Macroevolutionary patterns of defenseand pollination in Dalechampia vines:Adaptation, exaptation, and evolutionary noveltyW. Scott Armbrustera,b,c,1, Joongku Leed,2, and Bruce G. Baldwind

aSchool of Biological Sciences, University of Portsmouth, Portsmouth PO1 2DY, United Kingdom; bInstitute of Arctic Biology, University of Alaska, Fairbanks,AK 99775-7000; cDepartment of Biology, Norwegian University of Science and Technology, NO-7491 Trondheim, Norway; and dDepartment of IntegrativeBiology and Jepson Herbarium, University of California, Berkeley, CA 94720-2465

Edited by Anurag A. Agrawal, Cornell University, Ithaca, NY, and accepted by the Editorial Board August 29, 2009 (received for review June 25, 2009)

We conducted phylogenetically informed comparative analyses of81 taxa of Dalechampia (Euphorbiaceae) vines and shrubs to assessthe roles of historical contingency and trait interaction in theevolution of plant-defense and pollinator-attraction systems. Weasked whether defenses can originate by exaptation from preex-isting pollinator attractants, or vice versa, whether plant defensesshow escalation, and if so, whether by enhancing one line ofdefense or by adding new lines of defense. Two major patternsemerged: (i) correlated evolution of several complementary lines ofdefense of flowers, seeds, and leaves, and (ii) 5 to 6 losses of theresin reward, followed by redeployment of resin for defense ofmale flowers in 3 to 4 lineages, apparently in response to herbi-vore-mediated selection for defense of staminate flowers uponrelaxation of pollinator-mediated selection on resin. In all cases,redeployment of resin involved reversion to the inferred ancestralarrangement of flowers and resiniferous bractlets. Triterpene resinhas also been deployed for defense of leaves and developingseeds. Other unique defenses against florivores include nocturnalclosure of large involucral bracts around receptive flowers andpermanent closure around developing fruits (until opening againupon dehiscence). Escalation in one major clade occurred throughan early dramatic increase in the number of lines of defense and inthe other major clade by more limited increases throughout thegroup’s evolution. We conclude that preaptations played impor-tant roles in the evolution of unique defense and attractionsystems, and that the evolution of interactions with herbivores canbe influenced by adaptations for pollination, and vice versa.

floral resin � florivory � plant defense � resin defense

I t is accepted that most plant species experience a variety ofantagonistic and mutualistic relationships with animals simul-

taneously or sequentially over the course of their lives. To date,however, most research has focused on only 1 type of interactionat a time (e.g., herbivory ignoring pollination or pollinationignoring herbivory) (1). Less frequently considered are theinteractions between these partnerships (e.g., how pollinationand herbivory might interact evolutionarily).

Interactive effects of herbivory and pollination on plantreproductive success have been detected in a number of micro-evolutionary studies; these reveal surprisingly strong effects andcomplex, sometime counterintuitive, responses (2, 3). For ex-ample, the evolution of flowers may be influenced by selectiongenerated as much by nonpollinating agents as by pollinators(4–6; but see ref. 7), in contrast to traditional expectations thatpollinators alone drive floral evolution (8). Research on inter-actions between various plant–animal relationships has naturallyfocused on ecology (e.g., the immediate growth and/or repro-ductive outcomes of complex interactions). Although an evolu-tionary perspective underlies these studies, few studies haveexplicitly considered the long-term evolutionary dynamics ofplant interactions with multiple partners (but see articles citedbelow).

One advantage of taking a long-term approach to evolutionaryanalysis is that it sometimes permits detection of the causes ofevolutionary novelty, such as invasion of new adaptive zones (9)or escape from enemies through novel defenses, which can leadto subsequent evolutionary radiation (10, 11). An explicitlyhistorical approach also allows evaluation of the role of historicalcontingency in evolutionary change (12)—for example, phylo-genetic lag, genetic constraint, and preaptations (13–15). Phy-logeny-based approaches allow tests of associations betweentraits or partnerships (16, 17) and whether particular traits orrelationships influence the evolution of others (18).

