Adaptive evolution of threonine deaminase in plant defense against insect herbivores

Gene duplication is a major source of plant chemical diversity that mediates plant-herbivore interactions. There is little direct evidence, however, that novel chemical traits arising from gene duplication reduce herbivory. Higher plants use threonine deaminase (TD) to catalyze the dehydration of threonine (Thr) to α-ketobutyrate and ammonia as the committed step in the biosynthesis of isoleucine (Ile). Cultivated tomato and related Solanum species contain a duplicated TD paralog (TD2) that is coexpressed with a suite of genes involved in herbivore resistance. Analysis of TD2-deficient tomato lines showed that TD2 has a defensive function related to Thr catabolism in the gut of lepidopteran herbivores. During herbivory, the regulatory domain of TD2 is removed by proteolysis to generate a truncated protein (pTD2) that efficiently degrades Thr without being inhibited by Ile. We show that this proteolytic activation step occurs in the gut of lepidopteran but not coleopteran herbivores, and is catalyzed by a chymotrypsin-like protease of insect origin. Analysis of purified recombinant enzymes showed that TD2 is remarkably more resistant to proteolysis and high temperature than the ancestral TD1 isoform. The crystal structure of pTD2 provided evidence that electrostatic interactions constitute a stabilizing feature associated with adaptation of TD2 to the extreme environment of the lepidopteran gut. These findings demonstrate a role for gene duplication in the evolution of a plant defense that targets and co-opts herbivore digestive physiology.     

Testing hypotheses of a coevolutionary key innovation reveals a complex suite of traits involved in defusing the mustard oil bomb

Coevolutionary interactions are responsible for much of the Earth’s biodiversity, with key innovations driving speciation bursts on both sides of the interaction. One persistent question is whether macroevolutionary traits identified as key innovations accurately predict functional performance and selection dynamics within species, as this necessitates characterizing their function, investigating their fitness consequences, and exploring the selection dynamics acting upon them. Here, we used CRISPR-Cas9 mediating nonhomologous end joining (NHEJ) in the butterfly species Pieris brassicae to knock out and directly assess the function and fitness impacts of nitrile specifier protein (NSP) and major allergen (MA). These are two closely related genes that facilitate glucosinolate (GSL) detoxification capacity, which is a key innovation in mustard feeding Pierinae butterflies. We find NSP and MA are both required for survival on plants containing GSLs, with expression differences arising in response to variable GSL profiles, concordant with detoxification performance. Importantly, this concordance was only observed when using natural host plants, likely reflecting the complexity of how these enzymes interact with natural plant variation in GSLs and myrosinases. Finally, signatures of positive selection for NSP and MA were detected across Pieris species, consistent with these genes’ importance in recent coevolutionary interactions. Thus, the war between these butterflies and their host plants involves more than the mere presence of chemical defenses and detoxification mechanisms, as their regulation and activation represent key components of complex interactions. We find that inclusion of these dynamics, in ecologically relevant assays, is necessary for coevolutionary insights in this system and likely others.

