Floral Defense Research Articles

Slippery flowers as a mechanism of defence against nectar-thieving ants.

The great diversity of floral characteristics among animal-pollinated plants is commonly understood to be the result of coevolutionary interactions between plants and pollinators. Floral antagonists, such as nectar thieves, also have the potential to exert an influence upon the selection of floral characteristics, but adaptation against floral antagonists has attracted comparatively little attention. We found that the corollas of hornet-pollinated Codonopsis lanceolata (Campanulaceae) and the tepals of bee-pollinated Fritillaria koidzumiana (Liliaceae) are slippery to nectar-thieving ants living in the plant's habitat; because the flowers of both species have exposed nectaries, slippery perianths may function as a defence against nectar-thieving ants. We conducted a behavioural experiment and observed perianth surface microstructure by scanning electron microscopy to investigate the mechanism of slipperiness. Field experiments were conducted to test whether slippery perianths prevent floral entry by ants, and whether ant presence inside flowers affects pollination. Scanning electron microscopy observations indicated that the slippery surfaces were coated with epicuticular wax crystals. The perianths lost their slipperiness when wiped with hexane. Artificial bridging of the slippery surfaces using non-slippery materials allowed ants to enter flowers more frequently. Experimental introduction of live ants to the Codonopsis flowers evicted hornet pollinators and shortened the duration of pollinator visits. However, no statistical differences were found in the fruit or seed sets of flowers with and without ants. Slippery perianths, most probably based on epicuticular wax crystals, prevent floral entry by ants that negatively affect pollinator behaviour. Experimental evidence of floral defence based on slippery surfaces is rare, but such a mode of defence may be widespread amongst flowering plants.

HY5, an integrator of light and temperature signals in the regulation of anthocyanins biosynthesis in Arabidopsis

Anthocyanins are well-known plant specialized metabolites and this group can be classified into the phenolic compound class based on their chemical structure characterizing by a C6-C3-C6 carbon framework. Anthocyanins have been identified to play various functions in plants, for example, pigmentation of floral organs, UV protection, and defense system. In addition to their functions in plant growth and development, anthocyanins are also considered as a good natural antioxidant which can be used for human. Because of important functions, the biosynthesis of anthocyanins is precisely regulated by a number of endogenous and exogenous factors. In the plant, light and temperature are critical environmental factors contributing to various developmental processes. From the first identification, ELONGATED HYPOCOTYL 5 (HY5) has been documented to play as an important transcription factor that is involved in a number of signal transduction ways including light and temperature pathways. The purpose of this review is to provide a precise overview of current research progress on the regulation of anthocyanins biosynthesis under the control of HY5 transcription factor.

Promoter activity of genes encoding the Specific Tissue protein family in the reproductive organs of Medicago truncatula

The "Specific Tissue" (ST) are proteins of unknown function present only in some plant families, mainly Fabaceae and Asteraceae. They are included in the PF10950 protein family and characterized by the presence of at least one domain of unknown function (DUF)2775. In this work we studied the involvement of the six members of the Medicago truncatula ST family (ST1 to ST6) in the development of flowers, fruits, and seeds by analysing the activity of their promoters (pST) after the construction of M. truncatula transgenic plants expressing the b-glucuronidase (GUS) reporter gene under the control of the six pSTs. The GUS activity was analysed in whole flowers and fruits and also in histological sections of these organs. The pST expression in the reproductive organs was mainly associated with the vascular bundles, especially throughout fruit development. These results pointed to an important role of ST proteins during the reproductive development stage, related to nutrient mobilization during the fruit and seed formation, that could be facilitated by their presence in the pod vascular bundles, as well as in the connective tissue of the anthers (ST3, ST4, ST6), in the placenta, the funiculus, and the outer parts of the developing seed (ST2, ST3, ST6). The observations made in this study were in agreement with the functions previously established for the three groups of M. truncatula ST proteins, as in the proposed function for ST1 in the transport and assimilation of nutrients, or the involvement of ST4, ST5, and ST6 in floral defence.

