions dominate the local adaptation literature relative to mutualistic ones, preventing an overall assessment of adaptation within interspecific interactions. Here, we tested whether the legume Medicago lupulina is adapted to the locally abundant species of mutualistic nitrogen-fixing rhizobial bacteria that vary in frequency across its eastern North American range. We reciprocally inoculated northern and southern M. lupulina genotypes with the northern (Ensifer medicae) or southern bacterium (E. meliloti) in a greenhouse experiment. Despite producing different numbers of root nodules (the structures in which the plants house the bacteria), neither northern nor southern plants produced more seeds, flowered earlier, or were more likely to flower when inoculated with their local rhizobia. We then used a pre-existing dataset to perform a genome scan for loci that showed elevated differentiation between field-collected plants that hosted different bacteria. None of the loci we identified belonged to the well-characterized suite of legume-rhizobia symbiosis genes, suggesting that the rhizobia do not drive genetic divergence between M. lupulina populations. Our results demonstrate that symbiont local adaptation has not evolved in this mutualism despite large-scale geographic variation in the identity of the interacting species.

Environmental effects on the structure of the G-matrix.

Genetic correlations between traits determine the multivariate response to selection in the short term, and thereby play a causal role in evolutionary change. Although individual studies have documented environmentally induced changes in genetic correlations, the nature and extent of environmental effects on multivariate genetic architecture across species and environments remain largely uncharacterized. We reviewed the literature for estimates of the genetic variance-covariance (G) matrix in multiple environments, and compared differences in G between environments to the divergence in G between conspecific populations (measured in a common garden). We found that the predicted evolutionary trajectory differed as strongly between environments as it did between populations. Between-environment differences in the underlying structure of G (total genetic variance and the relative magnitude and orientation of genetic correlations) were equal to or greater than between-population differences. Neither environmental novelty, nor the difference in mean phenotype predicted these differences in G. Our results suggest that environmental effects on multivariate genetic architecture may be comparable to the divergence that accumulates over dozens or hundreds of generations between populations. We outline avenues of future research to address the limitations of existing data and characterize the extent to which lability in genetic correlations shapes evolution in changing environments.

Surprisingly little population genetic structure in a fungus-associated beetle despite its exploitation of multiple hosts.

In heterogeneous environments, landscape features directly affect the structure of genetic variation among populations by functioning as barriers to gene flow. Resource-associated population genetic structure, in which populations that use different resources (e.g., host plants) are genetically distinct, is a well-studied example of how environmental heterogeneity structures populations. However, the pattern that emerges in a given landscape should depend on its particular combination of resources. If resources constitute barriers to gene flow, population differentiation should be lowest in homogeneous landscapes, and highest where resources exist in equal proportions. In this study, we tested whether host community diversity affects population genetic structure in a beetle (Bolitotherus cornutus) that exploits three sympatric host fungi. We collected B. cornutus from plots containing the three host fungi in different proportions and quantified population genetic structure in each plot using a panel of microsatellite loci. We found no relationship between host community diversity and population differentiation in this species; however, we also found no evidence of resource-associated differentiation, suggesting that host fungi are not substantial barriers to gene flow. Moreover, we detected no genetic differentiation among B. cornutus populations separated by several kilometers, even though a previous study demonstrated moderate genetic structure on the scale of a few hundred meters. Although we found no effect of community diversity on population genetic structure in this study, the role of host communities in the structuring of genetic variation in heterogeneous landscapes should be further explored in a species that exhibits resource-associated population genetic structure.

Phenotypic assortment mediates the effect of social selection in a wild beetle population.

Social interactions often have major fitness consequences, but little is known about how specific interacting phenotypes affect the strength of natural selection. Social influences on the evolutionary process can be assessed using a multilevel selection approach that partitions the effects of social partner phenotypes on fitness (referred to as social or group selection) from those of the traits of a focal individual (nonsocial or individual selection). To quantify the contribution of social selection to total selection affecting a trait, the patterns of phenotypic association among interactants must also be considered. We estimated selection gradients on male body size in a wild population of forked fungus beetles (Bolitotherus cornutus). We detected positive nonsocial selection and negative social selection on body size operating through differences in copulation success, indicating that large males with small social partners had highest fitness. In addition, we found that, in low-density demes, the phenotypes of focal individuals were negatively correlated with those of their social partners. This pattern reversed the negative effect of group selection on body size and led to stronger positive selection for body size. Our results demonstrate multilevel selection in nature and stress the importance of considering social selection whenever conspecific interactions occur nonrandomly.