From beetles to butterflies, plant-feeding insects account for around a quarter of all known macroscopic animals (1, 2). This vast diversity is thought to have been fueled by diffuse coevolutionary interactions with host plants, with key innovations driving speciation bursts and trait escalation on both sides of the plant–insect interaction (3, 4). However, our understanding of plant–insect systems still lacks strong mechanistic connections between gene regulation and function, microevolutionary processes, and macroevolutionary patterns, as these schools of research are rarely integrated in the testing of reciprocal hypotheses (5). Such integration is important for understanding the origins and evolutionary dynamics of coevolutionary key innovations, as such innovations are often complex phenotypes that arise from many interacting genes and their fine-tuned regulation. To fill this gap and advance the study of coevolutionary key innovations, here we use an ecological and evolutionary functional genomics approach (6) to test diverse predictions that emerge from studies of the coevolutionary interactions between Brassicales plants and their Pierinae butterfly parasites.Pierinae butterflies (Lepidoptera: Pieridae) have been studied for over a century (7) due to their ability to overcome the glucosinolate (GSL) defenses of their host plants in the order Brassicales. A defining trait of these plants is their two-component, activated chemical defense system, known as the mustard-oil bomb (8). The inactive bomb is comprised of the enzyme myrosinase and GSL compounds, which are stored separately from each other until activation (9). Upon tissue damage (e.g., by larval feeding), myrosinase hydrolyzes GSLs, producing diverse breakdown products (10, 11). For most insects, the breakdown products of the GSLs found in Brassicales act as strong feeding deterrents, greatly reducing fitness and survival (12, 13). In contrast, Pierinae larvae are largely unharmed by GSL defenses [(13, 14), but see ref. 15] and in fact, GSL compounds have generally been coopted as stimulants for larval feeding (7, 16–20) and female oviposition (20–25)An important component of Pierinae’s resistance to GSL defenses is conferred by larval gut-expressed proteins known as nitrile-specifier proteins (NSPs), which interact with myrosinases to divert the breakdown of GSL defenses away from the formation of highly toxic isothiocyanates (ITCs) to less-toxic nitriles instead (10, 14). Nearly 50 y ago, Ehrlich and Raven hypothesized that this ability of Pierinae butterflies to detoxify GSLs was a coevolutionary key innovation (3). The identification of NSPs as GSL-detoxification mechanisms was a major step forward in testing this hypothesis, providing a phenotype and gene for analyses (14). The birth of NSP and its enzymatic activity appears to have evolved shortly after basal Pierinae butterflies colonized Brassicales plants, with this colonization associated with an increased speciation rate in Pierinae (26). Subsequent study focused upon escalations in both the GSL compounds and the detoxification potential of NSP genes (27). In Brassicales, two major bouts of escalation in GSL complexity were associated with increased diversification rates, with the final bout giving rise to the Brassicaceae, the most speciose and GSL-diverse family of Brassicales. Analysis of detoxification evolution in Brassicales-feeding Pierinae revealed NSP to be part of a small gene family undergoing extensive gene birth–death dynamics. Importantly, the two lineages that independently colonized Brassicaceae used different NSP loci for detoxification and both lineages had increased species diversification rates compared with other lineages (27). Thus, the study of NSP gene function provides insights into the evolution of a key innovation.The NSP-like gene family consists of NSPs, major-allergen (MA) proteins, and the single-domain major-allergen (SDMA) proteins from which the two previous genes evolved via exon duplications at the base of the Pierinae (27, 28). Several lines of evidence suggest that both NSP and MA, but not the SDMA genes, have played a key role in Pierinae butterfly adaptation to host plant GSL defense, leading to the following four predictions.NSP and MA Are Necessary for GSL Detoxification, but SDMA Is Not.Whereas the GSL detoxifying capacity of NSP has been clearly demonstrated in Pieris species (14), the same cannot be said for MA. Previous work in Pieris species using heterologously expressed proteins found nitrile-forming activity only for NSP but not for MA, leading to the conclusion that MA was inactive against the tested GSLs (27). However, subsequent attempts to express functional NSP and MA proteins have been extremely challenging, to the point that we now conclude that absence of nitrile formation by heterologously expressed proteins should not be used to infer the absence of this enzymatic capacity (for more discussion refer to SI Appendix, Text 16). MA is a known GSL detoxifying protein in other Pieridae (27), and experiments using Pieris melete have shown that NSP and MA expression is only activated by the presence of GSLs (29). This suggests that both proteins may have detoxification function within Pieris. Furthermore, Pierinae lineages that have stopped feeding upon plants with GSLs have lost both NSP and MA, but not SDMA, suggesting that the former two genes are costly to maintain in the absence of GSL (26, 27).NSP and MA Differ in Function in Relation to GSL Variation among Plants. While some functional differences have been identified between MA and NSP, studies have mostly been limited to in vitro assays of gut extracts and have lacked a clear ecological context. However, NSP and MA have been found to be differentially expressed when P. melete larvae are reared on different natural host plants, suggesting that the specificity of NSP and MA’s expression may be in response to different GSL classes (29). No such expression changes have been found in SDMA (29).MA Detoxifies a Broader Range of GSLs Compared with NSP.This prediction is supported by the loss of NSP, but continued presence of MA in the Brassicaceae-feeding Anthocharis genus (27). Since Anthocharis feed upon host plants with diverse GSLs (30), MA likely has a broader detoxification function against GSLs compared with NSP. It has also been observed that within a butterfly species, the expression of MA appears to be induced by a broader range of GSLs than NSP (29).NSP and Potentially MA Commonly Experience Positive Selection.The aforementioned studies suggest that these genes play an important and ongoing role in mediating plant–insect interactions over evolutionary time, leading diverse studies to use molecular tests of selection to try detecting evidence of these dynamics. Using consensus sequences of species in codon-based tests of selection, signatures of positive selection in NSP and MA, but not SDMA genes, have been identified among divergent taxa with dN/dS > 1 (27, 31), while other analyses find only an increased dN/dS rates at NSP among Pieris species though the rate is still < 1, consistent with either relaxed constraint or bouts of positive selection (27, 28, 31). Using samples of many individuals within species, two microevolutionary studies of NSP-family genes have been conducted to date. In the first of these candidate gene studies (32), no evidence of positive selection on NSP or MA was found in populations of Pieris rapae. However, NSP had an exceptionally high number of nonsynonymous fixations compared with other genes, suggestive of positive selection (32). The second study (33) found evidence of positive selection in the NSP genes of Japanese Pieris napi and suggestions of balancing selection at NSP in P. melete, with no clear evolutionary trends detected at MA.In sum, while there are suggestions of positive selection at NSP and occasionally at MA among Pieris taxa, these variable results among combinations of taxa and methods complicate drawing conclusions from these findings. Additionally, while much of this variation is expected, as such tests detect departures from neutrality in different ways and over different time scales, their implementation has also varied in ways known to degrade estimates of positive selection (SI Appendix, Text 13). The codon-based tests used few taxa and were thus very underpowered (27, 28, 31), the population analyses used incomplete gene sequences for population analyses of NSP and MA (32, 33), and all these analyses failed to account for underlying population dynamics. We predict that using advances in molecular tests of selection that leverage the power of genome scale data, while accounting for demographic history, will help resolve these mixed findings.Here we tested the above predictions to better understand how key innovations work and evolve. We used CRISPR-Cas9 mediating nonhomologous end joining (NHEJ) to knock out the function of both NSP and MA. This enabled us to characterize detoxification performance, assess functional redundancy, and understand the fitness consequences of each protein in vivo (Fig. 1). We ultimately found that NSP and MA both play important, but different roles in diverting the breakdown of GSLs and that there is a striking concordance between their induction by, and ability to detoxify, different GSLs. Additionally, we found evidence of positive selection acting on both NSP and MA genes within species lineages, albeit at different strengths, consistent with the ongoing importance of these genes to their lineages’ evolutionary success. Our work represents a major step forward both for the study of the Pierinae–Brassicales system and for the field of coevolutionary biology; we demonstrate that the genes involved in a key innovation phenotype experience positive selection at a microevolutionary scale, with their regulation fine-tuned to produce plastic responses for host plant-specific detoxification.Open in a separate windowFig. 1.(A) In wild P. brassicae caterpillars, feeding on a GSL-myrosinase-defended plant causes no damage to the larva, as specific enzymes in the larval gut (NSP and MA) divert the breakdown of GSL compounds so that nitriles are formed instead of toxic ITCs. These nitriles are easily detectable in larval frass. (B) In this study, we stopped P. brassicae’s adaptive diversion of the GSL breakdown process by knocking out both NSP and MA genes with the CRISPR-Cas9 system. As a result of these KOs, we predicted toxic ITCs would form instead of nitriles, and KO larvae feeding on GSL-defended plants would be unable to survive.     

Macroevolution and the biological diversity of plants and herbivores

Terrestrial biodiversity is dominated by plants and the herbivores that consume them, and they are one of the major conduits of energy flow up to higher trophic levels. Here, we address the processes that have generated the spectacular diversity of flowering plants (>300,000 species) and insect herbivores (likely >1 million species). Long-standing macroevolutionary hypotheses have postulated that reciprocal evolution of adaptations and subsequent bursts of speciation have given rise to much of this biodiversity. We critically evaluate various predictions based on this coevolutionary theory. Phylogenetic reconstruction of ancestral states has revealed evidence for escalation in the potency or variety of plant lineages'' chemical defenses; however, escalation of defense has been moderated by tradeoffs and alternative strategies (e.g., tolerance or defense by biotic agents). There is still surprisingly scant evidence that novel defense traits reduce herbivory and that such evolutionary novelty spurs diversification. Consistent with the coevolutionary hypothesis, there is some evidence that diversification of herbivores has lagged behind, but has nevertheless been temporally correlated with that of their host-plant clades, indicating colonization and radiation of insects on diversifying plants. However, there is still limited support for the role of host-plant shifts in insect diversification. Finally, a frontier area of research, and a general conclusion of our review, is that community ecology and the long-term evolutionary history of plant and insect diversification are inexorably intertwined.     