LC-MS/MS based comparative proteomics of floral nectars reveal different mechanisms involved in floral defense of Nicotiana spp., Petunia hybrida and Datura stramonium

Tobacco floral nectar (FN) is a biological fluid produced by nectaries composed of sugars, amino acids and proteins called nectarins, involved in the floral defense. FN provides an ideal source of nutrients for microorganisms. Understanding the role of nectar proteins is essential to predict impacts in microbial growth, composition and plants-pollinators interactions. Using LC-MS/MS-based comparative proteomic analysis we identified 22 proteins from P. hybrida, 35 proteins from D. stramonium, and 144 proteins from 23 species of Nicotiana. The data are available at ProteomeXchance (PXD014760). GO analysis and secretory signal prediction demonstrated that defense/stress was the largest group of proteins in the genus Nicotiana. The Nicotiana spp. proteome consisted of 105 exclusive proteins such as lipid transfer proteins (LTPs), Nectar Redox Cycle proteins, proteases inhibitors, and PR-proteins. Analysis by taxonomic sections demonstrated that LTPs were most abundant in Undulatae and Noctiflora, while nectarins were more abundant in Rusticae, Suaveolens, Polydicliae, and Alata sections. Peroxidases (Pox) and chitinases (Chit) were exclusive to P. hybrida, while D. stramonium had only seven unique proteins. Biochemical analysis confirmed these differences. These findings support the hypothesis that, although conserved, there is differential abundance of proteins related to defense/stress which may impact the mechanisms of floral defense. SignificanceThis study represents a comparative proteomic analysis of floral nectars of the Nicotiana spp. with two correlated Solanaceous species. Significant differences were identified between the proteome of taxonomic sections providing relevant insights into the group of proteins related to defense/stress associated with Nectar Redox Cycle, antimicrobial proteins and signaling pathways. The activity of FNs proteins is suggested impact the microbial growth. The knowledge about these proteomes provides significant insights into the diversity of proteins secreted in the nectars and the array of mechanisms used by Nicotiana spp. in its floral defense.

Consequences of multiple flower-insect interactions for subsequent plant-insect interactions and plant reproduction.

Plants often interact simultaneously with multiple antagonists and mutualists that can alter plant traits at the phenotypic or genetic level, subsequent plant-insect interactions, and reproduction. Although many studies have examined the effects of single floral antagonisms on subsequent pollination and plant reproduction, we know very little about the combined, potentially non-additive effects of multiple flower-insect interactions. We simulated increased florivory, nectar robbing, and pollination on field-grown Impatiens capensis, which allowed us to determine interactive effects on five subsequent plant-insect interactions and 16 plant traits, including traits related to plant growth, floral attractiveness, floral defenses, and plant reproduction. All three manipulative treatments had significant non-additive effects on the behavior of subsequent floral visitors, indicating that the effect of floral visitors generally depended on the presence or behavior of others. Pollination increased visitation by both pollinators and nectar larcenists (robbers and thieves), while florivory reduced pollinator and larcenist visits. Surprisingly, supplemental pollination also increased leaf herbivory. Florivores often responded to manipulations in opposite ways than did nectar larcenists and pollinators, suggesting different mechanisms influencing visitors that consume nectar compared to floral tissue. While our treatments did not affect any floral trait measured, they non-additively impacted plant reproduction, with florivory having a larger overall impact than either nectar robbing or pollination. These results emphasize the importance of understanding the context in which flower-insect interactions occur because the composition of the interacting community can have large and non-additive impacts on subsequent insect behavior and plant reproduction.

Functional characterization and expression analysis of two terpene synthases involved in floral scent formation in Lilium 'Siberia'.

Floral scent formation in Lilium 'Siberia' is mainly due to monoterpene presence in the floral profile. LoTPS1 and LoTPS3 are responsible for the formation of (±)-linalool and β-ocimene in Lilium 'Siberia'. Lilium 'Siberia' is a perennial herbaceous plant belonging to Liliaceae family, cultivated both as a cut flower and garden plant. The snowy white flower emits a pleasant aroma which is mainly caused by monoterpenes present in the floral volatile profile. Previously terpene synthase (TPS) genes have been isolated and characterized from various plant species but less have been identified from Liliaceae family. Here, two terpene synthase genes (LoTPS1 and LoTPS3), which are highly expressed in sepals and petals of Lilium 'Siberia' flower were functionally characterized recombinant LoTPS1 specifically catalyzes the formation of (Z)-β-ocimene and (±)-linalool as its main volatile compounds from geranyl pyrophosphate (GPP), whereas LoTPS3 is a promiscuous monoterpene synthase which utilizes both GPP and farnesyl pyrophosphate (FPP) as a substrate to generate (±)-linalool and cis-nerolidol, respectively. Transcript levels of both genes were prominent in flowering parts, especially in sepals and petals which are the main source of floral scent production. The gas chromatography-mass spectrometry (GC-MS) and quantitative real-time PCR analysis revealed that the compounds were emitted throughout the day, prominently during the daytime and lower levels at night following a strong circadian rhythm in their emission pattern. Regarding mechanical wounding, both genes showed considerable involvement in floral defense by inducing the emission of (Z)-β-ocimene and (±)-linalool, elevating the transcript accumulation of LoTPS1 and LoTPS3. Furthermore, the subcellular localization experiment revealed that LoTPS1 was localized in plastids, whilst LoTPS3 in mitochondria. Our findings on these two TPSs characterized from Lilium 'Siberia' provide new insights into molecular mechanisms of terpene biosynthesis in this species and also provide an opportunity for biotechnological modification of floral scent profile of Lilium.

