Macroevolutionary Patterns of Defense and Pollination in Dalechampia

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Macroevolutionary patterns of defense SPECIAL FEATURE and pollination in Dalechampia vines: Adaptation, exaptation, and evolutionary novelty

W. 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 Integrative Biology 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 of One advantage of taking a long-term approach to evolutionary 81 taxa of Dalechampia (Euphorbiaceae) vines and shrubs to assess analysis is that it sometimes permits detection of the causes of the roles of historical contingency and trait interaction in the evolutionary novelty, such as invasion of new adaptive zones (9) evolution of plant-defense and pollinator-attraction systems. We or escape from enemies through novel defenses, which can lead asked whether defenses can originate by exaptation from preex- to subsequent evolutionary radiation (10, 11). An explicitly isting pollinator attractants, or vice versa, whether plant defenses historical approach also allows evaluation of the role of historical show escalation, and if so, whether by enhancing one line of contingency in evolutionary change (12)—for example, phylo- defense or by adding new lines of defense. Two major patterns genetic lag, genetic constraint, and preaptations (13–15). Phy- emerged: (i) correlated evolution of several complementary lines of logeny-based approaches allow tests of associations between defense of ﬂowers, seeds, and leaves, and (ii) 5 to 6 losses of the traits or partnerships (16, 17) and whether particular traits or resin reward, followed by redeployment of resin for defense of relationships influence the evolution of others (18). male ﬂowers in 3 to 4 lineages, apparently in response to herbi- Historical contingency is implicit in Ehrlich and Raven’s

vore-mediated selection for defense of staminate flowers upon escape-and-radiate hypothesis of plant–herbivore coevolution EVOLUTION relaxation of pollinator-mediated selection on resin. In all cases, (10) and defensive escalation (11). Other historically contingent redeployment of resin involved reversion to the inferred ancestral evolutionary scenarios include consistent sequences of trait arrangement of flowers and resiniferous bractlets. Triterpene resin change (‘‘ordered change’’) (18); exaptation (13) (e.g., coopting has also been deployed for defense of leaves and developing preexisting compounds for new defense or reward functions); seeds. Other unique defenses against florivores include nocturnal and evolutionary ‘‘novelty’’ through regulatory gene–based trait closure of large involucral bracts around receptive flowers and reversals (‘‘atavisms’’) (19–21). Previous macroevolutionary permanent closure around developing fruits (until opening again studies of plant–herbivore interactions have shown patterns upon dehiscence). Escalation in one major clade occurred through of escalation and decline in the intensity and effectiveness of an early dramatic increase in the number of lines of defense and in different defense systems (22–24) and specific sequences of the other major clade by more limited increases throughout the evolutionary change in plant defense systems (25, 26). group’s evolution. We conclude that preaptations played impor- Macroevolutionary studies of the interactions between plant tant roles in the evolution of unique defense and attraction defense and attraction systems are few, although the importance systems, and that the evolution of interactions with herbivores can of this link has long been recognized. In considering pollination be influenced by adaptations for pollination, and vice versa. of primitive angiosperms, Pellmyr and Thien (27) hypothesized that the secretion of essential oils by flowers originated as floral resin ͉ florivory ͉ plant defense ͉ resin defense defense in response to selection generated by herbivores and/or pathogens. These mostly toxic compounds now play roles in t is accepted that most plant species experience a variety of advertisement and attract pollinators. The origin of floral ad- Iantagonistic and mutualistic relationships with animals simul- vertisements by exaptation [sensu Gould and Vrba (13) and taneously or sequentially over the course of their lives. To date, Arnold (28)] has been invoked as a key innovation in angiosperm however, most research has focused on only 1 type of interaction evolution (27). It seems likely that many of these compounds at a time (e.g., herbivory ignoring pollination or pollination today play dual roles in attraction and defense [see also Lev- ignoring herbivory) (1). Less frequently considered are the Yadun (29)], that is, are ‘‘addition exaptations,’’ whereby a new interactions between these partnerships (e.g., how pollination function is added to, rather than replaces, the prior function (13, and herbivory might interact evolutionarily). 28). Later studies based on phylogenetic approaches have also Interactive effects of herbivory and pollination on plant suggested that protection or defense functions often precede the reproductive success have been detected in a number of micro- attractive functions of biosynthetic products (4, 7, 30). For evolutionary studies; these reveal surprisingly strong effects and example, previous work suggested that the resin-reward system complex, sometime counterintuitive, responses (2, 3). For ex- seen in Dalechampia vines and shrubs (Euphorbiaceae) and ample, the evolution of flowers may be influenced by selection