From the Cover: Cardenolides,toxicity, and the costs of sequestration in the coevolutionary interaction between monarchs and milkweeds

For highly specialized insect herbivores, plant chemical defenses are often co-opted as cues for oviposition and sequestration. In such interactions, can plants evolve novel defenses, pushing herbivores to trade off benefits of specialization with costs of coping with toxins? We tested how variation in milkweed toxins (cardenolides) impacted monarch butterfly (Danaus plexippus) growth, sequestration, and oviposition when consuming tropical milkweed (Asclepias curassavica), one of two critical host plants worldwide. The most abundant leaf toxin, highly apolar and thiazolidine ring–containing voruscharin, accounted for 40% of leaf cardenolides, negatively predicted caterpillar growth, and was not sequestered. Using whole plants and purified voruscharin, we show that monarch caterpillars convert voruscharin to calotropin and calactin in vivo, imposing a burden on growth. As shown by in vitro experiments, this conversion is facilitated by temperature and alkaline pH. We next employed toxin-target site experiments with isolated cardenolides and the monarch’s neural Na⁺/K⁺-ATPase, revealing that voruscharin is highly inhibitory compared with several standards and sequestered cardenolides. The monarch’s typical >50-fold enhanced resistance to cardenolides compared with sensitive animals was absent for voruscharin, suggesting highly specific plant defense. Finally, oviposition was greatest on intermediate cardenolide plants, supporting the notion of a trade-off between benefits and costs of sequestration for this highly specialized herbivore. There is apparently ample opportunity for continued coevolution between monarchs and milkweeds, although the diffuse nature of the interaction, due to migration and interaction with multiple milkweeds, may limit the ability of monarchs to counteradapt.

Although coevolutionary interactions are often portrayed as simplified arms races of reciprocal defense and offense evolution, the dynamics are decidedly more complex. For example, how do plants respond to highly specialized herbivores, and are such adapted consumers immune to plant defenses? On average, specialists are less impacted by particular plant defense compounds than generalists (1, 2), but does this mean that further coevolution is not possible? Even highly specialized herbivores must contend with plant defenses if coevolutionary interactions are proceeding (3). For any herbivorous insect, larval feeding, protection from enemies, and adult oviposition are each key points in the life cycle where plant chemistry plays a role in the outcome. Thus, the typical cornucopia of chemical compounds in an individual plant presents opportunities for both plant resistance and co-option of this defense by specialist herbivores (4–6).Thus, it is unclear how often coevolutionary interactions reach equilibrium or “stalemate,” as it were (7). Nonetheless, several conditions are predicted to slow or suppress the endless arms race. First, the more specialized an interaction, the greater the investments required and potential challenges to innovation. Second, when different life stages of herbivores are subject to distinct selection pressures (8–10), continued coevolution may be restricted because of conflicting selection. Finally, when aspects of the population biology of the species involved reduce local adaptation, such as gene flow and the presence of alternate hosts, asymmetry may emerge in the coevolutionary match between plants and herbivores (11, 12). In the interaction between milkweed plants and monarch butterflies, cardenolides have played a central role in our understanding of coevolutionary specialization, larval feeding, sequestration, and, to a lesser extent, oviposition (13). Although monarchs are abundant across a broad geographical range, substantial phenotypic and genetic analyses have failed to reveal population differentiation (14, 15). A lack of local adaptation is likely due to the four-generation annual cycle where butterflies feed on diverse milkweed species and yet intermix during migration and overwintering (13).There is some evidence that cardenolides can be a burden for monarch caterpillars (16–20), although costs of sequestration have not been demonstrated. Nonetheless, many assays, even across >10-fold concentrations of cardenolides, fail to show negative effects of cardenolides on monarchs (21). More mechanistic in vitro work with the monarch’s highly resistant sodium-potassium pump (Na⁺/K⁺-ATPase), the cellular target of cardenolides, demonstrated that some milkweed cardenolides are strong inhibitors of monarch neural physiology (22). Thus, work with specific compounds that are variable in plants is needed to pinpoint agents of resistance. For sequestration of cardenolides, a model proposed by Nelson (23) and supported in a review of early work (24) and new research (25) suggests that monarchs selectively sequester more polar cardenolides, some compounds are metabolized (modification or detoxification), and others are transported via carriers (20, 26–28). Finally, observational work indicated that monarchs tend to oviposit on intermediate cardenolide concentration plants (29, 30), suggesting the hypothesis that adult butterflies minimize toxic exposure to larvae while optimizing sequestration of plant poisons.Asclepias curassavica is surprisingly understudied in its interactions with monarch butterflies, despite being a critical hostplant worldwide (second only to Asclepias syriaca) (13). Attack of A. curassavica by monarchs can be strong and therefore a likely source of selection for plant defense. The species is weedy throughout the tropics and has a plethora of cardenolides, including relatively uncommon compounds, some of which may be detrimental to monarch performance (20, 28, 31–34). In particular, voruscharin is a long-known cardenolide containing a thiazolidine heterocycle (having both nitrogen and sulfur; Fig. 1), yet its previous study was hampered by solubility issues and the inability of thin layer chromatography to separate it from related compounds (20, 28, 31, 35). In terms of sequestration, it was demonstrated decades ago that monarchs preferentially sequester two cardenolides, calotropin and calactin, especially when feeding on A. curassavica (20, 31, 33, 36). For oviposition, two flavonol glycosides were isolated from A. curassavica leaves that stimulate egg laying (37). Nonetheless, the relative importance of quercetin glycosides versus cardenolides in oviposition is unknown, and specific cardenolides that impact larval growth and sequestration have not been well-studied. If A. curassavica defends itself against this specialized herbivore in a coevolutionary interaction mediated by plant chemistry, connecting specific toxins to their target site in the context of sequestration and oviposition is critical. In particular, we hypothesized that specific cardenolides modulate a trade-off between benefits of specialization and costs of coping with toxicity.Open in a separate windowFig. 1.Chemical conversion of milkweed cardenolides by monarch caterpillars. (A) A visualization of metabolomic data showing the differences in the chemical composition across sample groups (n = 4 per group; significance tested by PERMANOVA). After data curation, over 7,000 chemical features (m/z) were generated with MS data collected in positive ionization mode and visualized with a Bray–Curtis distance matrix. Ellipses represent the region of 95% confidence. (B) Voruscharin was converted to calactin and calotropin when fed to monarch caterpillars. Shown are means ± SE concentrations as determined by UV-HPLC (n = 3 to 9). Data bars very close to zero had no detectable cardenolides. Note that the caterpillars fed A. curassavica were reared on this diet from hatching and had an order of magnitude higher cardenolides than other treatments which were dosed only during the fourth instar. (C) The basic skeleton of cardenolides and the chemical structures of calactin, calotropin, and voruscharin.Here, we identify cardenolides produced by A. curassavica and address which compounds are sequestered by monarchs, followed by asking four questions: 1) Do specific cardenolides reduce monarch larval growth, or does sequestration impose a burden for larvae? 2) Using in vivo and in vitro assays, do monarchs detoxify or convert particular cardenolides to less toxic forms? 3) What is the relative toxicity (measured as in vitro inhibition of the cellular target, the monarch’s Na⁺/K⁺-ATPase) of nonsequestered cardenolides, those sequestered intact, and those modified during sequestration? And, finally, 4) do monarch oviposition decisions minimize toxicity and optimize sequestration of cardenolides, or are oviposition stimulants (flavonol glycosides) drivers of oviposition?     

