Plants and their herbivores constitute more than half of the organisms in tropical forests. Therefore, a better understanding of the evolution of plant defenses against their herbivores may be central for our understanding of tropical biodiversity. Here, we address the evolution of antiherbivore defenses and their possible contribution to coexistence in the Neotropical tree genus Inga (Fabaceae). Inga has >300 species, has radiated recently, and is frequently one of the most diverse and abundant genera at a given site. For 37 species from Panama and Peru we characterized developmental, ant, and chemical defenses against herbivores. We found extensive variation in defenses, but little evidence of phylogenetic signal. Furthermore, in a multivariate analysis, developmental, ant, and chemical defenses varied independently (were orthogonal) and appear to have evolved independently of each other. Our results are consistent with strong selection for divergent defensive traits, presumably mediated by herbivores. In an analysis of community assembly, we found that Inga species co-occurring as neighbors are more different in antiherbivore defenses than random, suggesting that possessing a rare defense phenotype increases fitness. These results imply that interactions with herbivores may be an important axis of niche differentiation that permits the coexistence of many species of Inga within a single site. Interactions between plants and their herbivores likely play a key role in the generation and maintenance of the conspicuously high plant diversity in the tropics. plant defenses ͉ community assembly ͉ phylogenetic signal ͉ herbivory ͉ tropical diversity

Plant secondary metabolites play important ecological and evolutionary roles, most notably in the deterrence of natural enemies. The classical theory explaining the evolution of plant chemical diversity is that new defences arise through a pairwise co-evolutionary arms race between plants and their specialized natural enemies. However, plant species are bombarded by dozens of different herbivore taxa from disparate phylogenetic lineages that span a wide range of feeding strategies and have distinctive physiological constraints that interact differently with particular plant metabolites. How do plant defence chemicals evolve under such multiple and potentially contrasting selective pressures imposed by diverse herbivore communities? To tackle this question, we exhaustively characterized the chemical diversity and insect herbivore fauna from 31 sympatric species of Amazonian Protieae (Burseraceae) trees. Using a combination of phylogenetic, metabolomic and statistical learning tools, we show that secondary metabolites that were associated with repelling herbivores (1) were more frequent across the Protieae phylogeny and (2) were found in average higher abundance than other compounds. Our findings suggest that generalist herbivores can play an important role in shaping plant chemical diversity and support the hypothesis that chemical diversity can also arise from the cumulative outcome of multiple diffuse interactions.

Herbivores are often implicated in the generation of the extraordinarily diverse tropical flora. One hypothesis linking enemies to plant diversification posits that the evolution of novel defenses allows plants to escape their enemies and expand their ranges. When range expansion involves entering a new habitat type, this could accelerate defense evolution if habitats contain different assemblages of herbivores and/or divergent resource availabilities that affect plant defense allocation. We evaluated this hypothesis by investigating two sister habitat specialist ecotypes of Protium subserratum (Burseraceae), a common Amazonian tree that occurs in white-sand and terra firme forests. We collected insect herbivores feeding on the plants, assessed whether growth differences between habitats were genetically based using a reciprocal transplant experiment, and sampled multiple populations of both lineages for defense chemistry. Protium subserratum plants were attacked mainly by chrysomelid beetles and cicadellid hemipterans. Assemblages of insect herbivores were dissimilar between populations of ecotypes from different habitats, as well as from the same habitat 100 km distant. Populations from terra firme habitats grew significantly faster than white-sand populations; they were taller, produced more leaf area, and had more chlorophyll. White-sand populations expressed more dry mass of secondary compounds and accumulated more flavone glycosides and oxidized terpenes, whereas terra firme populations produced a coumaroylquinic acid that was absent from white-sand populations. We interpret these results as strong evidence that herbivores and resource availability select for divergent types and amounts of defense investment in white-sand and terra firme lineages of Protium subserratum, which may contribute to habitat-mediated speciation in these trees.