Floral-induced and constitutive defense against florivory: a comparison of chemical traits in 12 herb species

Little is known about how plants protect flowers—their reproductive organs—against florivory. Additionally, the induced floral defense system has been examined in only a few species. We tested the inducibility of putative floral defenses and investigated the relationship between natural florivory and the floral defenses of 12 naturally growing plant species. The relationships between florivory and four chemical traits (nitrogen, phosphorus, total phenolics, and condensed tannins) were investigated in 12 plant species. We also studied whether flowers induce changes in chemical defenses in response to artificial damage in 10 plant species. A higher concentration of floral nitrogen was associated with a decreasing frequency of florivore attacks. Among the four traits of the 10 plant species studied, no trait changed in response to the artificial damage. We suggest that induced defense systems may not be advantageous for flowers, although it is also possible that these species simply do not use induced defense in any of their plant parts.

Recurved Taraxacum phyllaries function as a floral defense: experimental evidence and its implication for Taraxacum evolutionary history

AbstractFlorivores directly decrease the reproductive success of plants by consuming pollen and seeds; thus, plants often have defense mechanisms against florivory. Here, we show that the recurved phyllaries of an agamospermous hybrid dandelion, Taraxacum japonicum × officinale, function as a physical barrier to slug florivory. We allowed Lehmannia valentiana, a European slug naturalized in Japan, to feed on eight pairs of the hybrid dandelion and T. japonicum, a Japanese species with erect phyllaries. Consequently, the slugs only damaged T. platycarpum flowers. The slugs either moved back from recurved phyllaries or spent more than twice as long on recurved than on erect phyllaries. When we removed the recurved phyllaries, the slugs stayed on hybrid and T. japonicum phyllaries for equal lengths of time. In addition to recurved outer phyllaries, the hybrid dandelion has erect inner phyllaries that are longer than those of T. japonicum, which effectively conceal the florets at night protecting them from slug florivory. Using the taxonomic literature, we have confirmed that recurved phyllaries evolved in many species in several parts of Europe, but are rare in East Asia. These findings suggest that European dandelions acquired recurved phyllaries as a defense mechanism under antagonistic coevolution with florivorous slugs, but this coevolution did not occur in East Asia.

Floral damage induces resistance to florivory in Impatiens capensis

Many plants produce defense chemicals that are induced in response to damage. In spite of the tight links between floral tissue and plant reproduction, very little is known about whether floral defenses are induced in response to floral damage. We manipulated Impatiens capensis flowers to determine whether floral damage reduces subsequent florivory, whether it induces anthocyanins or condensed tannins in floral tissues, and whether responses are localized or systemic. We damaged one flower per plant at one of three damage levels (0, 30, or 60 % tissue removal), collected subsequent flowers at set time intervals and branch locations, and measured whole-plant florivory for 3 weeks following damage. We also observed a flower color polymorphism and analyzed responses separately for red- and yellow-flowered plants. Moderate damage to a single flower reduced subsequent whole-plant florivory, but heavy damage did not. Moderate damage to a focal flower also increased anthocyanins in subsequent flowers on the same branch of red-flowered plants, but decreased anthocyanins on parallel-branch flowers of yellow-flowered plants. Damage did not affect floral tannins. Because the reduction in florivory was systemic and induced anthocyanins were not consistently induced systemically, there may be other secondary compounds not measured in this study that were systemically induced, or effects of visual or olfactory cues of damage itself that reduced subsequent florivory. This is the first study demonstrating that damage to a single flower can reduce subsequent whole-plant florivory in the field, indicating that initial damage can have cascading effects on subsequent interactions.