Historical contingency is implicit in Ehrlich and Raven’sescape-and-radiate hypothesis of plant–herbivore coevolution(10) and defensive escalation (11). Other historically contingentevolutionary scenarios include consistent sequences of traitchange (‘‘ordered change’’) (18); exaptation (13) (e.g., cooptingpreexisting compounds for new defense or reward functions);and evolutionary ‘‘novelty’’ through regulatory gene–based traitreversals (‘‘atavisms’’) (19–21). Previous macroevolutionarystudies of plant–herbivore interactions have shown patternsof escalation and decline in the intensity and effectiveness ofdifferent defense systems (22–24) and specific sequences ofevolutionary change in plant defense systems (25, 26).

Macroevolutionary studies of the interactions between plantdefense and attraction systems are few, although the importanceof this link has long been recognized. In considering pollinationof primitive angiosperms, Pellmyr and Thien (27) hypothesizedthat the secretion of essential oils by flowers originated asdefense in response to selection generated by herbivores and/orpathogens. These mostly toxic compounds now play roles inadvertisement and attract pollinators. The origin of floral ad-vertisements by exaptation [sensu Gould and Vrba (13) andArnold (28)] has been invoked as a key innovation in angiospermevolution (27). It seems likely that many of these compoundstoday play dual roles in attraction and defense [see also Lev-Yadun (29)], that is, are ‘‘addition exaptations,’’ whereby a newfunction is added to, rather than replaces, the prior function (13,28). Later studies based on phylogenetic approaches have alsosuggested that protection or defense functions often precede theattractive functions of biosynthetic products (4, 7, 30). Forexample, previous work suggested that the resin-reward systemseen in Dalechampia vines and shrubs (Euphorbiaceae) and

Author contributions: W.S.A. designed research; W.S.A., J.L., and B.G.B. performed re-search; W.S.A. and B.G.B. analyzed data; and W.S.A. and B.G.B. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. A.A.A. is a guest editor invited by the EditorialBoard.

1To whom correspondence should be addressed. E-mail: scott.armbruster@port.ac.uk.

2Present address: Korea Research Institute of Bioscience and Biotechnology, #52 Eoeun-dong, Yuseong-gu, Daejeon 305–333, South Korea.

This article contains supporting information online at www.pnas.org/cgi/content/full/0907051106/DCSupplemental.

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Clusia trees (Clusiaceae) evolved by exaptation, whereby resinsecretion originated for defense and secondarily took on anattractive function (31). Armbruster et al. (32) provided someexperimental support for this hypothesis. Working in the samesystem, Armbruster (33) also hypothesized that some antiher-bivore defenses in Dalechampia vines and shrubs had theirorigins in pollinator attraction, although these conclusions wererestricted to neotropical species and based on a morphologicphylogeny slightly at odds with our current knowledge (cf. 34).

Here we use new molecular–phylogenetic, morphologic, andchemical data from a worldwide sample of 81 neo- and paleo-tropical taxa of Dalechampia (Euphorbiaceae) to address thefollowing questions: (i) Have some defense systems originated byexaptation from pollinator attractants, and some attractantsoriginated from defense? (ii) Has selection for greater defenseled to broad-sense escalation (22–24); if so, has this been throughrefinement of single lines of defense or, instead, by addition ofnew lines of defense? More specifically, we ask: (iii) Have newfloral defense systems evolved subsequent to resin being rede-ployed as a pollinator reward rather than floral defense (transferexaptation; 28, 33)? (iv) What happens when resin ceases to bea reward; is resin ever used again in floral defense; if so, how doesthis come about? (v) Have floral attraction and defense systemsinfluenced the evolution of foliar defense systems?

