Selection for cheating across disparate environments in the legume-rhizobium mutualism.

The primary dilemma in evolutionarily stable mutualisms is that natural selection for cheating could overwhelm selection for cooperation. Cheating need not entail parasitism; selection favours cheating as a quantitative trait whenever less-cooperative partners are more fit than more-cooperative partners. Mutualisms might be stabilised by mechanisms that direct benefits to more-cooperative individuals, which counter selection for cheating; however, empirical evidence that natural selection favours cheating in mutualisms is sparse. We measured selection on cheating in single-partner pairings of wild legume and rhizobium lineages, which prevented legume choice. Across contrasting environments, selection consistently favoured cheating by rhizobia, but did not favour legumes that provided less benefit to rhizobium partners. This is the first simultaneous measurement of selection on cheating across both host and symbiont lineages from a natural population. We empirically confirm selection for cheating as a source of antagonistic coevolutionary pressure in mutualism and a biological dilemma for models of cooperation.

Specialization-generalization trade-off in a Bradyrhizobium symbiosis with wild legume hosts.

BACKGROUND: Specialized interactions help structure communities, but persistence of specialized organisms is puzzling because a generalist can occupy more environments and partake in more beneficial interactions. The "Jack-of-all-trades is a master of none" hypothesis asserts that specialists persist because the fitness of a generalist utilizing a particular habitat is lower than that of a specialist adapted to that habitat. Yet, there are many reasons to expect that mutualists will generalize on partners.Plant-soil feedbacks help to structure plant and microbial communities, but how frequently are soil-based symbiotic mutualistic interactions sufficiently specialized to influence species distributions and community composition? To address this question, we quantified realized partner richness and phylogenetic breadth of four wild-grown native legumes (Lupinus bicolor, L. arboreus, Acmispon strigosus and A. heermannii) and performed inoculation trials to test the ability of two hosts (L. bicolor and A. strigosus) to nodulate (fundamental partner richness), benefit from (response specificity), and provide benefit to (effect specificity) 31 Bradyrhizobium genotypes. RESULTS: In the wild, each Lupinus species hosted a broader genetic range of Bradyrhizobium than did either Acmispon species, suggesting that Acmispon species are more specialized. In the greenhouse, however, L. bicolor and A. strigosus did not differ in fundamental association specificity: all inoculated genotypes nodulated both hosts. Nevertheless, A. strigosus exhibited more specificity, i.e., greater variation in its response to, and effect on, Bradyrhizobium genotypes. Lupinus bicolor benefited from a broader range of genotypes but averaged less benefit from each. Both hosts obtained more fitness benefit from symbionts isolated from conspecific hosts; those symbionts in turn gained greater fitness benefit from hosts of the same species from which they were isolated. CONCLUSIONS: This study affirmed two important tenets of evolutionary theory. First, as predicted by the Jack-of-all-trades is a master of none hypothesis, specialist A. strigosus obtained greater benefit from its beneficial symbionts than did generalist L. bicolor. Second, as predicted by coevolutionary theory, each test species performed better with partner genotypes isolated from conspecifics. Finally, positive fitness feedback between the tested hosts and symbionts suggests that positive plant-soil feedback could contribute to their patchy distributions in this system.

How might bacteriophages shape biological invasions?

Invasions by eukaryotes dependent on environmentally acquired bacterial mutualists are often limited by the ability of bacterial partners to survive and establish free-living populations. Focusing on the model legume-rhizobium mutualism, we apply invasion biology hypotheses to explain how bacteriophages can impact the competitiveness of introduced bacterial mutualists. Predicting how phage-bacteria interactions affect invading eukaryotic hosts requires knowing the eco-evolutionary constraints of introduced and native microbial communities, as well as their differences in abundance and diversity. By synthesizing research from invasion biology, as well as bacterial, viral, and community ecology, we create a conceptual framework for understanding and predicting how phages can affect biological invasions through their effects on bacterial mutualists.

Precision of host sanctions in the fig tree-fig wasp mutualism: consequences for uncooperative symbionts.

