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.

Coevolutionary arms race versus host defense chase in a tropical herbivore-plant system.

Coevolutionary models suggest that herbivores drive diversification and community composition in plants. For herbivores, many questions remain regarding how plant defenses shape host choice and community structure. We addressed these questions using the tree genus Inga and its lepidopteran herbivores in the Amazon. We constructed phylogenies for both plants and insects and quantified host associations and plant defenses. We found that similarity in herbivore assemblages between Inga species was correlated with similarity in defenses. There was no correlation with phylogeny, a result consistent with our observations that the expression of defenses in Inga is independent of phylogeny. Furthermore, host defensive traits explained 40% of herbivore community similarity. Analyses at finer taxonomic scales showed that different lepidopteran clades select hosts based on different defenses, suggesting taxon-specific histories of herbivore-host plant interactions. Finally, we compared the phylogeny and defenses of Inga to phylogenies for the major lepidopteran clades. We found that closely related herbivores fed on Inga with similar defenses rather than on closely related plants. Together, these results suggest that plant defenses might be more evolutionarily labile than the herbivore traits related to host association. Hence, there is an apparent asymmetry in the evolutionary interactions between Inga and its herbivores. Although plants may evolve under selection by herbivores, we hypothesize that herbivores may not show coevolutionary adaptations, but instead "chase" hosts based on the herbivore's own traits at the time that they encounter a new host, a pattern more consistent with resource tracking than with the arms race model of coevolution.

Dispersal assembly of rain forest tree communities across the Amazon basin.

We investigate patterns of historical assembly of tree communities across Amazonia using a newly developed phylogeny for the species-rich neotropical tree genus Inga We compare our results with those for three other ecologically important, diverse, and abundant Amazonian tree lineages, Swartzia, Protieae, and Guatteria Our analyses using phylogenetic diversity metrics demonstrate a clear lack of geographic phylogenetic structure, and show that local communities of Inga and regional communities of all four lineages are assembled by dispersal across Amazonia. The importance of dispersal in the biogeography of Inga and other tree genera in Amazonian and Guianan rain forests suggests that speciation is not driven by vicariance, and that allopatric isolation following dispersal may be involved in the speciation process. A clear implication of these results is that over evolutionary timescales, the metacommunity for any local or regional tree community in the Amazon is the entire Amazon basin.

Phenolics lie at the centre of functional versatility in the responses of two phytochemically diverse tropical trees to canopy thinning.

Saplings in the shade of the tropical understorey face the challenge of acquiring sufficient carbon for growth as well as defence against intense pest pressure. A minor increase in light availability via canopy thinning may allow for increased investment in chemical defence against pests, but it may also necessitate additional biochemical investment to prevent light-induced oxidative stress. The shifts in secondary metabolite composition that increased sun exposure may precipitate in such tree species present an ideal milieu for evaluating the potential of a single suite of phenolic secondary metabolites to be used in mitigating both abiotic and biotic stressors. To conduct such an evaluation, we exposed saplings of two unrelated species to a range of light environments and compared changes in their foliar secondary metabolome alongside corresponding changes in the abiotic and biotic activity of their secondary metabolite suites. Among the numerous classes of secondary metabolites found in both species, phenolics accounted for the majority of increases in antioxidant and UV-absorbing properties as well as activity against an invertebrate herbivore and a fungal pathogen. Our results support the hypothesis that phenolics contribute to the capacity of plants to resist co-occurring abiotic and biotic stressors in resource-limited conditions.

Chemocoding as an identification tool where morphological- and DNA-based methods fall short: Inga as a case study.

The need for species identification and taxonomic discovery has led to the development of innovative technologies for large-scale plant identification. DNA barcoding has been useful, but fails to distinguish among many species in species-rich plant genera, particularly in tropical regions. Here, we show that chemical fingerprinting, or 'chemocoding', has great potential for plant identification in challenging tropical biomes. Using untargeted metabolomics in combination with multivariate analysis, we constructed species-level fingerprints, which we define as chemocoding. We evaluated the utility of chemocoding with species that were defined morphologically and subject to next-generation DNA sequencing in the diverse and recently radiated neotropical genus Inga (Leguminosae), both at single study sites and across broad geographic scales. Our results show that chemocoding is a robust method for distinguishing morphologically similar species at a single site and for identifying widespread species across continental-scale ranges. Given that species are the fundamental unit of analysis for conservation and biodiversity research, the development of accurate identification methods is essential. We suggest that chemocoding will be a valuable additional source of data for a quick identification of plants, especially for groups where other methods fall short.