Dalechampia comprises �120 species of monoecious twiningvines and (rarely) shrubs, occurring throughout most of the neo-and paleotropics, west of Wallace’s Line. Unisexual f lowers aresecondarily united into functionally bisexual, blossom inflores-cences, usually subtended by 2 large, showy involucral bracts(Fig. 1), and are pollinated mostly by resin-collecting megachilid(Hymenoptera: Megachilidae) or euglossine (Apidae) bees (35).Approximately 10 species in the neotropics are pollinated byfragrance-collecting male euglossine bees (35), and approxi-mately 10 species in Madagascar and 2 in the neotropics arepollinated by pollen-feeding beetles and/or pollen-collectingbees (34). The foliage of neotropical species is fed upon by thelarvae of specialist nymphalid butterflies (primarily Hamadryasand Ectima but also Catonephele, Myscelia, Mestra, and Biblis; ref.36), as well as generalists, such as leaf-cutting ants, Atta (Hy-menoptera: Formicidae). Foliage of paleotropical species iseaten by the larvae of Byblia (Nymphalidae). Floral parts are fed

upon by specialist butterfly larvae, Dynamine spp. in the neo-tropics and Neptidopsis in the paleotropics, as well as generalists,such as tettigoniid grasshoppers/katydids (Orthoptera: Tettigo-niidae), especially at night (36). Rewards produced for pollina-tors include pollen (34), oxygenated triterpenes secreted by acondensed resin gland associated with the staminate subinflo-rescence (31, 32), and monoterpene fragrances secreted by eitherthe stigmatic surface of the pistillate flowers (37) or a glandhomologous with the resin gland (38, 39).

ResultsBayesian posterior probabilities and maximum-parsimony boot-strap values from phylogenetic analyses of chloroplast DNA(cpDNA), internal transcribed spacer (ITS), and external tran-scribed spacer (ETS) sequence data provided strong support fornumerous species groups and 2 major clades: species with 4branches in the male subinflorescence (‘‘4-armed clade’’) andspecies with 5 branches (‘‘5-armed clade’’; Fig. 2 and supportinginformation Fig. S1). Maximum-likelihood optimization of traitsonto the ultrametric Bayesian trees resampled from 22,500retained ITS trees of the posterior distribution indicated 1 to 2origins of stinging crystalliferous trichomes on vegetative parts(depending on whether Tragia and Dalechampia share this traitby common descent; see Fig. 2, first line of defense). Optimi-zation also indicated that secretion of triterpene resin by floralstructures originated once, sometime after the divergence ofDalechampia from Tragia. We cannot ascertain whether resinplayed an initial role in defense of staminate flowers or inrewarding pollinators, although the former seems more reason-able. Resin ‘‘immediately’’ took on the latter function (i.e., nodescendants of the inferred basal state exist), however, and thisfunction persists throughout most of the genus today. One lineof defense based on a resin chemically similar to the rewardoriginated early in the evolution of Dalechampia (but after theshift from resin defense of flowers to resin reward for pollina-tors): deployment of resin by sepals in defense of developingfruits [2 to 3 origins inferred from maximum-likelihood (ML)optimization; Table 1]. A second use of the same resin evolvedmuch later: deployment of resin by stipules, leaves, and involu-cral bracts (one origin: Dalechampia stipulacea and relatives). Inone lineage, secretion of resin from pistillate sepals is augmentedby deployment of barbed, detaching, ‘‘glochidial’’ spines on thepistillate sepals. Although the order of trait gain cannot bedetected [Discrete Ordered-Change test, likelihood ratio (LR) �0.65 � 0.07, P � 0.5; Table S1], resin glands and glochidia on thesepals show strong evidence of correlated evolution (DiscreteOmnibus test, LR � 19.62 � 0.15, P � 0.001; Table S1).Deployment of defensive spines and resin glands on pistillatesepals (Fig. 1B) evolved in concert with sepal size (LR � 19.62 �0.15, P � 0.001 and LR � 8.29 � 0.08, P � 0.08, respectively;Table S1), usually originating after the sepals were large enoughto envelop the ovaries and developing fruits (but LR � 0.65 �0.07, P � 0.50 in ordered-change test; Table S1). The combina-tion of inflated bracts enclosing a mass of sharp, barbed,detachable spines seems to be a particularly effective defensecombination (at least against humans), protecting developingseeds and fruits (presumably against granivorous mammals andbirds, rather than botanists), and there is evidence of correlatedevolution of these 2 traits (LR � 13.82 � 1.21, P � 0.01; TableS1).

