Diversity and divergence: evolution of secondary metabolism in the tropical tree genus Inga.

Plants are widely recognized as chemical factories, with each species producing dozens to hundreds of unique secondary metabolites. These compounds shape the interactions between plants and their natural enemies. We explore the evolutionary patterns and processes by which plants generate chemical diversity, from evolving novel compounds to unique chemical profiles. We characterized the chemical profile of one-third of the species of tropical rainforest trees in the genus Inga (c. 100, Fabaceae) using ultraperformance liquid chromatography-mass spectrometry-based metabolomics and applied phylogenetic comparative methods to understand the mode of chemical evolution. We show: each Inga species contain structurally unrelated compounds and high levels of phytochemical diversity; closely related species have divergent chemical profiles, with individual compounds, compound classes, and chemical profiles showing little-to-no phylogenetic signal; at the evolutionary time scale, a species' chemical profile shows a signature of divergent adaptation. At the ecological time scale, sympatric species were the most divergent, implying it is also advantageous to maintain a unique chemical profile from community members; finally, we integrate these patterns with a model for how chemical diversity evolves. Taken together, these results show that phytochemical diversity and divergence are fundamental to the ecology and evolution of plants.

Divergent Secondary Metabolites and Habitat Filtering Both Contribute to Tree Species Coexistence in the Peruvian Amazon.

Little is known about the mechanisms promoting or limiting the coexistence of functionally divergent species in hyperdiverse tropical tree genera. Density-dependent enemy attacks have been proposed to be a major driver for the local coexistence of chemically divergent congeneric species. At the same time, we expect local soil conditions to favor the coexistence of species sharing similar functional traits related to resource use strategies, while environmental heterogeneity would promote the diversity of these traits at both local and large spatial scales. To test how these traits mediate species coexistence, we used functional trait data for 29 species from the tree genus Protium (Burseraceae), collected in 19 plots (2 ha each) in the Peruvian Amazon. We characterized the presence-absence of 189 plant secondary metabolites (SM) for 27 of these species, and 14 functional traits associated with resource use strategies (RUT) for 16 species. Based on these data, we found that SM were significantly more dissimilar than null expectations for species co-occurring within plots, whereas RUT were significantly more similar. These results were consistent with the hypothesis that density-dependent enemy attacks contribute to the local coexistence of congeneric species displaying divergent chemical defenses, whereas local habitat conditions filter species with similar RUT. Using measurements of nine soil properties in each plot, we also found a significant turnover of RUT traits with increasing dissimilarity of soil texture and nutrient availabilities, providing support for the hypothesis that soil heterogeneity maintains functional diversity at larger spatial scales (from 500 m up to ca. 200 km) in Protium communities. Our study provides new evidence suggesting that density-dependent enemy attacks and soil heterogeneity both contribute to maintaining high species richness in diverse tropical forests.

Origin and maintenance of chemical diversity in a species-rich tropical tree lineage.

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.

Insect herbivores, chemical innovation, and the evolution of habit specialization in Amazonian trees.

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.

Solid-state NMR characterization of the molecular conformation in disordered methyl α-L-rhamnofuranoside.

A combination of solid-state (13)C NMR tensor data and DFT computational methods is utilized to predict the conformation in disordered methyl α-L-rhamnofuranoside. This previously uncharacterized solid is found to be crystalline and consists of at least six distinct conformations that exchange on the kHz time scale. A total of 66 model structures were evaluated, and six were identified as being consistent with experimental (13)C NMR data. All feasible structures have very similar carbon and oxygen positions and differ most significantly in OH hydrogen orientations. A concerted rearrangement of OH hydrogens is proposed to account for the observed dynamic disorder. This rearrangement is accompanied by smaller changes in ring conformation and is slow enough to be observed on the NMR time scale due to severe steric crowding among ring substituents. The relatively minor differences in non-hydrogen atom positions in the final structures suggest that characterization of a complete crystal structure by X-ray powder diffraction may be feasible.

An oxidized squalene derivative from Protium subserratum Engl. (Engl.) growing in Peru.

Protium subserratum (Burseraceae) is a neotropical tree species that is comprised of several habitat-specific ecotypes having distinct defense chemical profiles. A previously unknown triterpene, 25,30-dicarboxy-26,27,28,29-tetraacetoxy-10,11,14,15-tetrahydrosqualene, was isolated from P. subserratum young leaf tissue of one ecotype growing in Peru. The structure of 1 was determined by spectroscopic study, including 1 and 2D nuclear magnetic resonance experiments.

The evolution of antiherbivore defenses and their contribution to species coexistence in the tropical tree genus Inga.

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.

Galloyl depsides of tyrosine from young leaves of Inga laurina.

