j | i - | | The evolution of antiherbivore defenses and their contribution to species coexistence in the tropical tree genus Inga ‘Thomas A. Kursaré®*, Kyle G. Dexters, John Lokvam*, R. Toby Pennington®, James E. Richardson, Marjorie G. Weber’, Eric T. Murakami*, Camilla Drake®, Ruth McGregor’, and Phyllis D. Coley** *oepertment of ology, University of Utah, Salt Lake City, UT 84112; Smithsonian Tropical Research Insitute, Box 0843-03092, Balbo, Panes; “Laboratoire Evolution et Diverse ioiogique, Unite Mixte de Recherche 5176, Centre National ea Rechercne Slenifique, Universit Paul Sabatier, Batiment 483, $1062 Toulouse, France and Royal Botanic Garden, Edinburgh EM SLR, United Kingdom Edited by Anurag A. Agrawal, Cornell University, ithaca, NY, and accepted by the Editorial Board July 27, 2009 (recived for review May 1, 2008) 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 phy- logenetic signal. Furthermore, in a muitivariate analysis, develop ‘mental, ant, and chemical defenses varied independently (were forthogonal) and appear to have evolved independently of each other. Our results are consistent with strong selection for diver: gent 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 nga 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 | phylogeneticsignal | herbivory tropical diversity sause plants and herbivores constitute more than half ofthe macroscopic diversity on Earth, their interactions play a fundamental role in biodiversity and ecosystem function. Two central issues are how defenses against herbivores have evolved ‘and how such variation in defenses among species might regulate plant community composition (1-6). Here, we use a phyloge- netic approach to investigate the diversification of antiherbivore defenses and their role in community assembly. Several paradigms dominate our understanding of plant/ herbivore macroevolution. Ehrlich and Raven (7) observed that related plants host similar herbivores and suggested that plant herbivore coevolution is driven by changes in plant secondary ‘metabolites. Their hypothesis predicts that more closely related species should have more similar chemistry (8,9). An alternative hypothesis is that natural selection caused by herbivores may result in rapid trait evolution such that closely related species have divergent defenses. Another distinct, and widely accepted, proposition suggests that species coexisting at a single site are likely to differ in key ecological niche dimensions (10, 11). Here, we examine the diversity of antiherbivores defenses, their phylogenetic signal, and their contribution to species coexistence in the tree genus Inga (Fabaceae: Mimosoideae). ‘Inga diversified rapidly during the last 2 million to 10 million yn pa 1a /i/Si/10.1073/9n95 0804706108 years and now has >300 species distributed throughout the Neotropics (2, 13). Furthermore, Inga is one of the most species-rich and abundant genera in local communities (14) with 43 species comprising 6,0% of the stems recorded from 25 ha in Ecuador (15). In S0 ha in Panama, Inga comprises 7% of all toe species (16). ‘We measured antiherbivore defenses in Inga for 12 species in Panama and 31 species in Peru (six species are shared). We focused our study on the defenses of young, expanding leaves because their rate of damage is ~100-fold higher than for mature leaves and they receive >80% of the damage accrued throughout the lifetime of a leaf (17, 18). During the Very short period (1-3 \weeks) of leaf expansion inthe Inga study species, 25% ofthe leat area was eaten in Panama and 37% was eaten in Peru (Fig. 14). Therefore, we argue that the defense traits under the strongest ‘natural selection are those of young leaves. Chemical defenses are likely important for young leaves as Inga species invest up to ‘50% dry weight (DW) of young leaves in secondary metabolites (19). Young leaves are also defended against herbivory through ‘developmental defenses such as rapid leaf expansion or delayed chloroplast development (2023). Finally, Inga leaves have ex- tafloral nectaries that produce nectar and attract leaf-defending ants only during leaf expansion (Fig, 1B and refs. 2426). Thus, ‘we