In 3 of 5 lineages, loss of the resin-reward function, related toshifts to pollination by fragrance- or pollen-collecting insects, wasclosely associated with redeployment of the resiniferous staminatebractlets in a fashion that promotes defense of the staminateflowers (Fig. 3 and Fig. S2). The evidence for correlated evolutionof reward function (loss of resin-bee pollination), and bractlet/flower arrangement (defensive redeployment) was highly signifi-cant (LR � 21.04 � 1.16, P � 0.001), although there was no

Fig. 1. Blossom inflorescences (pseudanthia) in flower and fruit. (A) Recep-tive pseudanthium of D. stipulacea, a species that is pollinated by resin-collecting euglossine bees. Pseudanthia have resin glands (yellow arrow)formed by asymmetrical clusters of resiniferous staminate bractlets. The floralresin gland secretes a mixture of oxygenated triterpene ketones and alcohols(32). This species and its relatives also secrete the same oxygenated triterpenealcohols from capitate glands along the margins of stipules (green arrow),leaves, and involucral bracts (white arrow). (B) Capsular fruits of D. scandens,showing capitate glands (white arrow) on pistillate sepals, which secreteoxygenated-triterpene resin. Note also the involucral bracts, which are par-tially closed around the fruit. As the fruits mature these bracts begin to open(as here) in preparation for explosive dispersal of the seeds.

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evidence for one trait changing before, or influencing evolution ofthe other (Table S1). In the 2 lineages that do not conform to thispattern (D. brownsbergensis and D. spathulata-D. magnoliifolia), thestaminate bractlets do not secrete resin and instead are vestigial orsecrete monoterpene fragrance, respectively.

Another line of defense of flowers and seeds involves noc-turnal closure of enveloping bracts; this originated after theorigin of large, showy bracts adjacent to the flowers (Fig. 1 A).In one lineage (the 5-armed clade; Fig. 2, right branch) nocturnalclosure originated early in the lineage’s diversification, withvirtually all extant species exhibiting this feature. In the 4-armedclade (Fig. 2, left branch), nocturnal closure originated severaltimes rather late, such that approximately half the extant specieshave large bracts that do not close at night.

Examination of the sequence of changes in all defensive traitsacross the phylogeny of Dalechampia reveals a general pattern ofearly escalation in defensive systems in the 5-armed clade (1 lineof defense increasing to approximately 7), followed by a numberof minor reversals (deescalation: 7 lines of defense decreasing to6 or 5; Fig. 2). In the 4-armed clade, in contrast, escalation ismuch more limited, with apparently less deescalation (Fig. 2).Analysis of the number of lines of defense with BayesTraits-Continuous revealed a strong phylogenetic signal in the evolu-tion of defense systems and a tendency for defensive traits toevolve more slowly on long branches than on short branches andto show early radiation rather than later species-specific adap-tation (Table 2). Over most of the evolutionary history of thegroup, defense systems have accumulated, being added morerapidly than lost (Fig. 2). Due to the repeated, small-scalereversals in the overall trend, however, the Continuous test for

general evolutionary trends (escalation, in this case) was notsignificant (Table 2).

Species of Dalechampia show a distinctly nonnormal distribu-tion with respect to the number of lines of defense: species tendto cluster at the upper or (secondarily) lower end of thedistribution (Fig. S3). There thus seem to be 2 syndromes: (i)highly defended species, which are also largely ‘‘pioneer’’ speciesof secondary scrub (35), and (ii) poorly defended species, whichare largely late-succession species of forests. Members of these2 syndromes are, however, largely associated with differentclades. Highly defended species all belong to the 5-armed clade(right branch, Fig. 2), and most poorly defended species belongto the 4-armed clade (left branch, Fig. 2). Thus, the apparentassociation between habitat and defense may reflect sharedphylogenetic history instead of convergence. Nevertheless, phylo-genetically controlled analysis with BayesTraits-Continuousshowed a significant (although weak) association between defenseand habitat (R2 � 0.05, LR � 5.37 � 0.48, P � 0.05).