In addition to the free protein amino acid l-tyrosine, the expanding young leaves of Inga laurina accumulate high concentrations of three new depsides, galloyl, m-digalloyl, and m-trigalloyl l-tyrosine (1, 2, and 3). The structures of these compounds were determined on the basis of their spectroscopic properties and through degradation and derivatization experiments. They occur in young leaves at the following dry-weight mass percentages: tyrosine, 10.4%; 1, 3.1%; 2, 5.0%; 3, 1.3%. These concentrations are most consistent with chemical defense during the vulnerable expansion stage of leaf development. Neither free tyrosine nor its galloyl depsides are present in mature leaves.

Contrasting mechanisms of secondary metabolite accumulation during leaf development in two tropical tree species with different leaf expansion strategies.

Young leaves of most species experience remarkably higher herbivore attack rates than mature leaves. Considerable theoretical effort has focused on predicting optimal defense and tradeoffs in defense allocation during leaf expansion. Among others, allocation to secondary chemistry may be dependent on growth constraints. We studied flavanoid production during leaf development in two species of Inga (Fabaceae: Mimosoideae) with different expansion strategies: Inga goldmanii, a species with slowly expanding young leaves, and Inga umbellifera, a species with fast-expanding young leaves. In these two species, the most abundant and toxic class of defensive compounds is flavanoids (which include tannins). We measured their concentration by leaf dry weight, their total content per leaf, their HPLC chemical profile and their toxicity to a generalist herbivore at different expansion levels. Although in both species the flavanoid concentration decreased with increasing leaf expansion, that decrease was twice as pronounced for I. umbellifera as it was for I. goldmanii. I. umbellifera leaves produced flavanoids only during the first half of their development while I. goldmanii leaves continued production throughout. The changes in flavanoid HPLC profiles and toxicity were also more dramatic for I. umbellifera, which had different flavanoids in young than in mature leaves. Relative to I. umbellifera, I. goldmanii showed smaller changes in both flavanoid composition and toxicity in the transition from young to mature leaves. These results indicate that, even though young leaves suffer higher rates of attack and are predicted to have better chemical defenses than mature leaves, growth constraints may modulate defense allocation and thus, evolution of defense strategies.

Allelochemic function for a primary metabolite: the case of l-tyrosine hyper-production in Inga umbellifera (Fabaceae).

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.

Divergence in structure and activity of phenolic defenses in young leaves of two co-occurring Inga species.

The leaves of tropical forest trees are most likely to suffer herbivore damage during the period of expansion. Herbivore selection on young leaves has given rise to a variety of leaf developmental strategies and age-specific chemical defense modes. We are studying correlations between leaf developmental types and chemical defenses in the Neotropical genus Inga. We have characterized defense metabolites in Inga goldmanii and Inga umbellifera, two species that co-occur in the lowland moist forest of Panama. These congeners have markedly different young-leaf developmental phenotypes but suffer approximately equal rates of herbivory. Bioassays of whole and fractionated leaf extracts using larvae of Heliothis virescens show that I. goldmanii chemical defenses are nearly three times more inhibitory than those of I. umbellifera. In both species, most of the inhibitory activity resides in complex mixtures of monomeric and polymeric flavan-3-ols. This group comprises >30% of young leaf dry weight in both I. goldmanii and I. umbellifera. The species' phenolic chemistry differs markedly, however, both in the structure of the monomeric units and in the distribution of polymer sizes. The differences in chemical structure have pronounced effects on their bioactivities, with I. goldmanii flavans being twice as inhibitory to H. virescens larvae as I. umbellifera flavans, and more than three times more efficient at protein binding. Given the extraordinarily high polyphenol concentrations that are found in the young leaves of these species, protein precipitation could be an important mechanism of growth inhibition. Nevertheless, our data show that another mode of phenolic action, possibly oxidative stress, occurs simultaneously.

Cinnamoyl glucosides of catechin and dimeric procyanidins from young leaves of Inga umbellifera (Fabaceae).

The rapidly growing, nearly achlorophyllous, young leaves of Inga umbellifera express high concentrations of mono and dimeric 3-O-gluco-cinnamoyl catechin/epicatechin, rare forms of substituted flavan-3-ols. Here we present structures for five novel compounds in this class: three monomers [catechin-3-O-beta-D-gluco(2-cinnamoyl)pyranoside, catechin-3-O-beta-D-gluco(6-cinnamoyl) pyranoside, catechin-3-O-beta-D-gluco(2,6-biscinnamoyl)pyranoside] and two dimeric procyanidins [catechin-3-O-beta-D-glucopyrano-(4alpha-->8)-catechin-3-O-beta-D-gluco(2-cinnamoyl)pyranoside and catechin-3-O-beta-D-glucopyrano-(4alpha-->8)-epicatechin-3-O-beta-D-gluco(6-cinnamoyl)pyranoside]. The young leaves of Inga umbellifera express high concentrations of 3-O-(cinnamoyl)glucosides of catechin and epicatechin.

Anti-bacterial function in the sexually dimorphic pollinator rewards of Clusia grandiflora (Clusiaceae).

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.