Host sanctions that reduce the relative fitness of uncooperative symbionts provide a mechanism that can limit cheating and thus stabilise mutualisms over evolutionary timescales. Sanctions have been demonstrated empirically in several mutualisms. However, if multiple individual symbionts interact with each host, the precision with which individual cheating symbionts are targeted by host sanctions is critical to their short- and long-term effectiveness. No previous empirical study has directly addressed this issue. Here, we report the precision of host sanctions in the mutualism between fig trees and their pollinating wasps. Using field experiments and molecular parentage analyses, we show that sanctions in Ficus nymphaeifolia act at the level of entire figs (syconia), not at the level of the individual flowers within. Such fig-level sanctions allow uncooperative wasps, which do not bring pollen, to avoid sanctions in figs to which other wasps bring pollen. We discuss the relevance of sanction precision to other mutualisms.

Transcriptomic insights into mechanisms of symbiotic cooperation.

Intraspecific genetic variation can affect community structure and ecosystem processes (Bolnick et al. 2011). It can also influence phenotypic expression by genotypes within other species to produce genotype-by-genotype (G × G) interaction (Falconer & Mackay 1996). Evolution of one species drives correlated evolution of others when it causes G × G for fitness (Thompson 2005). However, the mechanisms by which species interact also influence evolutionary outcomes (Kummel & Salant 2006; Golubski & Klausmeier 2010; Akçay & Simms 2011; Grman et al. 2012). To identify genes and putative functional mechanisms underlying G × G interactions, Heath et al. (2012) analysed natural variation in the symbiotic transcriptome of the mutualistic nutritional symbiosis between a legume host Medicago truncatula and the facultative endosymbiotic rhizobium Sinorhizobium meliloti. Using twelve microarrays, the authors simultaneously measured host and symbiont gene expression in root nodules from four factorial pairings of host and symbiont genotypes that produced G × G in host fitness (Fig. 1, upper panel). Rhizobium gene expression was influenced by rhizobium and plant genotype and the G × G interaction (Fig. 1, lower panel), whereas plant gene expression was influenced primarily by plant genotype. The authors identified rhizobium genes that might contribute to G × G in host plant fitness. Heath et al. (2012) have moved beyond the constraints of single organism analysis towards a more realistic understanding of plants and bacteria as organisms inextricably linked with symbioses that affect even basic patterns of gene expression.

Negotiation, sanctions, and context dependency in the legume-Rhizobium mutualism.

Two important questions about mutualisms are how the fitness costs and benefits to the mutualist partners are determined and how these mechanisms affect the evolutionary dynamics of the mutualism. We tackle these questions with a model of the legume-rhizobium symbiosis that regards the mutualism outcome as a result of biochemical negotiations between the plant and its nodules. We explore the fitness consequences of this mechanism to the plant and rhizobia and obtain four main results. First, negotiations permit the plant to differentially reward more-cooperative rhizobia--a phenomenon termed "plant sanctions"--but only when more-cooperative rhizobia also provide the plant with good outside options during negotiations with other nodules. Second, negotiations may result in seemingly paradoxical cases where the plant is worse off when it has a "choice" between two strains of rhizobia than when infected by either strain alone. Third, even when sanctions are effective, they are by themselves not sufficient to maintain cooperative rhizobia in a population: less cooperative strains always have an advantage at the population level. Finally, partner fidelity feedback, together with genetic correlations between a rhizobium strain's cooperativeness and the outside options it provides, can maintain cooperative rhizobia. Our results show how joint control over the outcome of a mutualism through the proximate mechanism of negotiation can affect the evolutionary dynamics of interspecific cooperation.

Selection on herbivory resistance and growth rate in an invasive plant.