Abstract. In the recently radiated genus Inga (Fabaceae), few nucleotide substitutions have accumulated among species, yet large divergences have occurred in defensive phenotypes, suggesting strong selection by herbivores. We compared herbivory and defenses of young leaves for I. goldmanii, a more derived species that follows a ''defense'' strategy, and I. umbellifera, a more basal species that follows an ''escape'' strategy. The two species suffered similar rates of herbivory (22% of the leaf area eaten during expansion) but were attacked by different communities of herbivores. I. goldmanii relied heavily on extra-floral nectaries and on a diversity of effective secondary metabolites, while I. umbellifera minimized damage through rapid leaf expansion and synchronous flushing. The major classes of secondary compounds in both species were flavanoids and non-protein amino acids; however, there were large differences in structure, biosynthetic pathways, and efficacy against herbivores. Growth rates of lepidopteran larvae were significantly lower when fed artificial diets with crude extracts from I. goldmanii as compared to I. umbellifera. Flavanoids accounted for the majority of growth reduction in both species. I. umbellifera had more unusual flavanoids and a non-protein amino acid not reported from plants, but the more common flavanoids found in I. goldmanii were more bioactive against herbivores. I. goldmanii also had greater ant visitation to extrafloral nectaries, suggesting that there was no trade-off between biotic and chemical defenses. In contrast, young leaves of I. umbellifera expanded more rapidly, minimizing the window of vulnerability before toughening. Resources for rapid expansion may have been reallocated from chloroplast development as I. umbellifera delayed the greening process until after full leaf expansion. Leaves were also produced synchronously, which can satiate herbivores and reduce damage. These defense differences are reflected in almost completely nonoverlapping herbivore faunas and the more frequent occurrence of generalists on I. umbellifera. To understand why defenses have evolved, it is important to view them in light of the herbivore community as well as in the context of the other co-occurring traits. We hypothesize that the effectiveness of chemical defenses determines whether a species follows the evolutionary path of ''defense'' or ''escape'' strategies.

Abstract.A combination of solid-state 13 C NMR tensor data and DFT computational methods are utilized to predict conformation in disordered methyl α-Lrhamnofuranoside. This previously uncharacterized solid is found to be crystalline and consists of at least six distinct conformations that exchange on the kHz timescale. A total of 66 model structures were evaluated and six were identified as being consistent with and is slow enough to be observed on the NMR timescale due to severe steric crowding among ring substituents. The relatively minor heavy atom differences in the final structures suggest that characterization of a complete crystal structure by x-ray powder diffraction may be feasible.

Young leaves of tropical forest trees experience far higher herbivory pressure than mature leaves of the same species. Selection on young leaves has led to diverse forms of defense chemical expression. Though most allelochemicals are secondary metabolites, allelochemic function for a primary metabolite remains a possibility. We recently observed this phenomenon in the young leaves of Inga umbellifera, which accumulate the protein amino acid l-tyrosine to very high levels. We isolated l-tyrosine from young leaves of trees in Panama and characterized it using spectroscopic and chemical means. We chromatographically quantified leaf l-tyrosine levels across a range of developmental stages, showing that it was present in the youngest leaves and that its concentration increased throughout the period of expansion, reaching an average maximum of ca 10% of leaf dry mass in late-stage young leaves. This chemical phenotype was seen to be highly leaf-age specific: Free tyrosine was only present in mature leaves at very low levels. In bioassays with larvae of the noctuid moth H. virescens, l-tyrosine proved to be a potent growth inhibitor when added to artificial diet at 10% of dry mass. This suggests that a rarely observed defense strategy occurs in young I. umbellifera leaves, a hyper-produced primary metabolite functioning as an allelochemical.

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Many species of the dioecious, neo-tropical plant genus Clusia secrete a viscous, hydrophobic resin from glandular tissues in both male and female flowers. This substance is readily gathered by meliponine and euglossine bees for whom it most often serves as the sole pollinator reward. Bees use Clusia resin as a nest-building material. As such, resin clearly serves an indispensable mechanical function. However, resins with antimicrobial properties may also serve to reduce the risk of pathogenesis in the nest. If resin-gathering apids benefit from antimicrobial properties in nesting materials and are able to discern these characteristics in the forage they gather, one might predict that the resin reward presented in Clusia could have evolved under selection for both mechanical and antimicrobial properties. In dioecious species, where females and males each present a resin reward, selection regimes may differ between the sexes with the result that resin form and function diverge. We investigated both the form and function of the male and female pollinator reward resins of Clusia grandiflora. Using thin-layer chromatography (TLC), we compared the chemical compositions of floral resins from five widely separated populations of this species growing in southeastern Venezuela. We found that male and female resins exhibited a marked chemical dimorphism, with females having two major TLC-resolvable fractions and males having seven. This dimorphism was stable: there were no component differences between populations in either sex. Using a disk-diffusion technique, we surveyed the same resins for antimicrobial activity using assay microorganisms isolated from eusocial meliponine bees. Both male and female Clusia grandiflora resins had pronounced but relatively directed antimicrobial activity: both were toxic to 10 of 11 Gram-positive bacteria, 7 of 15 Gram-negative or variably-staining bacteria, 0 of 3 yeasts, and 0 of 3 filamentous fungi. Again with the disk-diffusion technique, we performed more detailed tests of resin bioactivity using two Gram-positive honeybee associates, Paenibacillus larvae and P. alvei, as model pathogens. Both male and female C. grandiflora resins were highly toxic to these honeybee pathogens. Female resin, however, produced zones of inhibition with more than twice the mean diameter of those produced by the male resin. This divergence in form and function of the C. grandiflora pollinator reward resins could be in response to different selective regimes as mediated by the pollinating insects.