Pseudomonas syringae manipulates systemic plant defenses against pathogens and herbivores

Many pathogens are virulent because they specifically interfere with host defense responses and therefore can proliferate. Here, we report that virulent strains of the bacterial phytopathogen Pseudomonas syringae induce systemic susceptibility to secondary P. syringae infection in the host plant Arabidopsis thaliana. This systemic induced susceptibility (SIS) is in direct contrast to the well studied avirulence/R gene-dependent resistance response known as the hypersensitive response that elicits systemic acquired resistance. We show that P. syringae-elicited SIS is caused by the production of coronatine (COR), a pathogen-derived functional and structural mimic of the phytohormone jasmonic acid (JA). These data suggest that SIS may be a consequence of the previously described mutually antagonistic interaction between the salicylic acid and JA signaling pathways. Virulent P. syringae also has the potential to induce net systemic susceptibility to herbivory by an insect (Trichoplusia ni, cabbage looper), but this susceptibility is not caused by COR. Rather, consistent with its role as a JA mimic, COR induces systemic resistance to T. ni. These data highlight the complexity of defense signaling interactions among plants, pathogens, and herbivores.     

From the Cover: Defense mutualisms enhance plant diversification

The ability of plants to form mutualistic relationships with animal defenders has long been suspected to influence their evolutionary success, both by decreasing extinction risk and by increasing opportunity for speciation through an expanded realized niche. Nonetheless, the hypothesis that defense mutualisms consistently enhance plant diversification across lineages has not been well tested due to a lack of phenotypic and phylogenetic information. Using a global analysis, we show that the >100 vascular plant families in which species have evolved extrafloral nectaries (EFNs), sugar-secreting organs that recruit arthropod mutualists, have twofold higher diversification rates than families that lack species with EFNs. Zooming in on six distantly related plant clades, trait-dependent diversification models confirmed the tendency for lineages with EFNs to display increased rates of diversification. These results were consistent across methodological approaches. Inference using reversible-jump Markov chain Monte Carlo (MCMC) to model the placement and number of rate shifts revealed that high net diversification rates in EFN clades were driven by an increased number of positive rate shifts following EFN evolution compared with sister clades, suggesting that EFNs may be indirect facilitators of diversification. Our replicated analysis indicates that defense mutualisms put lineages on a path toward increased diversification rates within and between clades, and is concordant with the hypothesis that mutualistic interactions with animals can have an impact on deep macroevolutionary patterns and enhance plant diversity.Ever since the key innovation hypothesis was first proposed in the 1940s (1, 2), the origination of novel traits has been a popular yet controversial explanation for the exceptional disparity in species richness observed across clades in the tree of life. Despite decades of research linking traits to diversification, we have remarkably few examples of traits that have been convincingly demonstrated to spur diversification repeatedly across independent, distantly related groups. Notable exceptions include a number of ecologically important traits mediating interactions between plants and animals (3–6), suggesting that these interactions may be particularly important drivers of macroevolutionary patterns. Here, we test the hypothesis that plant defense mutualisms, a widespread and classically studied ecological interaction whereby plants provide food rewards to arthropod bodyguards in return for protection against natural enemies (7), increase the evolutionary diversification rate of the plant lineages that participate in them. The morphological traits that mediate defense mutualisms represent well-studied examples of characters hypothesized to expand a plant’s niche via interactions with mutualists and influence species success in various environmental contexts (8). Although the costs and benefits of participating in defense mutualisms are well studied (9), the hypothesis that the ecological impact of defense mutualisms leaves a predictable macroevolutionary signature, increasing lineage diversification within and among clades of plants, has only been examined in a single genus (10).Defense mutualisms may have an impact on plant speciation and extinction rates via several mechanisms. Unlike the evolution of traits related to reproduction, which, more intuitively, could have an impact on lineage diversification (e.g., refs. 5, 11), the direct mechanism by which defense mutualisms are hypothesized to influence diversification is less obvious. One direct mechanism is a decreased incidence of damage and disease due to an enhanced defensive repertoire, which may allow for increased population sizes and, in turn, lower extinction rates (6). Additionally, by expanding the realized niche of a plant (12), defense mutualisms may broaden the range of habitats a plant can occupy (10), thereby increasing instances of allopatric speciation.However, in addition to these direct mechanisms, the evolution of mutualistic traits may facilitate diversification indirectly. First, if niche expansion results in the successful occupation of more environments, mutualistic traits may increase the probability a lineage will encounter conditions ripe with ecological opportunity (e.g., new adaptive zones), which, in turn, will drive increases in diversification. In other words, the evolution of a trait may enable subsequent diversification via increasing exposure to new environments, some of which will harbor external drivers of radiation, such as the uplift of a mountain range or unoccupied niches. Second, the evolution of defense mutualisms may free up resources for the plant, and thereby facilitate the evolution of other innovative traits that subsequently enhance diversification. These indirect effects need not be contingent on the existence of the direct effects mentioned above, and represent a largely overlooked hypothesis concerning how traits can affect diversification (13–15).We suggest that indirect impacts of trait evolution on diversification should be reflected in a phylogenetic pattern in which the origination of a trait is followed by an increased probability of subsequent, downstream rate shifts relative to clades that lack the trait (Fig. 1). Because the indirect effect of the trait is contingent upon additional conditions (e.g., ecological opportunity, the evolution of another trait), there may be a substantial lag between the origin of the trait and rate shifts. Alternatively, a direct effect of the trait on the diversification rate is consistent with a pattern whereby a sustained rate shift occurs concomitantly with, or on the same branch as, the origin of the trait on the phylogeny (Fig. 1). Direct and indirect patterns are not mutually exclusive, and both patterns may be detectable on a single phylogeny (Fig. 1).Open in a separate windowFig. 1.A conceptualization of phylogenetic patterns consistent with direct or indirect effects of EFNs (or any trait) on lineage diversification. A net change in diversification may be due to direct or indirect mechanisms. In the Upper Right, a rate shift occurs concomitantly with the origin of EFNs, consistent with a direct effect. If one or more shifts occur with some delay (Lower), this is consistent with a hypothesis that a trait has an indirect or context-dependent effect on diversification rates.We focus on the macroevolutionary consequences of the repeated origination of extrafloral nectaries (EFNs), nectar-secreting glands found on nonfloral plant tissues that provide food for a wide array of beneficial arthropod bodyguards (16). EFNs are well studied ecologically, and their only known function is defense against herbivores and microbial pathogens by attracting natural enemies (17). Such features have evolved hundreds of times and occur in about a quarter of all vascular plant families (18). Here, we first ask whether, across all vascular plants, families containing species with EFNs are associated with higher diversification rates than families without EFNs. We then focus in on the phylogenetic history and evolution of EFNs in six distantly related plant clades to evaluate whether EFNs are linked, directly or indirectly, to increased lineage diversification rates. As such, this study represents a replicated, multiscale test of the macroevolutionary consequences of a convergently evolved and ecologically important mutualistic trait.     