Stereoselectivity of chalcone isomerase with chalcone derivatives: a computational study

Chalcone isomerase (CHI) catalyzes the intramolecular cyclization of chalcones into flavonoids. The activity of CHI is essential for the biosynthesis of flavonoids precursors of floral pigments and phenylpropanoid plant defense compounds. In the present study, we explored the detailed binding structures and binding free energies for two different active site conformations of CHI with s-cis/s-trans conformers of three chalcone compounds by performing molecular dynamics (MD) simulations and binding free energy calculations. The computational results indicate that s-cis/s-trans conformers of chalcone compounds are orientated in the similar binding position in the active site of CHI and stabilized by the different first hydrogen bond network and the same second hydrogen bond network. The first hydrogen bond network results in much lower binding affinity of s-trans conformer of chalcone compound with CHI than that of s-cis conformer. The conformational change of the active site residue T48 from indirectly interacting with the substrate via the second hydrogen bond network to directly forming the hydrogen bond with the substrates cannot affect the binding mode of both conformers of chalcone compounds, but remarkably improves the binding affinity. These results show that CHI has a strong stereoselectivity. The calculated binding free energies for three chalcone compounds with CHI are consistent with the experimental activity data. In addition, several valuable insights are suggested for future rational design and discovery of high-efficiency mutants of CHI.

Why do florivores prefer hermaphrodites over females in Nemophila menziesii (Boraginaceae)?

Although florivores can destroy significant amounts of sexual tissues and indirectly affect pollination, little is known about their preferences, which could shape the evolution of floral traits or defense. In this study, we used a gynodioecious plant Nemophila menziesii, and its main florivore Platyprepia virginalis, to test which floral characteristics are associated with florivory in the field and with florivore choice in the laboratory. Hermaphrodite flowers consistently received more damage than nearby females in the field. In the laboratory setting, florivores also preferred unmanipulated hermaphrodites versus unmanipulated females. Systematic evaluation of hermaphrodite traits, such as corolla size, anther presence, and corolla color, revealed that corolla diameter was the main determinant of florivore preference in this system. Here, we discuss the implications of both pollinator and florivore choice in the evolution of corolla size and sex ratio in gynodioecious species with cytoplasmic male sterility and emphasize the need for more information on the preferences of florivores.

The major volatile organic compound emitted from Arabidopsis thaliana flowers, the sesquiterpene (E)‐β‐caryophyllene, is a defense against a bacterial pathogen

Flowers have a high risk of pathogen attack because of their rich nutrient and moisture content, and high frequency of insect visitors. We investigated the role of (E)-β-caryophyllene in floral defense against a microbial pathogen. This sesquiterpene is a common volatile compound emitted from flowers, and is a major volatile released from the stigma of Arabidopsis thaliana flowers. Arabidopsis thaliana lines lacking a functional (E)-β-caryophyllene synthase or constitutively overexpressing this gene were challenged with Pseudomonas syringae pv. tomato DC3000, which is a bacterial pathogen of brassicaceous plants. Flowers of plant lines lacking (E)-β-caryophyllene emission showed greater bacterial growth on their stigmas than did wild-type flowers, and their seeds were lighter and misshapen. By contrast, plant lines with ectopic (E)-β-caryophyllene emission from vegetative parts were more resistant than wild-type plants to pathogen infection of leaves, and showed reduced cell damage and higher seed production. Based on in vitro experiments, (E)-β-caryophyllene seems to act by direct inhibition of bacterial growth, rather than by triggering defense signaling pathways. (E)-β-Caryophyllene thus appears to serve as a defense against pathogens that invade floral tissues and, like other floral volatiles, may play multiple roles in defense and pollinator attraction.

Hawaiian ant–flower networks: nectar-thieving ants prefer undefended native over introduced plants with floral defenses