generated as much by nonpollinating agents as by pollinators Author contributions: W.S.A. designed research; W.S.A., J.L., and B.G.B. performed re- (4–6; but see ref. 7), in contrast to traditional expectations that search; W.S.A. and B.G.B. analyzed data; and W.S.A. and B.G.B. wrote the paper. pollinators alone drive floral evolution (8). Research on inter- The authors declare no conﬂict of interest. actions between various plant–animal relationships has naturally This article is a PNAS Direct Submission. A.A.A. is a guest editor invited by the Editorial focused on ecology (e.g., the immediate growth and/or repro- Board. ductive outcomes of complex interactions). Although an evolu- 1To whom correspondence should be addressed. E-mail: [email protected]. tionary perspective underlies these studies, few studies have 2Present address: Korea Research Institute of Bioscience and Biotechnology, #52 Eoeun- explicitly considered the long-term evolutionary dynamics of dong, Yuseong-gu, Daejeon 305–333, South Korea. plant interactions with multiple partners (but see articles cited This article contains supporting information online at www.pnas.org/cgi/content/full/ below). 0907051106/DCSupplemental.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0907051106 PNAS ͉ October 27, 2009 ͉ vol. 106 ͉ no. 43 ͉ 18085–18090 Downloaded by guest on September 27, 2021 upon by specialist butterfly larvae, Dynamine spp. in the neotropics and Neptidopsis in the paleotropics, as well as generalists, such as tettigoniid grasshoppers/katydids (Orthoptera: Tettigo- niidae), especially at night (36). Rewards produced for pollinators include pollen (34), oxygenated triterpenes secreted by a condensed resin gland associated with the staminate subinflorescence (31, 32), and monoterpene fragrances secreted by either the stigmatic surface of the pistillate flowers (37) or a gland homologous with the resin gland (38, 39). Results Bayesian posterior probabilities and maximum-parsimony bootstrap values from phylogenetic analyses of chloroplast DNA (cpDNA), internal transcribed spacer (ITS), and external tran- Fig. 1. Blossom inflorescences (pseudanthia) in flower and fruit. (A) Recep- scribed spacer (ETS) sequence data provided strong support for tive pseudanthium of D. stipulacea, a species that is pollinated by resin- numerous species groups and 2 major clades: species with 4 collecting euglossine bees. Pseudanthia have resin glands (yellow arrow) branches in the male subinflorescence (‘‘4-armed clade’’) and formed by asymmetrical clusters of resiniferous staminate bractlets. The floral species with 5 branches (‘‘5-armed clade’’; Fig. 2 and supporting resin gland secretes a mixture of oxygenated triterpene ketones and alcohols information Fig. S1). Maximum-likelihood optimization of traits (32). This species and its relatives also secrete the same oxygenated triterpene alcohols from capitate glands along the margins of stipules (green arrow), onto the ultrametric Bayesian trees resampled from 22,500 leaves, and involucral bracts (white arrow). (B) Capsular fruits of D. scandens, retained ITS trees of the posterior distribution indicated 1 to 2 showing capitate glands (white arrow) on pistillate sepals, which secrete origins of stinging crystalliferous trichomes on vegetative parts oxygenated-triterpene resin. Note also the involucral bracts, which are par- (depending on whether Tragia and Dalechampia share this trait tially closed around the fruit. As the fruits mature these bracts begin to open by common descent; see Fig. 2, first line of defense). Optimi- (as here) in preparation for explosive dispersal of the seeds. zation also indicated that secretion of triterpene resin by floral structures originated once, sometime after the divergence of Dalechampia from Tragia. We cannot ascertain whether resin Clusia trees (Clusiaceae) evolved by exaptation, whereby resin played an initial role in defense of staminate flowers or in secretion originated for defense and secondarily took on an rewarding pollinators, although the former seems more reason- attractive function (31). Armbruster et al. (32) provided some able. Resin ‘‘immediately’’ took on the latter function (i.e., no experimental support for this hypothesis. Working in the same descendants of the inferred basal state exist), however, and this system, Armbruster (33) also hypothesized that some antiher- function persists throughout most of the genus today. One line bivore defenses