To assess whether innovations in defense or pollination hadeffects on rates of speciation, extinction, or net diversification,we estimated these parameters with BiSSE (see Materials andMethods). We could not, however, detect heterogeneity inspeciation, extinction, or net diversification rates associated withany of the various innovations and changes in defense andpollination characters.

Five of seven evolutionary innovations in defense seem to havebeen the result of exaptation (origin of a trait by a change infunction of a preexisting trait; Table 1). Changes to new func-tions were followed by further adaptive ‘‘fine tuning.’’ Initialfunctions were usually related to attracting pollinators. One

Fig. 2. Escalatory and deescalatory evolution of lines of defense in Dalechampia, treating defense as a single ordinal trait (number of lines of defense). Thetree is a typical representative of the 30,000 post–burn-in, nonultrametric Bayesian trees estimated from analysis of ITS sequence data, with branch lengthsproportional to ITS divergence. The number of lines of defense was optimized across 100 trees sampled regularly from the posterior distribution, using MPRs(proportional most-parsimonious reconstruction tracing), ordered-trait evolution, and the map-across-all-trees options in Mesquite (63). The cross-sectionalproportion of colors on a branch indicates the joint proportion of trees and maximally parsimonious reconstructions with that ancestor state at the node. Thewidth of the red shading indicates the proportion of trees lacking that node. Scale on right indicates branch length.

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innovation in pollination probably drew on a preexisting defensefeature: secretion of resin in blossoms (Table 1).

DiscussionThe evolutionary histories of plant-defense and pollinator-attraction systems in Dalechampia of both the neo- and paleo-tropics have been intertwined, linked by the mechanical andchemical commonalities of plant–animal interactions. There arerepeated examples of features originating apparently in responseto selection generated by one function (pollination or defense)and later being coopted for the other, often several timesindependently. This underscores the historical contingency of

the evolution of plant–animal interactions and the importance ofphysical and chemical links between plants and insects.

The most puzzling feature of Dalechampia evolution revealedby the molecular phylogeny is repeated reversal in full detail tothe inferred ancestral arrangement of the staminate flowers andresiniferous bractlets (Fig. 3 and Fig. S2). This may be explainedby strong selection for protection of pollen (genomic copies)against f lorivores. The inferred ancestral arrangement of stami-nate flowers and bractlets (based on comparisons with candidateoutgroups) was diffuse deployment of bractlets around thestaminate flowers arranged in 4 to 5 fertile branches (Fig. 3). Inthis arrangement, male floral buds were protected by a layer ofresin. After resin became a reward for pollinators, the resin-secreting bractlets became rearranged into a gland-like struc-ture. The staminate flowers were then located below the gland,near the pistillate flowers, creating bilateral symmetry, enhanc-ing the consistent positioning of the pollinators and thus floralprecision and accuracy (Fig. 3; refs. 40–42). However, thisresulted in resin no longer protecting the staminate flowers frompollen-feeding insects (� ‘‘transfer exaptation,’’ Table 1; refs. 28,33). Resin was later replaced by pollen or fragrance as thepollinator reward 5 to 6 times; in 3 to 4 of these, the resiniferousbractlets were ‘‘immediately’’ (intermediates not extant) rede-ployed in the diffuse fashion that protects the staminate flowers.These reversals suggest that florivores that consume male ga-metes generate strong selection and that the easiest line ofevolutionary response was to reactivate preexisting developmen-tal information for the arrangement of the bractlets, informationthat was still in the genome but suppressed by regulatory genes.Support for this hypothesis derives from the fact that theseapparent atavisms have occurred in identical detail indepen-dently 3 to 4 times on both deep and shallow branches in bothSouth America and Madagascar. Additionally, the reversal in-cludes rearrangement of the male flowers to the inferred an-

Table 1. Novel defense and attraction systems in Dalechampia (/Tragia), with inferred modes of origin

Defense/pollination trait Current function Type of novelty Source/processBasis of inference for

function

Diffusely deployed resiniferousbractlets envelopingstaminate flowers

Defense of male flowers andpollen from florivores

(i) Adaptation (inferred in ancestor);(ii) exaptation due to preexistingbractlets attracting pollinators andgenetic information in extant sppfor distributed arrangement(atavistic reversal)

In (ii), regulatory gene changeleading to reexpression of�suppressed� genetic information(atavistic reversal)?