Consequences of interspecific variation in defenses and herbivore host choice for the ecology and evolution of Inga, a speciose rainforest tree.

We summarize work on a speciose Neotropical tree genus, Inga (Fabaceae), examining how interspecific variation in anti-herbivore defenses may have evolved, how defenses shape host choice by herbivores and how they might regulate community composition and influence species radiations. Defenses of expanding leaves include secondary metabolites, extrafloral nectaries, rapid leaf expansion, trichomes, and synchrony and timing of leaf production. These six classes of defenses are orthogonal, supporting independent evolutionary trajectories. Moreover, only trichomes show a phylogenetic signature, suggesting evolutionary lability in nearly all defenses. The interspecific diversity in secondary metabolite profiles does not arise from the evolution of novel compounds, but from novel combinations of common compounds, presumably due to changes in gene regulation. Herbivore host choice is determined by plant defensive traits, not host phylogeny. Neighboring plants escape each other's pests if their defenses differ enough, thereby enforcing the high local diversity typical of tropical forests. Related herbivores feed on hosts with similar defenses, implying that there are phylogenetic constraints placed on the herbivore traits that are associated with host use. Divergence in defensive traits among Inga appears to be driven by herbivore pressure. However, the lack of congruence between herbivore and host phylogeny suggests that herbivores are tracking defenses, choosing hosts based on traits for which they already have adaptations. There is, therefore, an asymmetry in the host-herbivore evolutionary arms race.

The global distribution of diet breadth in insect herbivores.

Understanding variation in resource specialization is important for progress on issues that include coevolution, community assembly, ecosystem processes, and the latitudinal gradient of species richness. Herbivorous insects are useful models for studying resource specialization, and the interaction between plants and herbivorous insects is one of the most common and consequential ecological associations on the planet. However, uncertainty persists regarding fundamental features of herbivore diet breadth, including its relationship to latitude and plant species richness. Here, we use a global dataset to investigate host range for over 7,500 insect herbivore species covering a wide taxonomic breadth and interacting with more than 2,000 species of plants in 165 families. We ask whether relatively specialized and generalized herbivores represent a dichotomy rather than a continuum from few to many host families and species attacked and whether diet breadth changes with increasing plant species richness toward the tropics. Across geographic regions and taxonomic subsets of the data, we find that the distribution of diet breadth is fit well by a discrete, truncated Pareto power law characterized by the predominance of specialized herbivores and a long, thin tail of more generalized species. Both the taxonomic and phylogenetic distributions of diet breadth shift globally with latitude, consistent with a higher frequency of specialized insects in tropical regions. We also find that more diverse lineages of plants support assemblages of relatively more specialized herbivores and that the global distribution of plant diversity contributes to but does not fully explain the latitudinal gradient in insect herbivore specialization.

Mycoleptodiscins A and B, cytotoxic alkaloids from the endophytic fungus Mycoleptodiscus sp. F0194.

Two novel reddish-orange alkaloids, mycoleptodiscin A (1) and mycoleptodiscin B (2), were isolated from liquid cultures of the endophytic fungus Mycoleptodiscus sp. that had been isolated from Desmotes incomparabilis in Panama. Elucidation of their structures was accomplished using 1D and 2D NMR spectroscopy in combination with IR spectroscopic and MS data. These compounds are indole-terpenes with a new skeleton uncommon in nature. Mycoleptodiscin B (2) was active in inhibiting the growth of cancer cell lines with IC50 values in the range 0.60-0.78 µM.

Leaf metabolic traits reveal hidden dimensions of plant form and function.

The metabolome is the biochemical basis of plant form and function, but we know little about its macroecological variation across the plant kingdom. Here, we used the plant functional trait concept to interpret leaf metabolome variation among 457 tropical and 339 temperate plant species. Distilling metabolite chemistry into five metabolic functional traits reveals that plants vary on two major axes of leaf metabolic specialization-a leaf chemical defense spectrum and an expression of leaf longevity. Axes are similar for tropical and temperate species, with many trait combinations being viable. However, metabolic traits vary orthogonally to life-history strategies described by widely used functional traits. The metabolome thus expands the functional trait concept by providing additional axes of metabolic specialization for examining plant form and function.

Coibanoles, a new class of meroterpenoids produced by Pycnoporus sanguineus.