The evolution of increased competitive ability (EICA) hypothesis proposes that invasive species evolve decreased defense and increased competitive ability following natural enemy release. Previous tests of EICA examined the result of evolution by comparing individuals from home and introduced ranges, but no previous study of this hypothesis has examined the process of evolution by analyzing patterns of selection. On the basis of EICA, there should be selection for competitive ability without herbivores and selection for defense with herbivores. Selection on competitive ability should be stronger for genotypes accustomed to herbivores (home range genotypes), and selection on defense should be stronger for genotypes unaccustomed to herbivores (introduced range genotypes). Using a field experiment, we tested these hypotheses for the invasive plant Melaleuca quinquenervia. There was a negative genetic correlation between resistance and growth, indicating a trade-off. However, selection for stem elongation (an indicator of competitive ability) was always positive, and selection on resistance was always negative and did not depend on genotype source or the presence of herbivores. The patterns of selection found in this study contrast with predictions from EICA and accurately predict the lack of evolutionary change in growth and resistance following the introduction of this species from Australia to Florida.

Invasive legumes can associate with many mutualists of native legumes, but usually do not.

Mutualistic interactions can strongly influence species invasions, as the inability to form successful mutualisms in an exotic range could hamper a host's invasion success. This barrier to invasion may be overcome if an invader either forms novel mutualistic associations or finds and associates with familiar mutualists in the exotic range. Here, we ask (1) does the community of rhizobial mutualists associated with invasive legumes in their exotic range overlap with that of local native legumes and (2) can any differences be explained by fundamental incompatibilities with particular rhizobial genotypes? To address these questions, we first characterized the rhizobial communities naturally associating with three invasive and six native legumes growing in the San Francisco Bay Area. We then conducted a greenhouse experiment to test whether the invasive legume could nodulate with any of a broad array of rhizobia found in their exotic range. There was little overlap between the Bradyrhizobium communities associated with wild-grown invasive and native legumes, yet the invasive legumes could nodulate with a broad range of rhizobial strains under greenhouse conditions. These observations suggest that under field conditions in their exotic range, these invasive legumes are not currently associating with the mutualists of local native legumes, despite their potential to form such associations. However, the promiscuity with which these invading legumes can form mutualistic associations could be an important factor early in the invasion process if mutualist scarcity limits range expansion. Overall, the observation that invasive legumes have a community of rhizobia distinct from that of native legumes, despite their ability to associate with many rhizobial strains, challenges existing assumptions about how invading species obtain their mutualists. These results can therefore inform current and future efforts to prevent and remove invasive species.

An empirical test of partner choice mechanisms in a wild legume-rhizobium interaction.

Mutualisms can be viewed as biological markets in which partners of different species exchange goods and services to their mutual benefit. Trade between partners with conflicting interests requires mechanisms to prevent exploitation. Partner choice theory proposes that individuals might foil exploiters by preferentially directing benefits to cooperative partners. Here, we test this theory in a wild legumerhizobium symbiosis. Rhizobial bacteria inhabit legume root nodules and convert atmospheric dinitrogen (N2) to a plant available form in exchange for photosynthates. Biological market theory suits this interaction because individual plants exchange resources with multiple rhizobia. Several authors have argued that microbial cooperation could be maintained if plants preferentially allocated resources to nodules harbouring cooperative rhizobial strains. It is well known that crop legumes nodulate non-fixing rhizobia, but allocate few resources to those nodules. However, this hypothesis has not been tested in wild legumes which encounter partners exhibiting natural, continuous variation in symbiotic benefit. Our greenhouse experiment with a wild legume, Lupinus arboreus, showed that although plants frequently hosted less cooperative strains, the nodules occupied by these strains were smaller. Our survey of wild-grown plants showed that larger nodules house more Bradyrhizobia, indicating that plants may prevent the spread of exploitation by favouring better cooperators.

Quantitative trait loci affecting delta13C and response to differential water availibility in Arabidopsis thaliana.