measured the following defensive traits on young leaves: the presence or absence of chemical compounds from classes that are relevant to antiherbivore defense, the rate of leaf expansion, chloroplast development (chlorophyll content), and the average ‘number of ants per extrafloral nectary. ‘The primary goal of this study is to determine how interspe- cific variation in defenses may contribute to our understanding ‘ofthe origin and maintenance of tropical diversity. Therefore, we also constructed a phylogenetic hypothesis for all of the focal species by using plastid DNA sequence data. We also assessed coexistence in the ficld by using data on the abundance and richness of Inga from the $0-ha forest dynamics plot in Panama ‘and a network of tree plots in southern Peru, These data allowed ‘he athor socae no conte of ine “hi arco 3 PUAS Die Submiion. KAA. gle eter ites bythe Est ut depeston-Thesauencesrepreinthspaperhavebeendepstedin the Geran ‘stator lacoon noe snes, Fats, Panate,H7alen, NTA, PEE, fiavast, Panu, Havana, Fanta, aesam BSN, PTAs, ATR Fisnara, Henao, fisaasa, Fsna%n, F7e675, Hspaae, Firsts, FSO", Fisso3s, GaverGs Ga 088, Ge? 28)-GORTT27, ad COWS). “Townon cepondence ould be deed. al: kur biology utah ey, “Th ati contains supporting information enine at win ona erent cpocest0sbcSuplener. PNAS | exaber 27,2008 | vol. 105 | no. <3 | 18073-19078Fig. 1. Young leaves of J. poeppigiana being damaged by a hemipteran (ete photo by Robyn Burnham) and young leaves of 1. thidaudlana with teraforal nectaries being visited by the ant Ectatomma (Right: photo by Sania Brenes-arguede) us to test the above-mentioned hypotheses that pertain to defense evolution (7-9) and community assembly (10, 11) by asking whether or not (j) defense traits are phylogenetically conserved and (ii) coexisting species differ more in defense strategies than would be expected by chance. Results and Discussion Chemical defenses vary considerably among Inga species and include compounds belonging to several distinct classes that are known for their antiherbivore effects (27). Secondary metabo- Iites include phenolic, saponins and nonprotein amino/imino acids, and an overexpressed protein amino acid, tyrosine (28 30). Twelve types of metabolites were identified that arise from branch points in the shikimic acid, phenylpropanoid and fla- vonoid pathways (Fig. Sl; referred to as “phenols”. These include quinic,galic, and cinnamie acid derivatives and tyrosine and tyramine, either in pure or conjugated form. The most common and widespread phenolics were the flavan-3-ol, the polymeric forms of which are called condensed tannins. ‘Thirty-two species contained one or more forms of phenolic metabolite; 12 species contained saponins. In all, the 37 study species were distributed among 13 distinct chemotypes, each a ‘unique combination of phenolics and saponins (Table $1). The ‘most common chemotype, polymers of the flavan-3-ol, gallocat- echin-epigalocatechin gallate, was shared by 11 species. In addition to nga, tis lass of metabolites is known from at least two genera of mimosoid legumes Sinphnodendron (31) and Cojoba (Table $2), which suggests that it may be the ancestral chemical defense in the genus. All other chemotypes. were considerably less common: nine of the 13 chemotypes were represented by only one or two species (Table SI). leven species from Panama were also examined for protein’ nonprotein aminovimino acid content, a class characteristic of legumes (32). There were 17 different compounds (Table Si), 11 of which are derived from five different amino acid precursors (G3), with the remainder being uncharacterized. No two species had the same suite of compounds. ‘The rate of lea? expansion, a Key developmental defense, determines how long a leaf is tender and vulnerable to herbi- vores, This rate varied widely among species, ranging from 16% to 80% per day (Fig. 2), equivalent to the variation among all non-inga species sampled (34, 35). The chlorophyll content of ‘young leaves also varied (Fig, 2), with some species having almost ‘white young leaves (20 mgm?) and others with normal gree (186 mgm), There was a significant negative relationship between expansion and chlorophyll within Znga (Fig. 2), which was true for each site independently Panama, = 0.71, P< 18074 | wo. srasora/), = 050, P= 0.0001). The dotted diagonal line separates species into two ‘equalsized groups designated as defense and escape. 