Cucumber Mosaic Virus-Induced Systemic Necrosis in Arabidopsis thaliana: Determinants and Role in Plant Defense

Effector-triggered immunity (ETI) is one of the most studied mechanisms of plant resistance to viruses. During ETI, viral proteins are recognized by specific plant R proteins, which most often trigger a hypersensitive response (HR) involving programmed cell death (PCD) and a restriction of infection in the initially infected sites. However, in some plant–virus interactions, ETI leads to a response in which PCD and virus multiplication are not restricted to the entry sites and spread throughout the plant, leading to systemic necrosis. The host and virus genetic determinants, and the consequences of this response in plant–virus coevolution, are still poorly understood. Here, we identified an allelic version of RCY1—an R protein—as the host genetic determinant of broad-spectrum systemic necrosis induced by cucumber mosaic virus (CMV) infection in the Arabidopsis thaliana Co-1 ecotype. Systemic necrosis reduced virus fitness by shortening the infectious period and limiting virus multiplication; thus, this phenotype could be adaptive for the plant population as a defense against CMV. However, the low frequency (less than 1%) of this phenotype in A. thaliana wild populations argues against this hypothesis. These results expand current knowledge on the resistance mechanisms to virus infections associated with ETI in plants.     

Receptor-like kinase SOBIR1/EVR interacts with receptor-like proteins in plant immunity against fungal infection

The plant immune system is activated by microbial patterns that are detected as nonself molecules. Such patterns are recognized by immune receptors that are cytoplasmic or localized at the plasma membrane. Cell surface receptors are represented by receptor-like kinases (RLKs) that frequently contain extracellular leucine-rich repeats and an intracellular kinase domain for activation of downstream signaling, as well as receptor-like proteins (RLPs) that lack this signaling domain. It is therefore hypothesized that RLKs are required for RLPs to activate downstream signaling. The RLPs Cf-4 and Ve1 of tomato (Solanum lycopersicum) mediate resistance to the fungal pathogens Cladosporium fulvum and Verticillium dahliae, respectively. Despite their importance, the mechanism by which these immune receptors mediate downstream signaling upon recognition of their matching ligand, Avr4 and Ave1, remained enigmatic. Here we show that the tomato ortholog of the Arabidopsis thaliana RLK Suppressor Of BIR1-1/Evershed (SOBIR1/EVR) and its close homolog S. lycopersicum (Sl)SOBIR1-like interact in planta with both Cf-4 and Ve1 and are required for the Cf-4– and Ve1-mediated hypersensitive response and immunity. Tomato SOBIR1/EVR interacts with most of the tested RLPs, but not with the RLKs FLS2, SERK1, SERK3a, BAK1, and CLV1. SOBIR1/EVR is required for stability of the Cf-4 and Ve1 receptors, supporting our observation that these RLPs are present in a complex with SOBIR1/EVR in planta. We show that SOBIR1/EVR is essential for RLP-mediated immunity and propose that the protein functions as a regulatory RLK of this type of cell-surface receptors.     

Navigating natural variation in herbivory-induced secondary metabolism in coyote tobacco populations using MS/MS structural analysis