Phenotypic plasticity is an important response mechanism of plants to environmental heterogeneity. Here, we explored the genetic basis of plastic responses of Arabidopsis thaliana to water deficit by experimentally mapping quantitative trait loci (QTL) in two recombinant inbred populations (Cvi x Ler and Ler x Col). We detected genetic variation and significant genotype-by-environment interactions for many traits related to water use. We also mapped 26 QTL, including six for carbon isotope composition (delta13C). Negative genetic correlations between fruit length and fruit production as well as between flowering time and branch production were corroborated by QTL colocalization, suggesting these correlations are due to pleiotropy or physical linkage. Water-limited plants were more apically dominant with greater root:shoot ratios and higher delta13C (higher water-use efficiency) when compared to well-watered plants. Many of the QTL effects for these traits interacted significantly with the irrigation treatment, suggesting that the observed phenotypic plasticity is genetically based. We specifically searched for epistatic (QTL-QTL) interactions using a two-dimensional genome scan, which allowed us to detect epistasis regardless of additive genetic effects. We found several significant QTL-QTL interactions including three that exhibited environmental dependence. These results provide preliminary evidence for proposed genetic mechanisms underlying phenotypic plasticity.

Sanctions and mutualism stability: why do rhizobia fix nitrogen?

Why do rhizobia expend resources on fixing N(2) for the benefit of their host plant, when they could use those resources for their own reproduction? We present a series of theoretical models which counter the hypotheses that N(2) fixation is favoured because it (i) increases the exudation of useful resources to related rhizobia in the nearby soil, or (ii) increases plant growth and therefore the resources available for rhizobia growth. Instead, we suggest that appreciable levels of N(2) fixation are only favoured when plants preferentially supply more resources to (or are less likely to senesce) nodules that are fixing more N(2) (termed plant sanctions). The implications for different agricultural practices and mutualism stability in general are discussed.

Pathogen-induced systemic resistance in Ipomoea purpurea.

Infection of Ipomoea purpurea by anthracnose, the disease caused by the fungal pathogen Colletotrichum dematium, increases resistance to subsequent infections on previously uninfected leaves. Fungal isolates varied in their levels of virulence but not in the extent to which they induced resistance. Induced resistance was equally effective against all isolates tested. Plant lines varied in the baseline level of resistance expressed in newly emerging leaves. In some lines, new leaves were poorly defended but developed resistance with maturity, even in the absence of infection. In those lines, induced resistance could not prevent anthracnose damage to young leaves, and this damage reduced plant fitness by increasing juvenile mortality and decreasing juvenile growth rates. In contrast, anthracnose damage to well-defended older leaves had no effect on juvenile growth rates. In at least one line, new leaves were well-defended, regardless of disease experience. This line did not experience reduced growth from anthracnose infection of either young or mature leaves, suggesting that lines with higher baseline levels of resistance are more fit than those dependent upon induced resistance. These results suggest that induced resistance cannot substitute for baseline local resistance in this I. purpurea population.

Effect of seed predation on seed bank size and seedling recruitment of bush lupine (Lupinus arboreus).

Whether seed consumers affect plant establishment is an important unresolved question in plant population biology. Seed consumption is ubiquitous; at issue is whether seedling recruitment is limited by safe-sites or seeds. If most seeds inhabit sites unsuitable for germination, post-dispersal seed consumption primarily removes seeds that would otherwise never contribute to the population and granivory has minimal impacts on plant abundance. Alternatively, if most seeds ultimately germinate before they lose viability, there is greater potential for seed consumption to affect plant recruitment. Of the many studies on seed consumption, few ask how seed loss affects seedling recruitment for species with long-lived seed banks. We examined post-dispersal seed predation and seedling emergence in bush lupine (Lupinus arboreus), a woody leguminous shrub of coastal grasslands and dunes in California. We followed the fate of seeds in paired experimental seed plots that were either protected or exposed to rodent granivores in grassland and dune habitats. Significantly more seeds were removed by rodents in dunes than grasslands. In dunes, where rodent granivory was greatest (65% and 86% of seeds removed from plots by rodents in two successive years), there is a sparse seed bank (6.6 seeds m-2), and granivory significantly reduced seedling emergence (in the same two years, 18% and 19.4% fewer seedlings emerged from exposed versus protected plots), suggesting seed rather than safe-site limited seedling recruitment. In contrast, rodents removed an average of 6% and 56% of seeds from grassland plots during the same two years, and the grassland seed bank is 43-fold that of the dunes (288 seeds m-2). Even high seed consumption in the second year of the study only marginally influenced recruitment because seeds that escaped predation remained dormant. Burial of seeds in both habitats significantly reduced the percentage of seeds removed by rodents. Results suggest that granivores exert strong but habitat-dependent effects on lupine seed survival and seedling emergence.