0.001; Peru, = 0.41, P < 0,001) and all species together ( = 050, P < 0.001. "To test whether the correlations between expansion and chlorophyll could be caused by convergence, we examined their phylogenetically independent contrasts (PICs) (36). There was a significant negative correlation between PICS for the two traits (across 200 randomly selected Bayesian trees: = 0.37, P (0.0001, P < 0105 for 99% of trees). We have argued that this Widespread tradeoff may be caused by an unavoidable physio- logical constraint that restricts simultaneous allocation of re- sources to both rapid expansion and the costly photosynthetic system (21-23). A second, although not mutually exclusive, explanation is that this association could arise because of pleiotropic effects. ‘Ant abundance, a biotic defense, also varied among Inga species. Ant visitation to the extrafloral nectaris of young leaves ilfered 20-fold among species and on average was 2.3 times higher in Peru compared with Panama (test, < 0.001; Fig. S2). ‘We tested the hypothesis that ant visitation could be associated ith developmental defenses. Using nonphylogenetic regres- sions and PICs, we found no significant correlation between expansion rates and ant visitation (? = O01, P = 0.72, Fi S2s PICS: 2 = 0001, P = 048) or between chlorophyll and ant visitation (# = 0.01, P = 051; PICs: = 0.01, P = 0.45). The chemical, nectary, and leat-development traits discussed above vary considerably among species. The broad distribution fof waits seen in Inga could result from two quite distinct ‘evolutionary trajectories. The species with dissimilar defenses could be the most phylogenetically distant (high phylogenetic Signal), pattern consistent with Ehrlich and Raven's (7) view of plant-hetbivore macroevolution. Alternatively, close relatives may have divergent defenses (low phylogenetic signal, an un- ‘expected pattern that would suggest distinct macroevotutionary processes. ‘Are More Closely Related Species More Similar in Their Defenses? The assumption that more closely related species have similar de- fenses. has dominated coevolutionary theory (7). However, within the genus Inga, we inferred that defense strategy shows little phylogenetic signal. Our phylogenetic hypothesis for Inga species, based on ~6,000 bp of plastid DNA sequence and inferred by using Bayesian approaches, resolves several clades with strong support (posterior probabilities 95%; Fig. 3). This phylogenetic tree shows more resolution than that of Richardson ct al. (13), who sampled fewer Jnga species and used only ~ 1,500 bp of DNA sequence. ‘We quantified the chemical distances between species based ure eaFig. 3. Phylogenetic tree (Left: 50% majority rule consensus tree from Bayesian analysis of 6.000 bps of plastid DNA) and chemistry dendrogram (ight) for Inga species, Thickened branches on the phylogeny represent 50.95 posterior probability forthe acjacent node. The chemistry sendrogram wae ganerated by using hierarchical Curtaring of presenclabsence data for 13 defense chemicals ghenolis and saponins, weighted equal). Thickened branches are adjacent to nodes with P< 0.1 according to multacale boot strapping analysis (approximately >90% bootstrap suppor see 5! Te Species in bot are classified as cefeme (ee Fg, 2), onc other spec Alasifeg as escape. Gray species are unclasifed because of lack of data, on the number of compounds for which species differed (see ‘Materials and Methods). We found a weak correlation betwe phylogenetic distance and chemical distance between species, based on combining data on saponin and phenolic compounds and weighting them equally (Mantel Tests across 200 Bayesian trees, 7 = 0.13, P = 041, P'< 0105 for 70% of trees; Fig. $3). ‘The weak phylogenetic signal for total chemical distance derives, from moderate similarity in phenolic chemistry of closely related species (Mantel Tests,7 ~ 0.23, P = 0.021, P < 005 for 95% of ttees). There was no relationship between phylogenetic distance and saponin chemical distance (Mantel Tests,r = 0.03, = 0.31), and for Panamanian species, there was no relationship between phylogenetic and aminovimino acid chemical distances (Mantel ‘Tests, = =0.04, P= 0.51), ‘We also compared the phylogeny with a dendrogram of the chemical similarities among species of Inga (Fig. 3). Inspection of Fig. 3 showed weak congruence. Some closely related species (. capitata, morphosp.12 and 1. morphosp.35) shared similar chemistry, but more frequently, close relatives were chemically dissimilar. For example, the clade containing 1. umbelifra, I brevipes, morphosp.36, and I. heterophyla exhibited chemical profiles'that spanned the entire chemical space. For the five species having only saponins, two, I. morphosp.59 and /. mor- phosp.13, are sister in the phylogeny, whereas the other three species, I. alba, I sapindoides, and I. tomentosa, are placed in thre other clades. We conclude that, for Inga, related species are more divergent in their secondary metabolite composition than expected by chance. “To test for a phylogenetic signal for developmental and ant defenses, we first conducted @ principal component analysis (PCA) to determine the orthogonality of these two proposed defense strategies. ‘The first axis was highly correlated with expansion rate and chlorophyll content (development), whereas the second axis was highly correlated with ant visitation rates. “The frst ais showed significant phylogenetic signal (Blombera's across 200 Bayesian trees: K= 0.69, P= 0.04, P< 0105 for 73% kurar et. ‘of tees) (37) although stil lower than the expected value of one under pure Brownian evolution, The second axis showed no detectable phylogenetic signal (K = 0.42, P = 0.87). To further visualize the evolution of these characters, we elasified species {nto the conventional categories of “escape” vs. “defense” (35) based on species’ coordinates in Fig, 2 and as having high vs. iow ant visitation. These binary characters showed no detectable Phylogenetic signal when mapped onto 100 Bayesian trees sampled at stationarity (Fig. $4) (ref. 38 and http:// ‘mesquiteproject.org). We also show in Fig. 3 how species were classified with respect 10 escape vs. defense. Hence, we found significant phylogenetic signal only for developmental traits and ‘only in the ease when we treated these defenses as quantitative traits and analyzed them by PCA. ‘We also assessed Mantel correlations of the chemical dis- tances between species with the differences between species in the values along each of the two principal component axes representing developmental and ant defense. Neither was cor- ‘elated with chemistry (frst axis: Mantel r = ~003, P = 049; second axis: Mantel = ~0.01, P = 085), These results and the PCAs suggest that developmental, ant, and chemical profiles are independent defensive strategies that also have evolved indepen- dently of one another. Species in the cotton clade also showed independent evolution of ant and chemical defenses (39) ‘We have sampled only 37 species of Inga, and further sampling could result in subelades showing more uniformity in defensive strategies. However, that is unlikely for several reasons. Fest, ur sampling spans the taxonomic diversity ofthe genus, represent- ing of 14 Sections recognized by Pennington (12) Second, some fof the species included are definitively shown to be closely related but chemically divergent. For example, two species pairs show a profound lack of sequence divergence in the rapidly ‘volving plastid regions we have used. These are. goldmanti and 1 muljuga, identical for ~6,000 bp of plastid DNA, and 1 leiocabyeina and I. morphosp6, differing by two substitutions in 16,000 bp; and they nevertheless show contrasting chemical defenses (Fig. 3), ‘Although there is a significant association of developmental defense strategy with phylogeny, we find litle evidence for phylogenetic signal inthe other, independent defense traits, ants, ‘and chemistry. Our data show both extensive variation indefense traits and considerable differences among close relatives, a pattern that is