Natural variation can be extremely useful in unraveling the determinants of phenotypic trait evolution but has rarely been analyzed with unbiased metabolic profiling to understand how its effects are organized at the level of biochemical pathways. Native populations of Nicotiana attenuata, a wild tobacco species, have been shown to be highly genetically diverse for traits important for their interactions with insects. To resolve the chemodiversity existing in these populations, we developed a metabolomics and computational pipeline to annotate leaf metabolic responses to Manduca sexta herbivory. We selected seeds from 43 accessions of different populations from the southwestern United States—including the well-characterized Utah 30th generation inbred accession—and grew 183 plants in the glasshouse for standardized herbivory elicitation. Metabolic profiles were generated from elicited leaves of each plant using a high-throughput ultra HPLC (UHPLC)-quadrupole TOFMS (qTOFMS) method, processed to systematically infer covariation patterns among biochemically related metabolites, as well as unknown ones, and finally assembled to map natural variation. Navigating this map revealed metabolic branch-specific variations that surprisingly only partly overlapped with jasmonate accumulation polymorphisms and deviated from canonical jasmonate signaling. Fragmentation analysis via indiscriminant tandem mass spectrometry (idMS/MS) was conducted with 10 accessions that spanned a large proportion of the variance found in the complete accession dataset, and compound spectra were computationally assembled into spectral similarity networks. The biological information captured by this networking approach facilitates the mining of the mass spectral data of unknowns with high natural variation, as demonstrated by the annotation of a strongly herbivory-inducible phenolic derivative, and can guide pathway analysis.Elucidating the structure of metabolites underlying complex traits and the factors that maintain their variation in natural populations are important challenges in plant ecological studies (1). Many studies have notably shown that stress-responsive pathways that produce secondary metabolites are sporadically found across different plant taxa with extensive diversification (2). This important diversification suggests that particular metabolic systems have been recruited through natural selection when the set of compounds that they produce address specific ecological needs. Interactions with insects are important selection pressures that have sculpted plant metabolism, and many plant metabolites protect against herbivore attack and physical damage (3–5). The timely production of particular secondary metabolites in response to insect attack benefits plants by decreasing the costs of constitutive metabolite production. Trade-offs between defense metabolite productions and the intrinsic growth-related functions of central metabolic pathways likely provide important selection pressures that maintain the extensive metabolic polymorphisms commonly observed in natural populations.Gene discovery strategies exploiting natural variation in quantitative traits, including metabolite levels, have been extensively used in combination with genetic approaches (6–12). Analytical approaches applied in this research field are frequently focused on the quantification of individual or small families of compounds. Procedures such as liquid chromatography-mass spectrometry (LC-MS) and NMR have notably been used with both model and crop species to identify the genetic architecture of metabolic traits using quantitative trait locus mapping approaches (reviewed in ref. 13). Such approaches have been very successful in addressing genomic regions responsible for glucosinolate accumulation in Arabidopsis and related species (10, 14–16). Compared with modern sequencing and proteomics technologies, the profiling of entire plant metabolomes is, however, technically unfeasible with the existing analytical platforms, and, as a consequence, the analysis of metabolite natural variation has frequently been biased to secondary metabolite classes, for which a priori knowledge exists regarding their biological function, or to well-mapped parts of primary metabolism associated with energy and growth processes (17–19).Another critical aspect for exploiting natural variation in metabolism lies in the identification of unknown metabolites that exhibit significant associations with a phenotype of interest. Nontargeted approaches for rapidly collecting repertoires of tandem mass spectrometry (MS/MS) data can be extremely powerful in capturing the metabolic diversity expected to occur in natural populations (20). Indiscriminant or shotgun MS/MS strategies with high-resolution MS detectors offer many advantages in terms of rapidity and scale of analysis. Pipelines have been recently established to analyze such data (20). However, querying MS/MS data from the analysis of secondary metabolites from public databases is frequently unsuccessful because few standards are available for these compounds (21). An alternative is the use of comparative spectral analysis applied to experimental MS/MS datasets (22). This approach, termed molecular networking, is relatively new and aims at creating a map of mass spectral structural space in which molecules with related MS/MS spectra cluster together. Here, we combine the rapidly generated MS/MS data for all mass signals detected and molecular network construction in the analysis of the metabolic composition of natural plant populations.We applied our MS method to the natural variation in secondary metabolic profiles observed in accessions of the coyote tobacco, Nicotiana attenuata. This annual, native to the Great Basin Desert in the United States, primarily occurs in large ephemeral populations in post-fire habitats and smaller persistent populations found in washes (23). Dormant seeds of this species germinate from long-lived seed banks in sagebrush and pinyon-juniper ecosystems when fires pyrolize the litter layer, removing germination inhibitors and saturating the soils with smoke-derived germination cues (24, 25). This particular germination behavior affects the genetic structure of ephemeral monocultures produced by this species and results in relatively high within-population variation. N. attenuata populations represent a primary food source for insects that colonize the ecosystem after fires, and a vast array of genes and dependent metabolic pathways underlying resistance traits to native herbivores have been functionally characterized in this species. Among the major compound classes that contribute to the antiherbivore defense mechanisms of this plant is nicotine, a neurotoxin that functions synergistically with antidigestive plant proteins (26, 27), phenolic derivatives that exhibit strong tissue-specific responses to insect herbivory (28, 29), and 17-hydroxygeranyllinalool diterpene glycosides (HGL-DTGs) (30).Several studies have analyzed, with a high degree of spatial and temporal resolution, some of the metabolomic reconfigurations that are activated in plant tissues during biotic stresses (for a review, see ref. 31), including the attack of insects (32–34); but few of these studies have explored qualitative and quantitative variations of these metabolic adjustments across native populations. To systematically explore natural diversity patterns in the metabolic response to Manduca sexta herbivory of different N. attenuata populations, we conducted a glasshouse-based high-throughput MS-based metabolomics approach on 183 plants derived from seeds collected in Utah, Nevada, Arizona, and California. We then optimized an analytical and computational pipeline to assemble MS/MS data collected in a nontargeted manner and established mass spectral maps using a bioinformatics method to visualize metabolic branch-specific natural variation effects and annotate metabolites of interest.     

Population genetics of ecological communities with DNA barcodes: An example from New Guinea Lepidoptera

Comparative population genetics of ecological guilds can reveal generalities in patterns of differentiation bearing on hypotheses regarding the origin and maintenance of community diversity. Contradictory estimates of host specificity and beta diversity in tropical Lepidoptera (moths and butterflies) from New Guinea and the Americas have sparked debate on the role of host-associated divergence and geographic isolation in explaining latitudinal diversity gradients. We sampled haplotypes of mitochondrial cytochrome c oxidase I from 28 Lepidoptera species and 1,359 individuals across four host plant genera and eight sites in New Guinea to estimate population divergence in relation to host specificity and geography. Analyses of molecular variance and haplotype networks indicate varying patterns of genetic structure among ecologically similar sympatric species. One-quarter lacked evidence of isolation by distance or host-associated differentiation, whereas 21% exhibited both. Fourteen percent of the species exhibited host-associated differentiation without geographic isolation, 18% showed the opposite, and 21% were equivocal, insofar as analyses of molecular variance and haplotype networks yielded incongruent patterns. Variation in dietary breadth among community members suggests that speciation by specialization is an important, but not universal, mechanism for diversification of tropical Lepidoptera. Geographically widespread haplotypes challenge predictions of vicariance biogeography. Dispersal is important, and Lepidoptera communities appear to be highly dynamic according to the various phylogeographic histories of component species. Population genetic comparisons among herbivores of major tropical and temperate regions are needed to test predictions of ecological theory and evaluate global patterns of biodiversity.     