Ants are omnipresent in most terrestrial ecosystems, and plants have responded to their dominance by evolving traits that either facilitate positive interactions with ants or reduce negative ones. Because ants are generally poor pollinators, plants often protect their floral nectar against ants. Ants were historically absent from the geographically isolated Hawaiian archipelago, which harbors one of the most endemic floras in the world. We hypothesized that native Hawaiian plants lack floral features that exclude ants and therefore would be heavily exploited by introduced, invasive ants. To test this hypothesis, ant–flower interactions involving co-occurring native and introduced plants were observed in 10 sites on three Hawaiian Islands. We quantified the residual interaction strength of each pair of ant–plant species as the deviation of the observed interaction frequency from a null-model prediction based on available nectar sugar in a local plant community and local ant activity at sugar baits. As predicted, flowers of plants that are endemic or indigenous to Hawaii were more strongly exploited by ants than flowers of co-occurring introduced plants, which shared an evolutionary history with ants. We also found that the percentage of plant species with ant-visited flowers was much higher in Hawaii than in other island and continental systems, even reaching 100% in habitats dominated by endemic species. We showed experimentally that the absence of ants on flowers of most introduced and few native plants species was due to morphological barriers, repellent floral scents, and to a lesser extent, unpalatable nectar. Analysis of floral volatiles, however, revealed no consistent ant-repellent “syndrome” attributable to negative responses by ants, probably due to the high chemical variability within the floral scent bouquets. Results from a molecular phylogeny imply that floral defenses against ants were convergently lost in native Hawaiian plants. Exploitation of floral nectar by ants may be an important threat to Hawaiian ecosystems, reducing nectar resources available to native flower visitors and potentially reducing the reproductive success of the endangered endemic flora.

NPR1 and EDS11 contribute to host resistance against Fusarium culmorum in Arabidopsis buds and flowers

The cereal ear blight fungal pathogen Fusarium culmorum can infect Arabidopsis floral tissue, causing disease symptoms and mycotoxin production. Here we assessed the effect of seven mutants and one transgenic overexpression line, residing in either the salicylic acid (SA), jasmonic acid (JA) or ethylene (ET) defence signalling pathways, on the outcome of the Fusarium-Arabidopsis floral interaction. The bacterial susceptiblity mutant eds11 was also assessed. Flowering plants were spray inoculated with F. culmorum conidia to determine the host responses to initial infection and subsequent colonization. Enhanced susceptibility and higher concentrations of deoxynivalenol mycotoxin were observed in buds and flowers of the npr1 and eds11 mutants than in the wild-type Col-0 plants. An effect of the other two defence signalling pathways on disease was either absent (ET/JA combined), absent/minimal (ET) or inconclusive (JA). Overall, this study highlights a role for NPR1 and EDS11 in basal defence against F. culmorum in some floral organs. This is the first time that any of these well-characterized defence signalling mutations have been evaluated for a role in floral defence in any plant species.

Is the nectar redox cycle a floral defense against microbial attack?

Many angiosperms use a remarkable reproductive strategy that relies on attracting animals (insect, avian or mammalian pollinators) to transfer pollen between plants. Relying on other organisms for sexual reproduction seems evolutionarily untenable, but the great diversity of angiosperms illustrates how highly successful this strategy is. To attract pollinators, plants offer a variety of rewards. Perhaps the primary floral reward is floral nectar. Plant nectar has long been considered a simple sugar solution but recent work has demonstrated that nectar is a complex biological fluid containing significant and important biochemistry with the potential function of inhibiting microbial growth. These results lead the way to novel insights into the mechanisms of floral defense and the co-evolution of angiosperms and their pollinators.

Filogenias y método comparativo: identificación y evaluación de hipotésis evolutivas y establecimiento de criterios para conservar especies en riesgo.

El propósito principal de esta revisión es promover la discusión acerca del impacto que ha tenido el uso de las filogenias y el método comparativo en diversos campos de la biología moderna. La combinación entre el método comparativo y las filogenias es usada para (1) inferir los estados ancestrales de patrones morfológicos, ecológicos y conductuales para sugerir patrones evolutivos de cambio, (2) desarrollar hipótesis acerca de los procesos de cambio evolutivo, (3) probar asociación evolutiva entre dos o más caracteres que pudiésen sugerir procesos coevolutivos y de limitantes evolutivas, y (4) corregir problemas de independencia de datos correspondientes a especies (e.g., problemas estadísticos en análisis interespecíficos que no son de naturaleza evolutiva). Por otro lado, la manera de evaluar hipótesis de adaptación ha cambiado dramáticamente en las últimas dos décadas. A pesar de que los avances en técnicas moleculares y de reconstrucción filogenética que permiten evaluar hipótesis de adaptación de una manera robusta, se siguen invocando argumentos adaptacionistas o usando aproximaciones experimentales para explicar correlaciones evolutivas. Mi intención en este artículo es enfatizar el uso de filogenias en la interpretación de hipótesis evolutivas y de adaptación, hipótesis de origen y transición de caracteres, correlación evolutiva entre dos o más caracteres, coevolución, diversificación filogenética, y el papel de las filogenias y el método comparativo en la identificación de especies en riesgo y su conservación. Para ello, reviso ocho ejemplos que incluyen casos de estudio sobre la evolución: (1) de plantas dioicas con semillas dispersadas por animales, (2) del gregarismo en larvas de mariposas con una coloración aposemática, (3) de las alas reducidas entre macrolepidópteros de bosques estables, (4) del robo de néctar entre colibríes con tomios aserrados, (5) de flores con mecanismos de defensa en contra de "aprovechados", (6) de los cambios de hospedero por la química de las plantas, (7) de la nectarivoría entre colibríes y su radiación adaptativa, y (8) de la tendencia hacia la generalización de hospederos por tordos parásitos de cria. Los primeros dos sistemas son usados principalmente para explicar los problemas metodológicos en el uso de filogenias y los seis restantes conforman una revisión conceptual del uso de filogenias en la evaluación de hipótesis de adaptación y en biología/ecología evolutiva. Por último, reviso como las filogenias se pueden utilizar para establecer prioridades en la conservación de especies e identificación de procesos biológicos que están determinando distintos niveles de "susceptibilidad" a la extinción por cuestiones de origen filogenético.