Three new terpenoids of mixed biosynthetic origin were isolated from the culture filtrate of the endophytic fungus Pycnoporus sanguineus. Their structures were determined by extensive spectroscopic analyses. We have named these tricyclic and tetracyclic metabolites 'coibanoles A-C' in reference to Coiba Island and Coiba National Park, Panamá, from which the plant and endophyte were collected. The extract was inactive to the human parasites Trypanosoma cruzi, Leishmania donovani, and Plasmodium falciparum at a test concentration of 10 µg/mL.

Antifungal Depsidone Metabolites from Cordyceps dipterigena, an Endophytic Fungus Antagonistic to the Phytopathogen Gibberella fujikuroi.

Among thirty four endophytic fungal strains screened for in vitro antagonism, the endophytic fungus Cordyceps dipterigena was found to strongly inhibit mycelial growth of the plant pathogenic fungus Gibberella fujikuroi. Two new depsidone metabolites, cordycepsidone A (1) and cordycepsidone B (2), were isolated from the PDA culture extract of C. dipterigena and identified as being responsible for the antifungal activity. Elucidation of their chemical structures was carried out using 1D and 2D NMR spectroscopy in combination with IR and MS spectroscopic data. Cordycepsidone A displayed strong and dose-dependent antifungal activity against the plant pathogenic fungus Gibberella fujikuroi. The isolates were inactive in bioassays for malaria (Plasmodium falciparum), leishmaniasis (Leishmania donovani), Chagas's disease (Trypanosoma cruzi), and cytotoxicity at 10 µg/mL. The compounds were also found to be inactive against several bacterial strains at 50 µg/mL.

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.

Culturing and direct PCR suggest prevalent host generalism among diverse fungal endophytes of tropical forest grasses.

Most studies examining endophytic fungi associated with grasses (Poaceae) have focused on agronomically important species in managed ecosystems or on wild grasses in subtropical, temperate and boreal grasslands. However grasses first arose in tropical forests, where they remain a significant and diverse component of understory and forest-edge communities. To provide a broader context for understanding grass-endophyte associations we characterized fungal endophyte communities inhabiting foliage of 11 species of phylogenetically diverse C(3) grasses in the understory of a lowland tropical forest at Barro Colorado Island, Panama. Our sample included members of early-arising subfamilies of Poaceae that are endemic to forests, as well as more recently arising subfamilies that transitioned to open environments. Isolation on culture media and direct PCR and cloning revealed that these grasses harbor species-rich and phylogenetically diverse communities that lack the endophytic Clavicipitaceae known from diverse woodland and pasture grasses in the temperate zone. Both the incidence and diversity of endophytes was consistent among grass species regardless of subfamily, clade affiliation or ancestral habitat use. Genotype and phylogenetic analyses suggest that these endophytic fungi are predominantly host generalists, shared not only among distinctive lineages of Poaceae but also with non-grass plants at the same site.

Pests vs. drought as determinants of plant distribution along a tropical rainfall gradient.

Understanding the mechanisms that shape the distribution of organisms can help explain patterns of local and regional biodiversity and predict the susceptibility of communities to environmental change. In the species-rich tropics, a gradient in rainfall between wet evergreen and dry seasonal forests correlates with turnover of plant species. The strength of the dry season has previously been shown to correlate with species composition. Herbivores and pathogens (pests) have also been hypothesized to be important drivers of plant distribution, although empirical evidence is lacking. In this study we experimentally tested the existence of a gradient in pest pressure across a rainfall gradient in the Isthmus of Panama and measured the influence of pests relative to drought on species turnover. We established two common gardens on the dry and wet sides of the Isthmus using seedlings from 24 plant species with contrasting distributions along the Isthmus. By experimentally manipulating water availability and insect herbivore access, we showed that pests are not as strong a determinant of plant distributions as is seasonal drought. Seasonal drought in the dry site excluded wet-distribution species by significantly increasing their seedling mortality. Pathogen mortality and insect herbivore damage were both higher in the wet site, supporting the existence of a gradient in pest pressure. However, contrary to predictions, we found little evidence that dry-distribution species suffered significantly more pest attack than wet-distribution species. Instead, we hypothesize that dry-distribution species are limited from colonizing wetter forests by their inherently slower growth rates imposed by drought adaptations. We conclude that mechanisms limiting the recruitment of dry-distribution species in wet forests are not nearly as strong as those limiting wet-distribution species from dry forests.