in Dalechampia vines and shrubs had their of defense based on a resin chemically similar to the reward origins in pollinator attraction, although these conclusions were originated early in the evolution of Dalechampia (but after the restricted to neotropical species and based on a morphologic shift from resin defense of flowers to resin reward for pollina- phylogeny slightly at odds with our current knowledge (cf. 34). tors): deployment of resin by sepals in defense of developing Here we use new molecular–phylogenetic, morphologic, and fruits [2 to 3 origins inferred from maximum-likelihood (ML) chemical data from a worldwide sample of 81 neo- and paleo- optimization; Table 1]. A second use of the same resin evolved tropical taxa of Dalechampia (Euphorbiaceae) to address the much later: deployment of resin by stipules, leaves, and involu- following questions: (i) Have some defense systems originated by cral bracts (one origin: Dalechampia stipulacea and relatives). In exaptation from pollinator attractants, and some attractants one lineage, secretion of resin from pistillate sepals is augmented originated from defense? (ii) Has selection for greater defense by deployment of barbed, detaching, ‘‘glochidial’’ spines on the led to broad-sense escalation (22–24); if so, has this been through pistillate sepals. Although the order of trait gain cannot be refinement of single lines of defense or, instead, by addition of detected [Discrete Ordered-Change test, likelihood ratio (LR) ϭ new lines of defense? More specifically, we ask: (iii) Have new 0.65 Ϯ 0.07, P Ͼ 0.5; Table S1], resin glands and glochidia on the floral defense systems evolved subsequent to resin being rede- sepals show strong evidence of correlated evolution (Discrete ployed as a pollinator reward rather than floral defense (transfer Omnibus test, LR ϭ 19.62 Ϯ 0.15, P Ͻ 0.001; Table S1). exaptation; 28, 33)? (iv) What happens when resin ceases to be Deployment of defensive spines and resin glands on pistillate a reward; is resin ever used again in floral defense; if so, how does sepals (Fig. 1B) evolved in concert with sepal size (LR ϭ 19.62 Ϯ this come about? (v) Have floral attraction and defense systems 0.15, P Ͻ 0.001 and LR ϭ 8.29 Ϯ 0.08, P ϭ 0.08, respectively; influenced the evolution of foliar defense systems? Table S1), usually originating after the sepals were large enough Dalechampia comprises Ϸ120 species of monoecious twining to envelop the ovaries and developing fruits (but LR ϭ 0.65 Ϯ vines and (rarely) shrubs, occurring throughout most of the neo- 0.07, P Ͼ 0.50 in ordered-change test; Table S1). The combina- and paleotropics, west of Wallace’s Line. Unisexual flowers are tion of inflated bracts enclosing a mass of sharp, barbed, secondarily united into functionally bisexual, blossom inflores- detachable spines seems to be a particularly effective defense cences, usually subtended by 2 large, showy involucral bracts combination (at least against humans), protecting developing (Fig. 1), and are pollinated mostly by resin-collecting megachilid seeds and fruits (presumably against granivorous mammals and (Hymenoptera: Megachilidae) or euglossine (Apidae) bees (35). birds, rather than botanists), and there is evidence of correlated Approximately 10 species in the neotropics are pollinated by evolution of these 2 traits (LR ϭ 13.82 Ϯ 1.21, P Ͻ 0.01; Table fragrance-collecting male euglossine bees (35), and approxi- S1). mately 10 species in Madagascar and 2 in the neotropics are In 3 of 5 lineages, loss of the resin-reward function, related to pollinated by pollen-feeding beetles and/or pollen-collecting shifts to pollination by fragrance- or pollen-collecting insects, was bees (34). The foliage of neotropical species is fed upon by the closely associated with redeployment of the resiniferous staminate larvae of specialist nymphalid butterflies (primarily Hamadryas bractlets in a fashion that promotes defense of the staminate and Ectima but also Catonephele, Myscelia, Mestra, and Biblis; ref. flowers (Fig. 3 and Fig. S2). The evidence for correlated evolution 36), as well as generalists, such as leaf-cutting ants, Atta (Hy- of reward function (loss of resin-bee pollination), and bractlet/ menoptera: Formicidae). Foliage of paleotropical species is flower arrangement (defensive redeployment) was highly signifi- eaten by the larvae of Byblia (Nymphalidae). Floral parts are fed cant (LR ϭ 21.04 Ϯ 1.16, P Ͻ 0.001), although there was no