consistent with defense strategies in Inga being ‘evolutionarily labile (40) "This finding contrasts with the pre= vain hypothesis (Ehrlich and Raven (7)), which implies that closely related plant species should be similar in defense strat- ‘egies. Few studies have examined defenses across many related species, although terpenes in the genus Bursera (Burseraceae) showed low congruence between chemical dendrogram and a phylogeny (3) Cocristence and Defense Trait Similarity. Could antiherbivore de- fenses be an important mechanism of niche partitioning in community assembly? To address this question, we assessed phylogenetic and defense trait dispersion in Jnga communities. We used the inverse ofthe metrics of Webb (41) so that positive values represent overdispersion and negative values represent underdispersion. In the Sb-ha forest dynamics plot in Panama, we found that co-occurring Inga species are more distantly related than expected by chance. (overdispersion; Fig, 4; across 200 Bayesian trees, two-tailed £ tests: NRI = 1583, 2 < 0.00001, P< 0.05 for 100% of trees; NTI = 0.30, = 7.83, P 0.005, B < 0.5 for 99% trees). In contrast, in Peru we found phylogenetic underdispersion of Inga communities (Fig. 4; two- tailed tess: NRI = =0.66,1 = 5.08, P < 0.0002, P < 0.05 for 100% of trees, NTI = ~0.50,? = ~3.15, P = 0.021, P < 0.05 for 91% trees). This site difference likely reflects the different spatial and environmental scales that were sampled. In Panama, PNAS | October 27,2008 | vol. 106 | no. 43 | 18075! | i i | | ig EE 24 2 ae ee Defense Trait Dispersion 07 24 CNRENT NRE NTT Fig. 4 Phylogenetic and defense trait dispersion for Inga communities in Peru and Panama, Phylogenetic results are for one, randomly selected Bayer. lan te, Values given are the inverse of the net relatedness index (ND and nearest axon index (NT) following Webb (4). Valves > O indicate overds- person, and values <0 indieate undersepersion, The departure of comm rites from the null expectation (zero) of communities Being randomly si Sembled was evaluated by using twosided tet for phylogeneticdlspersion and onesided «tess for defense trait dispersion (¢,P =< 005; +», P= 0.0; ste, P0001), ‘we assessed co-occurrence within the homogenous habitat of the 50-ha plot (local spatial scale). In Peru, we sampled across a 150-%-200-km atea (regional spatial scale) and in two different habitat types, terra firme and floodplain forest (42). We suggest that @ phylogenetic signal for traits associated with habitat preference (cf. ref. 41) may underlie the contrasting phylogenetic structures. ‘AL both spatial scales, including Peru where neighbors were close relatives, the co-occurring species were more dissimilar than expected at random for a defense index combining the developmental, ant, and chemical waits (see Materials and ‘Methods; Fig. 4; Peru: one-tailed test: NRI = 0.32, = 2.26, 0.016; NTI = 0.40, = 3.56, P = 0,0007; Panama: one-tailed NRI = 0.60,¢ = 13.75, P < 0,00001; NTI = 1.06, = 17.35, P< (0.00001). We also assessed the dispersion of two leaf traits that are likely unrelated to herbivore defense, the presence vs. absence of wings on the rachis and the number of leaflets per leaf. We found no evidence for overdspersion of leaf traits in Panama (one-tailed test: NRI = ~0.68,0 = ~8.96, P< 0.00001; NTI = =0.05, ¢ = -0.74, P = 0.46) or Peru (NRI = 029, ¢ = 1.33, P = 0.196; NTI = ~0.04, 1 = -0.16, P = 0.87). "These results illustrate that, although nondefense traits such as leaflet number and presence of wings are randomly or under- dispersed, defensive traits at both sites are overdispersed. Di- ‘vergence in chemical defenses at single sites has also been shown for another speciose tree genus, Bursera (4). The co-occurrence of species having divergent defense strategies could be caused by herbivores preferentially foraging on individuals with common defense phenotypes. By such @ mechanism, herbivores may structure the assembly of rainforest tree communities. Conclusions (Our analysis of defenses is consistent with the idea that, in Inga, the arms race between plants and their herbivores has led to rapid and divergent trait evolution, The young