Herbivores as drivers of negative density dependence in tropical forest saplings.

Ecological theory predicts that the high local diversity observed in tropical forests is maintained by negative density-dependent interactions within and between closely related plant species. By using long-term data on tree growth and survival for coexisting Inga (Fabaceae, Mimosoideae) congeners, we tested two mechanisms thought to underlie negative density dependence (NDD): competition for resources and attack by herbivores. We quantified the similarity of neighbors in terms of key ecological traits that mediate these interactions, as well as the similarity of herbivore communities. We show that phytochemical similarity and shared herbivore communities are associated with decreased growth and survival at the sapling stage, a key bottleneck in the life cycle of tropical trees. None of the traits associated with resource acquisition affect plant performance, indicating that competition between neighbors may not shape local tree diversity. These results suggest that herbivore pressure is the primary mechanism driving NDD at the sapling stage.

Antileishmanial constituents of the Panamanian endophytic fungus Edenia sp.

Bioassay-directed fractionation of extracts from the fermentation broth and mycelium of the fungus Edenia sp. led tothe isolation of five antileishmanial compounds, preussomerin EG1 (1), palmarumycin CP2 (2), palmarumycin CP17 (3), palmarumycin CP18 (4), and CJ-12,371 (5). Compounds 3 and 4 are new natural products, and this is only the second report of compound 1. The structures of compounds 1-5 were established by spectroscopic analyses (HRMS and NMR). All metabolites caused significant inhibition of the growth of Leishmania donoVani in the amastigote form, with IC50 values of 0.12, 3.93, 1.34, 0.62, and 8.40 microM, respectively. Compounds 1-5 were inactive when tested against Plasmodium falciparum or Trypanasoma cruzi at a concentration of 10 microg/mL, indicating that they have selective activity against Leishmania parasites. Compounds 1-5 showed weak cytotoxicity to Vero cells (IC50 of 9, 162, 174, 152, and 150 microM, respectively); however, the therapeutic window of these compounds is quite significant with 75, 41, 130, 245, and 18 times (respectively) more antileishmanial activity than cytotoxicity.

Tracking of Host Defenses and Phylogeny During the Radiation of Neotropical Inga-Feeding Sawflies (Hymenoptera; Argidae).

Coevolutionary theory has long predicted that the arms race between plants and herbivores is a major driver of host selection and diversification. At a local scale, plant defenses contribute significantly to the structure of herbivore assemblages and the high alpha diversity of plants in tropical rain forests. However, the general importance of plant defenses in host associations and divergence at regional scales remains unclear. Here, we examine the role of plant defensive traits and phylogeny in the evolution of host range and species divergence in leaf-feeding sawflies of the family Argidae associated with Neotropical trees in the genus Inga throughout the Amazon, the Guiana Shield and Panama. Our analyses show that the phylogenies of both the sawfly herbivores and their Inga hosts are congruent, and that sawflies radiated at approximately the same time, or more recently than their Inga hosts. Analyses controlling for phylogenetic effects show that the evolution of host use in the sawflies associated with Inga is better correlated with Inga chemistry than with Inga phylogeny, suggesting a pattern of delayed host tracking closely tied to host chemistry. Finally, phylogenetic analyses show that sister species of Inga-sawflies are dispersed across the Neotropics, suggesting a role for allopatric divergence and vicariance in Inga diversification. These results are consistent with the idea that host defensive traits play a key role not only in structuring the herbivore assemblages at a single site, but also in the processes shaping host association and species divergence at a regional scale.

Food quality, competition, and parasitism influence feeding preference in a neotropical lepidopteran.