18086 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0907051106 Armbruster et al. Downloaded by guest on September 27, 2021 SPECIAL FEATURE EVOLUTION

Fig. 2. Escalatory and deescalatory evolution of lines of defense in Dalechampia, treating defense as a single ordinal trait (number of lines of defense). The tree is a typical representative of the 30,000 post–burn-in, nonultrametric Bayesian trees estimated from analysis of ITS sequence data, with branch lengths proportional 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-sectional proportion of colors on a branch indicates the joint proportion of trees and maximally parsimonious reconstructions with that ancestor state at the node. The width of the red shading indicates the proportion of trees lacking that node. Scale on right indicates branch length.

evidence for one trait changing before, or influencing evolution of general evolutionary trends (escalation, in this case) was not the other (Table S1). In the 2 lineages that do not conform to this significant (Table 2). pattern (D. brownsbergensis and D. spathulata-D. magnoliifolia), the Species of Dalechampia show a distinctly nonnormal distribu- staminate bractlets do not secrete resin and instead are vestigial or tion with respect to the number of lines of defense: species tend secrete monoterpene fragrance, respectively. to cluster at the upper or (secondarily) lower end of the Another line of defense of flowers and seeds involves noc- distribution (Fig. S3). There thus seem to be 2 syndromes: (i) turnal closure of enveloping bracts; this originated after the highly defended species, which are also largely ‘‘pioneer’’ species origin of large, showy bracts adjacent to the flowers (Fig. 1A). of secondary scrub (35), and (ii) poorly defended species, which In one lineage (the 5-armed clade; Fig. 2, right branch) nocturnal are largely late-succession species of forests. Members of these closure originated early in the lineage’s diversification, with 2 syndromes are, however, largely associated with different virtually all extant species exhibiting this feature. In the 4-armed clades. Highly defended species all belong to the 5-armed clade clade (Fig. 2, left branch), nocturnal closure originated several (right branch, Fig. 2), and most poorly defended species belong times rather late, such that approximately half the extant species to the 4-armed clade (left branch, Fig. 2). Thus, the apparent have large bracts that do not close at night. association between habitat and defense may reflect shared Examination of the sequence of changes in all defensive traits phylogenetic history instead of convergence. Nevertheless, phylo- across the phylogeny of Dalechampia reveals a general pattern of genetically controlled analysis with BayesTraits-Continuous early escalation in defensive systems in the 5-armed clade (1 line showed a significant (although weak) association between defense of defense increasing to approximately 7), followed by a number and habitat (R2 ϭ 0.05, LR ϭ 5.37 Ϯ 0.48, P Ͻ 0.05). of minor reversals (deescalation: 7 lines of defense decreasing to To assess whether innovations in defense or pollination had 6 or 5; Fig. 2). In the 4-armed clade, in contrast, escalation is effects on rates of speciation, extinction, or net diversification, much more limited, with apparently less deescalation (Fig. 2). we estimated these parameters with BiSSE (see Materials and Analysis of the number of lines of defense with BayesTraits- Methods). We could not, however, detect heterogeneity in Continuous revealed a strong phylogenetic signal in the evolu- speciation, extinction, or net diversification rates associated with tion of defense systems and a tendency for defensive traits to any of the various innovations and changes in defense and evolve more slowly on long branches than on short branches and pollination characters. to show early radiation rather than later species-specific adap- Five of seven evolutionary innovations in defense seem to have tation (Table 2). Over most of the evolutionary history of the been the result of exaptation (origin of a trait by a change in group, defense systems have accumulated, being added more function of a preexisting trait; Table 1). Changes to new func- rapidly than lost (Fig. 2). Due to the repeated, small-scale tions were followed by further adaptive ‘‘fine tuning.’’ Initial reversals in the overall trend, however, the Continuous test for functions were usually related to attracting pollinators. One