Experimental data (32)

Stinging crystalliferoustrichomes that injecthistamines

Defense of leaves frommammals? (and some insects?)

De novo adaptation Unknown Effect on humans

Nocturnal closure of involucralbracts

Protection of flower and pollen(from nocturnal insectherbivores)

Addition exaptation Secondary adaptive modificationof large, showy bracts

Experimental data (36)

Closed bracts in bud Protection of flower buds (frominsects)

Addition exaptation Secondary adaptive modificationof large, showy bracts

Extrapolation fromArmbruster and Mziray(36)

Closed bracts in fruit Protection of developing seedsand fruits (from insects)

Addition exaptation Secondary adaptive modificationof large, showy bracts

Unpublished experimentaldata

Resin secretion by pistillatesepals

Protection of ovules anddeveloping seeds (from insects)

Addition exaptation Expression of preexisting resinbiosynthetic system in newtissues

Experimental data (32)

Detaching glochidial spines onpistillate sepals

Protection of seeds beforedispersal (primarily from birdsand mammals?)

De novo adaptation Unknown Effect on humans

Resin secretion by stipules,leaves, and bracts

Protection of vegetative parts(from Atta ants and otherherbivores)

Addition exaptation Expression of preexisting resinbiosynthetic system in newtissues

Experimental data (32)

Resin reward Attraction and reward ofpollinating bees

Transfer exaptation Exaptation from resin defense(?) Comparison with outgroup,studies of other plantgroups, field observations

Showy involucral bracts Advertisement to pollinatingbees

Adaptation or transfer exaptation(defined broadly)

Adaptive modification of leaves Experimental data (40)

Fig. 3. Repeated reorganization of staminate flowers and resiniferousbractlets in the male subinflorescence (cymule) of Dalechampia (4-armedclade depicted), associated with (i) the inferred shift from defense of maleflowers with resin to attraction of pollinators with resin (left arrow), and (ii)the shift from pollination by resin-collecting bees to pollination by other kindsof insects and redeployment of bractlets in a defensive arrangement (rightarrow). The redeployment of flowers in the putative ancestral arrangementargues for the role of regulatory-gene changes in both transitions. Circlesrepresent staminate flowers and curved lines the resiniferous bractlets. Theorientation of the diagram is the same as the orientation of the inflorescencein nature (i.e., the top of the diagram is the top the inflorescence) (Fig. 1).

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cestral decussate position, even though there is no obviousadaptive reason for this to occur.

The 2 exceptions to this trend can be explained by the fact thatthe bractlets lost their ability to secrete resin in the process ofadapting to new pollinators, and as such were not influenced byselection for greater protection of the staminate flowers. In thelineage containing D. spathulata, the bractlets instead secretemonoterpene fragrances and remain asymmetrically clustered,orienting the pollinating bees as does the resin gland (38, 39).Hence, these species have not been ‘‘released’’ from pollinator-mediated selection against diffuse deployment. In the otherexceptional lineage (D. brownsbergensis), the resin gland isvestigial and nonsecretory (37).

Defensive escalation in Dalechampia has occurred throughincreases in the number of lines of defense rather than refine-ment of a single line. The 4-armed clade exhibits a pattern oflimited late escalation with few reversals. In contrast, the5-armed clade exhibits dramatic early escalation followed bynumerous minor losses or gains of defense systems much later(Fig. 2). One small clade, D. stipulacea and relatives, exhibits allbut 1 line of defense ‘‘invented’’ by Dalechampia over its entireevolutionary history, illustrating the tendency for additive ac-cumulation rather than substitution of lines of defense.