Permanent genetic resources added to Molecular Ecology Resources Database 1 October 2011-30 November 2011.

This article documents the addition of 139 microsatellite marker loci and 90 pairs of single-nucleotide polymorphism sequencing primers to the Molecular Ecology Resources Database. Loci were developed for the following species: Aglaoctenus lagotis, Costus pulverulentus, Costus scaber, Culex pipiens, Dascyllus marginatus, Lupinus nanus Benth, Phloeomyzus passerini, Podarcis muralis, Rhododendron rubropilosum Hayata var. taiwanalpinum and Zoarces viviparus. These loci were cross-tested on the following species: Culex quinquefasciatus, Rhododendron pseudochrysanthum Hay. ssp. morii (Hay.) Yamazaki and R. pseudochrysanthum Hayata. This article also documents the addition of 48 sequencing primer pairs and 90 allele-specific primers for Engraulis encrasicolus.

Pathways to mutualism breakdown.

Mutualisms are ubiquitous in nature despite the widely held view that they are unstable interactions. Models predict that mutualists might often evolve into parasites, can abandon their partners to live autonomously and are also vulnerable to extinction. Yet a basic empirical question, whether mutualisms commonly break down, has been mostly overlooked. As we discuss here, recent progress in molecular systematics helps address this question. Newly constructed phylogenies reveal that parasites as well as autonomous (non-mutualist) taxa are nested within ancestrally mutualistic clades. Although models have focused on the propensity of mutualism to become parasitic, such shifts appear relatively rarely. By contrast, diverse systems exhibit reversions to autonomy, and this might be a common and unexplored endpoint to mutualism.

Epistasis and genotype-environment interaction for quantitative trait loci affecting flowering time in Arabidopsis thaliana.

A major goal of evolutionary biology is to understand the genetic architecture of the complex quantitative traits that may lead to adaptations in natural populations. Of particular relevance is the evaluation of the frequency and magnitude of epistasis (gene-gene and gene-environment interaction) as it plays a controversial role in models of adaptation within and among populations. Here, we explore the genetic basis of flowering time in Arabidopsis thaliana using a series of quantitative trait loci (QTL) mapping experiments with two recombinant inbred line (RIL) mapping populations [Columbia (Col) x Landsberg erecta (Ler), Ler x Cape Verde Islands (Cvi)]. We focus on the response of RILs to a series of environmental conditions including drought stress, leaf damage, and apical damage. These data were explicitly evaluated for the presence of epistasis using Bayesian based multiple-QTL genome scans. Overall, we mapped fourteen QTL affecting flowering time. We detected two significant QTL-QTL interactions and several QTL-environment interactions for flowering time in the Ler x Cvi population. QTL-environment interactions were due to environmentally induced changes in the magnitude of QTL effects and their interactions across environments--we did not detect antagonistic pleiotropy. We found no evidence for QTL interactions in the Ler x Col population. We evaluate these results in the context of several other studies of flowering time in Arabidopsis thaliana and adaptive evolution in natural populations.

EXAMINING SELECTION ON THE MULTIVARIATE PHENOTYPE: PLANT RESISTANCE TO HERBIVORES.

Cost-benefit theory posits that stabilizing selection, produced by a trade-off between associated costs and benefits, often maintains phenotypic traits at intermediate equilibrium values. Measurement of selection on one type of trait, resistance to herbivory, should provide evidence to test this prediction. However, most plants host more than one species of herbivore, and resistance to various herbivores may be phenotypically correlated. Consequently, selection must be measured on a multivariate phenotype, which may produce a very complex selection gradient. Canonical analysis of the matrix of quadratic coefficients of the phenotypic selection gradient is presented as a method to simplify the interpretation of the estimated multidimensional adaptive surface. Because this analytical method finds the canonical axes of second-order effects on the selection surface, it shows where nonlinear, and possibly stabilizing, selection is strongest. The resulting index traits may generate hypotheses regarding underlying resistance traits to which more than one type of herbivore responds.