Thrips pollination of Mesozoic gymnosperms

Within modern gymnosperms, conifers and Ginkgo are exclusively wind pollinated whereas many gnetaleans and cycads are insect pollinated. For cycads, thrips are specialized pollinators. We report such a specialized pollination mode from Early Cretaceous amber of Spain, wherein four female thrips representing a genus and two species in the family Melanthripidae were covered by abundant Cycadopites pollen grains. These females bear unique ring setae interpreted as specialized structures for pollen grain collection, functionally equivalent to the hook-tipped sensilla and plumose setae on the bodies of bees. The most parsimonious explanation for this structure is parental food provisioning for larvae, indicating subsociality. This association provides direct evidence of specialized collection and transportation of pollen grains and likely gymnosperm pollination by 110-105 million years ago, possibly considerably earlier.     

Evidence for adaptive radiation from a phylogenetic study of plant defenses

One signature of adaptive radiation is a high level of trait change early during the diversification process and a plateau toward the end of the radiation. Although the study of the tempo of evolution has historically been the domain of paleontologists, recently developed phylogenetic tools allow for the rigorous examination of trait evolution in a tremendous diversity of organisms. Enemy-driven adaptive radiation was a key prediction of Ehrlich and Raven''s coevolutionary hypothesis [Ehrlich PR, Raven PH (1964) Evolution 18:586–608], yet has remained largely untested. Here we examine patterns of trait evolution in 51 North American milkweed species (Asclepias), using maximum likelihood methods. We study 7 traits of the milkweeds, ranging from seed size and foliar physiological traits to defense traits (cardenolides, latex, and trichomes) previously shown to impact herbivores, including the monarch butterfly. We compare the fit of simple random-walk models of trait evolution to models that incorporate stabilizing selection (Ornstein-Ulenbeck process), as well as time-varying rates of trait evolution. Early bursts of trait evolution were implicated for 2 traits, while stabilizing selection was implicated for several others. We further modeled the relationship between trait change and species diversification while allowing rates of trait evolution to vary during the radiation. Species-rich lineages underwent a proportionately greater decline in latex and cardenolides relative to species-poor lineages, and the rate of trait change was most rapid early in the radiation. An interpretation of this result is that reduced investment in defensive traits accelerated diversification, and disproportionately so, early in the adaptive radiation of milkweeds.     

Soil biotic legacy effects of extreme weather events influence plant invasiveness

Climate change is expected to increase future abiotic stresses on ecosystems through extreme weather events leading to more extreme drought and rainfall incidences [Jentsch A, et al. (2007) Front Ecol Environ 5(7):365–374]. These fluctuations in precipitation may affect soil biota, soil processes [Evans ST, Wallenstein MD (2012) Biogeochemistry 109:101–116], and the proportion of exotics in invaded plant communities [Jiménez MA, et al. (2011) Ecol Lett 14:1277–1235]. However, little is known about legacy effects in soil on the performance of exotics and natives in invaded plant communities. Here we report that drought and rainfall effects on soil processes and biota affect the performance of exotics and natives in plant communities. We performed two mesocosm experiments. In the first experiment, soil without plants was exposed to drought and/or rainfall, which affected soil N availability. Then the initial soil moisture conditions were restored, and a mixed community of co-occurring natives and exotics was planted and exposed to drought during growth. A single stress before or during growth decreased the biomass of natives, but did not affect exotics. A second drought stress during plant growth resetted the exotic advantage, whereas native biomass was not further reduced. In the second experiment, soil inoculation revealed that drought and/or rainfall influenced soil biotic legacies, which promoted exotics but suppressed natives. Our results demonstrate that extreme weather events can cause legacy effects in soil biota, promoting exotics and suppressing natives in invaded plant communities, depending on the type, frequency, and timing of extreme events.     

From the Cover: Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode of plant odor reception and defense

Plants receive volatile compounds emitted by neighboring plants that are infested by herbivores, and consequently the receiver plants begin to defend against forthcoming herbivory. However, to date, how plants receive volatiles and, consequently, how they fortify their defenses, is largely unknown. In this study, we found that undamaged tomato plants exposed to volatiles emitted by conspecifics infested with common cutworms (exposed plants) became more defensive against the larvae than those exposed to volatiles from uninfested conspecifics (control plants) in a constant airflow system under laboratory conditions. Comprehensive metabolite analyses showed that only the amount of (Z)-3-hexenylvicianoside (HexVic) was higher in exposed than control plants. This compound negatively affected the performance of common cutworms when added to an artificial diet. The aglycon of HexVic, (Z)-3-hexenol, was obtained from neighboring infested plants via the air. The amount of jasmonates (JAs) was not higher in exposed plants, and HexVic biosynthesis was independent of JA signaling. The use of (Z)-3-hexenol from neighboring damaged conspecifics for HexVic biosynthesis in exposed plants was also observed in an experimental field, indicating that (Z)-3-hexenol intake occurred even under fluctuating environmental conditions. Specific use of airborne (Z)-3-hexenol to form HexVic in undamaged tomato plants reveals a previously unidentified mechanism of plant defense.In response to herbivory, plants emit specific blends of volatiles (1). When undamaged plants are exposed to volatiles from neighboring herbivore-infested plants, they begin to defend against the impending infestation of herbivores (2, 3). This so-called “plant–plant signaling” has been reported in several plant species (4). For example, a study on the expression profiles of defense-related genes when Arabidopsis was exposed to several volatiles, including green leaf volatiles and a monoterpene, showed that the manner of induction varied with the gene monitored or the volatile used, suggesting that the plant responses were specific to the individual volatile compound (5). Kost and Heil (6) reported that the secretion of extrafloral nectar (an alternative food for carnivores) in undamaged lima bean plants was enhanced by volatiles from infested conspecific plants; this reaction was specific to (Z)-3-hexenyl acetate. Recently, Kikuta et al. (7) showed that wound-induced volatile organic compounds from Chrysanthemum cinerariaefolium induced the biosynthesis of pyrethrins in volatile-exposed neighboring plants. In this plant–plant signaling system, a blend of five compounds at specific concentrations was essential for the pyrethrin biosynthesis in receiver plants.These previous studies on plant–plant signaling raise questions about how different airborne volatiles are received by undamaged neighboring plants. Tamogami et al. (8) reported that airborne (E)-nerolidol was metabolized by Achyranthes bidentata plants into (E)-4,8-dimethyl-1,3,7-nonatriene. However, the mechanisms involved in the reception of airborne (E)-nerolidol in plants remained unclear. To date, only the receptor for ethylene, ETR1, a typical histidine kinase involved in a two-component regulatory system, has been identified (9, 10); no information exists on receptors for other volatile compounds in plants. In this study, we conducted comprehensive analyses of metabolic changes in intact tomato plants (Solanum lycopersicum) exposed to volatiles emitted from conspecifics infested with common cutworm (CCW; Spodoptera litura) and also conducted bioassays and biochemical analyses. We report that (Z)-3-hexenol emitted from herbivore-infested tomato plants is used by undamaged plants to form a glycoside with defensive function against CCW.     