Anther difference between the 4-armed and 5-armed clades isthat the latter species generally have higher levels of defense(Fig. 2) and tend to occupy secondary habitats. This would seemat first to contradict classic ideas (e.g., ref. 43) about fugitivespecies being less defended. However, it is possible that theleaves of late-succession species in the 4-armed clade are de-fended by structural carbohydrates and tannins or other pheno-lics (not measured), and a survey of these compounds in thefuture would be valuable.

In conclusion, a broad overview of this group’s evolutionaryhistory yields evidence of historically contingent evolution,including exaptation and the recurrence of developmental ata-visms. It also suggests that exaptation and atavisms have playedimportant roles in morphologic and chemical evolution of bothdefense and attraction systems.

Materials and MethodsPhylogenetic Analysis. A molecular tree based on concatenated cpDNA (3� trnKintrons/partial matK) and 18S–26S nuclear ribosomal ETS and ITS sequenceswas generated from single individuals of 81 taxa (88 populations) of Da-lechampia and 2 closely related outgroup species in Plukenetia and Tragiausing Bayesian inference (Table S2). Although taxon sampling involved less

than 70% of the species, those sampled represented all major subgenericgroups and all geographic regions in the global distribution, and capturedcritical diversity in pollinator reward, blossom morphology, and defensewithin Dalechampia (30–42, 44, 45). We expect that the effects of incompletesampling are minimal, perhaps resulting in lower power but not bias or type1 error. DNA extractions, PCR, and sequencing followed standard procedures(46–49), with design of an internal primer for ETS amplification and sequenc-ing (5�-caa ctg ctc tta ggg gtt gct gtt-3�). Sequences were aligned in SeaView4.0 (50) using MUSCLE (51) and adjusted manually. Four independent BayesianMarkov chain Monte Carlo (MCMC) analyses were conducted using MrBayes3.1 (52), each using 3 ‘‘heated’’ and 1 ‘‘cold’’ chain(s) and 3 data partitions (forITS, ETS, and cpDNA sequences). Best-fit evolutionary models for Bayesianphylogenetic analysis based on the Akaike information criterion in MrMod-eltest 2.3 (53) were GTR�I� for the ITS region; HKY� for the ETS, andGTR�G for cpDNA sequences. Each MCMC analysis was terminated at 10 106

generations (1 tree saved every 1,000 generations), when the average stan-dard deviation of split frequencies (SDSF) was �0.004 across runs. The first25% of generations (including all with SDSF �0.01) were discarded as burn-in.Posterior probabilities for each clade were obtained from a 50% majority-ruleconsensus of the approximately 40,000 retained trees. Nonparametric boot-strapping (10,000 replicates) of the full dataset using maximum parsimony(MP) was also implemented, using PAUP* (54), with addseq � simple, swap �TBR, and maxtrees � 1 (see ref. 55). Although the entire dataset was used toobtain the best estimate of phylogeny, only the ITS partition was used forcomparative analyses (all dependent on branch lengths), based on missing ETSand trnK data for some taxa in the dataset and evidence for relativelyclock-like evolution of ITS sequences in angiosperms (56). Unconstrainedbranch lengths were used for analyses involving Pagel’s Continuous programbecause model B of Continuous cannot be used with ultrametric trees (57–59).Ultrametric trees were used for analysis of binary traits under the assumptionthat molecular branch lengths involving neutral (or nearly neutral) substitu-tions in gene spacers or introns are most meaningful for studies of phenotypicevolution if they reflect relative time (i.e., are ultrametric). To obtain ultra-metric trees, ITS data were analyzed using BEAST v1.4 (60) under a relaxedclock (uncorrelated lognormal; ref. 61), Yule process of speciation, and best-fitmodel of sequence evolution for ITS (GTR�I�; see above), with 4 � categoriesand constrained monophyly of Dalechampia. Each of 4 independent MCMCanalyses was terminated at 30 106 generations (saving 1 tree per 4,000generations), when TRACER v1.4 (62) indicated that the effective sample sizeof the posterior distribution was �1,000 (1,183.8 to 1,876.5) across runs, witha burn-in of 25%. The 4 post–burn-in posterior distributions were combined(22,500 trees total) and resampled every 100,000 generations using LogCom-biner v1.4.8 (in BEAST package; ref. 60) in preparation for use in BayesTraits.To obtain nonultrametric trees for comparative analyses of continuous char-acters in BayesTraits, ITS data alone were analyzed using 4 independent runsof MrBayes, as in the combined-data analyses, with termination at 15 106