Partner choice in nitrogen-fixation mutualisms of legumes and rhizobia.

Mutualistic interactions are widespread and obligatory for many organisms, yet their evolutionary persistence in the face of cheating is theoretically puzzling. Nutrient-acquisition symbioses between plants and soil microbes are critically important to plant evolution and ecosystem function, yet we know almost nothing about the evolutionary dynamics and mechanisms of persistence of these ancient mutualisms. Partner-choice and partner-fidelity are mechanisms for dealing with cheaters, and can theoretically allow mutualisms to persist despite cheaters.Many models of cooperative behavior assume pairwise interactions, while most plant-microbe nutrient-acquisition symbioses involve a single plant interacting with numerous microbes. Market models, in contrast, are well suited to mutualisms in which single plants attempt to conduct mutually beneficial resource exchange with multiple individuals. Market models assume that one partner chooses to trade with a subset of individuals selected from a market of potential partners. Hence, determining whether partner-choice occurs in plant-microbe mutualisms is critical to understanding the evolutionary persistence and dynamics of these symbioses. The nitrogen-fixation/carbon-fixation mutualism between leguminous plants and rhizobial bacteria is widespread, ancient, and important for ecosystem function and human nutrition. It also involves single plants interacting simultaneously with several to many bacterial partners, including ineffective ("cheating") strains. We review the existing literature and find that this mutualism displays several elements of partner-choice, and may match the requirements of the market paradigm. We conclude by identifying profitable questions for future research.

THE EVOLUTION OF RESISTANCE TO HERBIVORY IN IPOMOEA PURPUREA. II. NATURAL SELECTION BY INSECTS AND COSTS OF RESISTANCE.

An important component of the process of coevolution between plants and their insect herbivores is the imposition of selection on plants by insects. Although such selection has been inferred from indirect evidence, little direct evidence for it exists. One goal of this study was to seek direct evidence by determining, for a single plant-herbivore system, whether insect herbivores impose selection on their host plants. A second goal was to determine whether costs are associated with genotypes that confer resistance to herbivores, as has been commonly postulated. The annual morning glory, Ipomoea purpurea, exhibits genetic variation in resistance to four different types of insects. For three of these types, most of the genetic variation is additive. Removal of insect herbivores increased the number of seeds produced by I. purpurea by 20% and eliminated additive genetic variation for seed number (fitness). This result implies that herbivores impose selection on some trait(s) of their host plants. Coupled with selection for decreased damage by corn earworms, as revealed by a negative additive genetic covariance between damage and fitness, this result suggests that insect herbivores impose selection on resistance to corn earworms in I. purpurea. Two types of cost of resistance to herbivores were sought in I. purpurea: 1) internal trade-offs in allocation of resources and 2) ecological trade-offs between resistances to different insects. No costs of either type were detected. This result suggests that cost-benefit arguments that attempt to predict the evolution of levels of resistance to herbivores are not applicable to I. purpurea.

THE EVOLUTION OF RESISTANCE TO HERBIVORY IN IPOMOEA PURPUREA. I. ATTEMPTS TO DETECT SELECTION.

In this study, we looked for evidence of directional or stabilizing/disruptive selection on plant size and on the level of damage (resistance) caused by four types of herbivores in the annual morning glory Ipomoea purpurea. Selection was estimated by standard phenotypic regression analysis and by regression on breeding values. The phenotypic regression analysis revealed directional selection for all five characters (i.e., plant size and resistance to four types of herbivores) and indicated that plant size and resistance to corn-earworm damage were subject to stabilizing selection. By contrast, the analysis using breeding values revealed directional selection only for plant size and resistance to corn earworms, while none of the characters examined indicated stabilizing or disruptive selection. These results suggest that intermediate levels of damage in I. purpurea are, in general, not maintained by stabilizing selection. Rather, they may reflect either 1) a transient state that exists while directional selection pushes the population toward complete resistance (or, in one case, total absence of resistance) or 2) the evolution of susceptibility to damage by genetic drift.