EXAPTATIONS LINK EVOLUTION OF PLANT–HERBIVORE AND PLANT–POLLINATOR INTERACTIONS: A PHYLOGENETIC INQUIRY

To evaluate possible evolutionary links between plant–herbivore and plant–pollinator relationships, defense and reward characteristics and pollination ecology were mapped onto a morphologically estimated phylogeny of 42 species of Dalechampia. This procedure generated detailed hypotheses about the evolution of anti-herbivore defense and pollination systems. These hypotheses were tested using the results of chemical analyses and bioassays of foliar and floral secretions. Multiple lines of defense appear to have evolved in sequence in Dalechampia. The first defense system to originate was deployment of triterpene resins to defend the staminate flowers. This feature was a preaptation (preadaptation) that allowed the evolution of a resin-based, pollinator-reward system. Thus pollination by resin-collecting bees originated as a “transfer exaptation” (a new function replaced the old). This hypothesis is supported by anti-herbivore activities of floral resins and by chemical similarity of floral defense and reward resins. After the resin defense of flowers was lost by conversion into a reward system, there followed (in evolutionary time) a sequence of defensive innovations. These included nocturnal closure of large, involucral bracts to protect both staminate and pistillate flowers. Phylogenetic analysis showed that the large bracts themselves probably originated as a floral advertisement system, and the bracts assumed a defensive function through “addition exaptation” (a new function was added to the old). Additional lines of defense to evolve were deployment of resin to defend the developing ovaries and seeds (an addition exaptation), deployment of sharp, detaching trichomes on enveloping sepals to defend developing seeds (apparent adaptations), closure of involucral bracts around the developing fruits and seeds (an addition exaptation), and deployment of resin to defend the leaves and growing shoot tips (also an addition exaptation). Support for this scenario also derives from the chemical similarities of sepal, foliar, and floral resins, and the anti-herbivore properties of foliar resins. It appears that at least one pollinator-reward system originated by modification of a defense system, and several defense systems originated by modification of pollinator reward and advertisement systems. Thus exaptations have been important in the origin of new defense and pollination systems, and each system has significantly influenced the evolution of the other on several occasions.

Do Biochemical Exaptations Link Evolution of Plant Defense and Pollination Systems? Historical Hypotheses and Experimental Tests with Dalechampia Vines

Mapping resin secretion and pollination ecology onto the estimated phylogeny of species of the euphorb vine Dalechampia generated two historical hypotheses: resin rewards offered to pollinators by Dalechampia flowers evolved by minor modification of a preexisting, resin-based, floral defense system, and resin defense of leaves in advanced species evolved by modification of the preexisting resin-reward system. From these hypotheses, we derived two predictions: floral reward resins are chemically similar to putative floral defense resins and exhibit antiherbivore activities, and foliar resins are chemically similar to reward resins and also exhibit antiherbivore activities. We tested these predictions by chemical analyses and by using a broad sample of Neotropical herbivorous insects in a series of bioassays. All floral and foliar resins were chemically similar. Tests with two generalist (Orophus tesselatus, Orthoptera: Tettigoniidae; Atta colombica, Hymenoptera: Formicidae) and four Dalechampia-specialist ...