generations (SDSF �0.006) and a burn-in of 32.5%.

Analyses of Trait Evolution. The sequence of evolutionary change in binarydefensive and pollination traits was estimated by optimizing traits using ML

Table 2. Evolutionary analysis of the number of lines of defense seen in Dalechampia species, using BayesTraits-Continuous (57–59),showing model parameters, likelihood ratios, and interpretations of results

Parameter Mean ML estimate (� SE) Mean likelihood ratio (� SE) P (mean) Interpretation

� 1.003 � 0.004 85.94 � 2.55 (against � � 0) �0.001 Strong phylogenetic signal in data; trait varianceexplained significantly by tree topology

� 2.834 � 0.586 —� �2.801 � 0.579 —� 0.317 � 0.024 5.608 � 0.586 (against � � 0) �0.05 Stasis on longer branches, some punctuated

divergence� 0.300 � 0.019 4.838 � 0.411 (against � � 1) �0.05 Early adaptation important (adaptive radiation),

evolution of defense is not purely gradualisticEvolutionary trend,

gradual model0.329 � 0.055 (against model A) NS No detected overall directional trend for

increase in number of lines of defenseEvolutionary trend,

speciation model1.359 � 0.105 (against model A) NS No detected overall directional trend for

increase in lines of defense

Results are reported as the mean � SE seen across 100 Bayesian trees regularly resampled from approximately 40,000 Bayesian trees retained from the posteriordistribution. Branch lengths used were based on ITS data and were not constrained to ultrametric, following program restrictions (57). � is the estimated rootvalue, and � is the directional change parameter, measuring the overall trait change against total path length from the root, in model B. Because model B wasnot significantly better than model A under either gradual- or speciation-change assumption, these two statistical parameters probably have no biologicmeaning.

Armbruster et al. PNAS � October 27, 2009 � vol. 106 � no. 43 � 18089

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optimization in Mesquite (63). MP optimization was used for multistate traits(Fig. 2) and graphic display of binary-trait change (Fig. S2) but not for analysisor interpretation. Statistical analyses of correlated trait evolution, order oftrait change, and the influence of the state of one trait on the probability ofevolutionary change in the other were based on the Ominibus, Order, andContingency tests, respectively, in BayesTraits-Discrete (18, 58). BayesTraits-Continuous (57, 59) was used to assess patterns of total defense evolution,whereby number of lines of defense was treated as a continuous trait (al-though actually ordinal, with 9 states). See Agrawal et al. (24) for a clearexplanation of analyses and interpretation of parameters.

To assess whether any defense or pollination innovations had effects onrates of speciation, extinction, or net diversification, we estimated these

parameters with BiSSE (64) as implemented in Mesquite (63). However, wefound no significant effects of character state on these parameter estimates.This nonsignificant result indicates that our BayesTraits analyses were unlikelyto have been compromised by biased sampling caused by diversification orextinction rates being strongly influenced by character state (see ref. 65).

ACKNOWLEDGMENTS. We thank Bridget Wessa for help in DNA sequencing;Michael Park and Wayne Pfeiffer for analytical assistance; numerous graduateand postdoctoral students for help in the field, greenhouse, and laboratory;and David Ackerly and 3 anonymous reviewers for comments on earlier drafts.Funding was provided by National Science Foundation Grants DEB-9020265,-9318640, -9596019, and -0444745).

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