leaves of Inga ‘make substantial investments in chemical defenses [up to 50% of DW (19)] and developmental defenses and extrafloral nectar, suggesting that herbivory is a strong selective agent. The chem- ical, biotic, and developmental defenses of young leaves showed considerable variation among species. The observations of Ehr- Tich and Raven (7) suggest that defenses evolve by small changes ‘W076 | wa. prasora/e/4o/10.1073/pnas0s04786106 (8,9) and low trait divergence also might be predicted from the recent radiation of Jnga (13). Instead we found a weak or no correlation of phylogeny with defensive traits. These results suggest divergent selection on, and rapid evolution of, antiher- bivore defenses in dnga (40) and thus are consistent with the recognized importance of the interactions between hosts and natural enemies in driving diversifying evolution in both plants and animals (43 We also argue that divergent defenses could be a principal ‘mechanism structuring community assembly. Most species of [Inga are restricted to similar habitats and may vary little in resource use: they also have similar flower and fruit morphol- ‘ogies (12), thus suggesting similar pollinator and dispersal syndromes. Therefore, the high number of species of Inga, up to 43, that coexist ata single site (15) presents an enigma, We found that, regardless of whether the co-occurring species of Inga are less’ related (Panama) or more closely related (Peru) than expected by chance, the assemblage of Inga ata single ste differs ‘more in defense strategy than random. In addition, most lepi- 1) by using analyses of Slomiberg's (37). We evaluated '2 posible evolutionary relationship between chemical defense strategy and these axes by using a Mantel test relating chemical stance to differences betoveen species along the fst two aes We evaluated the distance between speciesin defense waltsas the average ‘of distances forthe different chemical dase and for dlstances between ‘Species in ekcapeldetense (developmental) strategy and ant visitation rater ‘We standerdized each chemical or other defense clas or axis to vary from 0 and such that each was weighted equally inthe otal defense wat analyses ‘We evaluated the distance betveen species in leaf traits (wings and leaflet number) arthe average distance between speclerslang wo axerdarved rm 2 principal component anaysis ofthe lea alts All analyses were conducted inthe Rstatseal environment (R Core Development Tear 2008), and etal. ‘an be found ins Text ‘ACKNOWLEDGMENTS. We thank E. Leigh fr stimulating discussions and 0. ‘Acevedo and B Jimenez for moral and fogstial support atthe Smithsonian ‘Tropial Research institute, K: Rudolph, & Bromberg, D.Dvoret 5. Rng, Wee, and Mi. pps fo eld assstancein Panema W Pima fr intelectual Input and. Soldano, C. Lazo, ond Para for ele workin Peru officals rom ‘Autoridad Nacional del Ambiente of Panama and instuto Nacional de Re {uss Natufles of Peru for permision to Conduct field werk 8. Le and D. ‘Grapov for eistance wth the chemical characterization af the Unversity of Utahy officials a the Center for Tropial Forest Science ofthe Smithsonian ‘Tropical Reseoren Insitute for use ofthe data from the 5O-ha plot (Nps! iiss edutdatasetvb) and M. Lavin, M, Lemes, M. Hollingsworth and A. ‘ark for phylogenetic reearen, KD. was supported by National scence Foundation Grant OD1G-0608365 and grants rom the Amazon Conservation ‘Astociation, American Philosophical society, Duke Unversty, Explorers lub, Society for$ystematc Biology andSigna XiT.AK and? 0.C weresupported by National Scince Foundation Grants DEB.0232830 and DEB-0680650 and Research experience for Undergradatesupplements RTP. wassupperted by Leverhulme Trust Study Abad Fellowship 7220080182. 1. Valeri Baie ito GP (196 high te a dvenity in Amazonian Keune 15, Verein (2008 n Tropical Foret Obert and Oynamin: Fins fom 2 Larges lot Neer at ann EC Leigh Ei Chieaga Prem, Ca ob 16, Hubbel Foster 8B Conde, ao Peter (1985) Demographic Tre Data fom the Soha ar Clare lana Forest Dyramis Po 1982-195(Smehsonan suo, Washington, 0, TF Forest Dynami Plt Osta Sees D-4OM. 17. 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