We surveyed Lepidoptera found on 11 species of Inga (Fabaceae:Mimosoideae) co-existing on Barro Colorado Island, Panama, to evaluate factors influencing diet choice. Of the 47 species of caterpillars (747 individuals) recorded, each fed on a distinct set of Inga. In the field, 96% of the individuals were found on young leaves. Growth rates of caterpillars that were fed leaves in the laboratory were 60% higher on young leaves compared to mature leaves. When caterpillars were fed leaves of nonhost Inga, they grew more slowly. These data provide support for a link between preference and performance. However, among hosts on which larvae normally occurred, faster growth rates were not associated with greater host electivity (the proportion of larvae found on each host species in the field, corrected for host abundance). Growth rates on normal hosts were positively correlated with leaf expansion rates of the host, and fast expansion was associated with leaves with higher nutritional content. Detailed studies on a gelechiid leaf roller, the species with the largest diet breadth, allowed us to assess the importance of factors other than growth that could influence diet electivity. This species showed a 1.7-fold difference in growth rate among Inga hosts and faster growth on species with fast-expanding leaves. However, there was no correlation between caterpillar growth rate and abundance on different host species. Instead, abundance of the gelechiid on each Inga species was significantly correlated with the temporal predictability of food (synchrony of leaf flushing) and was negatively correlated with competition (amount of leaf area removed by species other than the gelechiid). Although rates of parasitism were high (23-43%), there were no differences among hosts. Parasitism was also not related to measures of escape, such as growth rates of caterpillars, leaf expansion rates, and synchrony of leaf production. Together, food availability, parasitism, and competition explained 84% of the variation in host preference by the gelechiid. We suggest that these ecological interactions may be particularly important in determining diet choice initially and that preferences may be reinforced by subsequent divergence in host chemistry and/or the herbivore's ability to tolerate the secondary metabolites.

The growth-defense trade-off and habitat specialization by plants in Amazonian forests.

Tropical forests include a diversity of habitats, which has led to specialization in plants. Near Iquitos, in the Peruvian Amazon, nutrient-rich clay forests surround nutrient-poor white-sand forests, each harboring a unique composition of habitat specialist trees. We tested the hypothesis that the combination of impoverished soils and herbivory creates strong natural selection for plant defenses in white-sand forest, while rapid growth is favored in clay forests. Recently, we reported evidence from a reciprocal-transplant experiment that manipulated the presence of herbivores and involved 20 species from six genera, including phylogenetically independent pairs of closely related white-sand and clay specialists. When protected from herbivores, clay specialists exhibited faster growth rates than white-sand specialists in both habitats. But, when unprotected, white-sand specialists outperformed clay specialists in white-sand habitat, and clay specialists outperformed white-sand specialists in clay habitat. Here we test further the hypothesis that the growth defense trade-off contributes to habitat specialization by comparing patterns of growth, herbivory, and defensive traits in these same six genera of white-sand and clay specialists. While the probability of herbivore attack did not differ between the two habitats, an artificial defoliation experiment showed that the impact of herbivory on plant mortality was significantly greater in white-sand forests. We quantified the amount of terpenes, phenolics, leaf toughness, and available foliar protein for the plants in the experiment. Different genera invested in different defensive strategies, and we found strong evidence for phylogenetic constraint in defense type. Overall, however, we found significantly higher total defense investment for white-sand specialists, relative to their clay specialist congeners. Furthermore, herbivore resistance consistently exhibited a significant trade-off against growth rate in each of the six phylogenetically independent species-pairs. These results confirm theoretical predictions that a trade-off exists between growth rate and defense investment, causing white-sand and clay specialists to evolve divergent strategies. We propose that the growth-defense trade-off is universal and provides an important mechanism by which herbivores govern plant distribution patterns across resource gradients.

High herbivore pressure favors constitutive over induced defense.

UNLABELLED: Theoretical and empirical studies show that, when past or current herbivory is a reliable cue of future attack and defenses are costly, defenses can be induced only when needed and thereby permit investment in other functions such as growth or reproduction. Theory also states that, in environments where herbivory is constantly high, constitutive defenses should be favored. Here, we present data to support the second aspect of the induced resistance hypothesis. We examined herbivore-induced responses for four species of Inga (Fabaceae), a common canopy tree in Neotropical forests. We quantified chemical defenses of expanding leaves, including phenolic, saponin and toxic amino acids, in experimental field treatments with and without caterpillars. Because young leaves lack fiber and are higher in protein than mature leaves, they typically lose >25% of their leaf area during the few weeks of expansion. We predicted that the high rates of attack would select for investment in constitutive defenses over induction. Our data show that chemical defenses were quite unresponsive to herbivory. We demonstrated that expanding leaves showed no or only small increases in investment in secondary metabolites, and no qualitative changes in the phenolic compound profile in response to herbivory. The proteinogenic amino acid tyrosine, which can be toxic at high concentrations, showed the greatest levels of induction. SYNTHESIS: These results provide some of the first support for theoretical predictions that the evolution of induced vs. constitutive defenses depends on the risk of herbivory. In habitats with constant and high potential losses to herbivores, such as tropical rainforests, high investments in constitutive defenses are favored over induction.