Plant mating system transitions drive the macroevolution of defense strategies

Understanding the factors that shape macroevolutionary patterns in functional traits is a central goal of evolutionary biology. Alternative strategies of sexual reproduction (inbreeding vs. outcrossing) have divergent effects on population genetic structure and could thereby broadly influence trait evolution. However, the broader evolutionary consequences of mating system transitions remain poorly understood, with the exception of traits related to reproduction itself (e.g., pollination). Across a phylogeny of 56 wild species of Solanaceae (nightshades), we show here that the repeated, unidirectional transition from ancestral self-incompatibility (obligate outcrossing) to self-compatibility (increased inbreeding) leads to the evolution of an inducible (vs. constitutive) strategy of plant resistance to herbivores. We demonstrate that inducible and constitutive defense strategies represent evolutionary alternatives and that the magnitude of the resulting macroevolutionary tradeoff is dependent on the mating system. Loss of self-incompatibility is also associated with the evolution of increased specificity in induced plant resistance. We conclude that the evolution of sexual reproductive variation may have profound effects on plant–herbivore interactions, suggesting a new hypothesis for the evolution of two primary strategies of plant defense.     

Revisiting Darwin's conundrum reveals a twist on the relationship between phylogenetic distance and invasibility

A key goal of invasion biology is to identify the factors that favor species invasions. One potential indicator of invasiveness is the phylogenetic distance between a nonnative species and species in the recipient community. However, predicting invasiveness using phylogenetic information relies on an untested assumption: that both biotic resistance and facilitation weaken with increasing phylogenetic distance. We test the validity of this key assumption using a mathematical model in which a novel species is introduced into communities with varying ecological and phylogenetic relationships. Contrary to what is generally assumed, we find that biotic resistance and facilitation can either weaken or intensify with phylogenetic distance, depending on the mode of interspecific interactions (phenotype matching or phenotype differences) and the resulting evolutionary trajectory of the recipient community. Thus, we demonstrate that considering the mechanisms that drive phenotypic divergence between native and nonnative species can provide critical insight into the relationship between phylogenetic distance and invasibility.Invasive species are a major cause of concern due to their large ecological, social, and economic consequences (1–3). In principle, future invasions could be avoided by preventing introductions of potential invaders into susceptible communities. Thus, research has focused on identifying the characteristics that predispose species to becoming invasive (4–7) and the properties that make communities susceptible to invasion (8, 9). Although generalities have been elusive, one approach that has recently been gaining interest is using phylogenetic distance (time since cladogenesis) between nonnative species and species in the recipient community as an indicator of invasion potential.Darwin was the first to suggest that the probability of establishment by introduced species depends on their relatedness to native species (10). However, as Darwin noted, the ecological similarity of related species can have opposing effects on their potential for coexistence (“Darwin’s naturalization conundrum”). On the one hand, establishment in regions with close relatives should be facilitated by favorable abiotic conditions and the presence of suitable prey, hosts, and mutualists (Fig. 1A). On the other hand, establishment in these regions should be inhibited by competition with the relatives themselves and exploitation by shared natural enemies (Fig. 1B). Citing observations by Alphonse de Candolle and Asa Gray that naturalized species are more frequently from nonnative genera, Darwin concluded that competition was the dominant factor and relatedness to native species should reduce establishment success (“Darwin’s naturalization hypothesis”).Open in a separate windowFig. 1.Conventional predictions for the relationship between phylogenetic distance and establishment probability. (A) Abiotic conditions, mutualists, and resource species (e.g., prey and hosts) are expected to favor establishment of related species. As time t since cladogenesis increases, these favorable effects decline, leading to reduced probability of establishment. (B) Competitors and natural enemies are expected to disfavor establishment of related species. As time since cladogenesis increases, these unfavorable effects decline, leading to increased probability of establishment.However, recent studies using statistical models, molecular phylogenetics, and experimental community assembly have revealed that the correlation between relatedness and establishment probability can be positive, negative, or zero (11). More fundamentally, doubt has been cast on the key assumption underlying both Darwin’s intuitive arguments and contemporary research: that the effects of native species are strongest when phylogenetic distance to the nonnative species is low (Fig. 1) (12). Although this assumption is supported by studies showing that the presence of closely related species in a community increases competition (13), attack by natural enemies (14–18), and visitation by mutualists (19), a number of recent studies demonstrate the opposite, i.e., that distantly related species can experience more intense competition (12, 20) and herbivory (21).

Table 1.

The effect of phylogenetic relatedness on the probability of establishment by nonnative species

The Rayleigh-Taylor condition for the evolution of irrotational fluid interfaces

For the free boundary dynamics of the two-phase Hele-Shaw and Muskat problems, and also for the irrotational incompressible Euler equation, we prove existence locally in time when the Rayleigh–Taylor condition is initially satisfied for a 2D interface. The result for water waves was first obtained by Wu in a slightly different scenario (vanishing at infinity), but our approach is different because it emphasizes the active scalar character of the system and does not require the presence of gravity.     

Tradeoffs associated with constitutive and induced plant resistance against herbivory

Several prominent hypotheses have been posed to explain the immense variability among plant species in defense against herbivores. A major concept in the evolutionary ecology of plant defenses is that tradeoffs of defense strategies are likely to generate and maintain species diversity. In particular, tradeoffs between constitutive and induced resistance and tradeoffs relating these strategies to growth and competitive ability have been predicted. We performed three independent experiments on 58 plant species from 15 different plant families to address these hypotheses in a phylogenetic framework. Because evolutionary tradeoffs may be altered by human-imposed artificial selection, we used 18 wild plant species and 40 cultivated garden-plant species. Across all 58 plant species, we demonstrate a tradeoff between constitutive and induced resistance, which was robust to accounting for phylogenetic history of the species. Moreover, the tradeoff was driven by wild species and was not evident for cultivated species. In addition, we demonstrate that more competitive species-but not fast growing ones-had lower constitutive but higher induced resistance. Thus, our multispecies experiments indicate that the competition-defense tradeoff holds for constitutive resistance and is complemented by a positive relationship of competitive ability with induced resistance. We conclude that the studied genetically determined tradeoffs are indeed likely to play an important role in shaping the high diversity observed among plant species in resistance against herbivores and in life history traits.