Armbruster et al. PNAS ͉ October 27, 2009 ͉ vol. 106 ͉ no. 43 ͉ 18087 Downloaded by guest on September 27, 2021 Table 1. Novel defense and attraction systems in Dalechampia (/Tragia), with inferred modes of origin

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

Diffusely deployed resiniferous Defense of male flowers and (i) Adaptation (inferred in ancestor); In (ii), regulatory gene change Experimental data (32) bractlets enveloping pollen from florivores (ii) exaptation due to preexisting leading to reexpression of staminate flowers bractlets attracting pollinators and ЉsuppressedЉ genetic information genetic information in extant spp (atavistic reversal)? for distributed arrangement (atavistic reversal) Stinging crystalliferous Defense of leaves from De novo adaptation Unknown Effect on humans trichomes that inject mammals? (and some insects?) histamines Nocturnal closure of involucral Protection of flower and pollen Addition exaptation Secondary adaptive modification Experimental data (36) bracts (from nocturnal insect of large, showy bracts herbivores) Closed bracts in bud Protection of flower buds (from Addition exaptation Secondary adaptive modification Extrapolation from insects) of large, showy bracts Armbruster and Mziray (36) Closed bracts in fruit Protection of developing seeds Addition exaptation Secondary adaptive modification Unpublished experimental and fruits (from insects) of large, showy bracts data Resin secretion by pistillate Protection of ovules and Addition exaptation Expression of preexisting resin Experimental data (32) sepals developing seeds (from insects) biosynthetic system in new tissues Detaching glochidial spines on Protection of seeds before De novo adaptation Unknown Effect on humans pistillate sepals dispersal (primarily from birds and mammals?) Resin secretion by stipules, Protection of vegetative parts Addition exaptation Expression of preexisting resin Experimental data (32) leaves, and bracts (from Atta ants and other biosynthetic system in new herbivores) tissues Resin reward Attraction and reward of Transfer exaptation Exaptation from resin defense(?) Comparison with outgroup, pollinating bees studies of other plant groups, field observations Showy involucral bracts Advertisement to pollinating Adaptation or transfer exaptation Adaptive modification of leaves Experimental data (40) bees (defined broadly)

innovation in pollination probably drew on a preexisting defense the evolution of plant–animal interactions and the importance of feature: secretion of resin in blossoms (Table 1). physical and chemical links between plants and insects. The most puzzling feature of Dalechampia evolution revealed Discussion by the molecular phylogeny is repeated reversal in full detail to The evolutionary histories of plant-defense and pollinator- the inferred ancestral arrangement of the staminate flowers and attraction systems in Dalechampia of both the neo- and paleo- resiniferous bractlets (Fig. 3 and Fig. S2). This may be explained tropics have been intertwined, linked by the mechanical and by strong selection for protection of pollen (genomic copies) chemical commonalities of plant–animal interactions. There are against florivores. The inferred ancestral arrangement of stami- repeated examples of features originating apparently in response nate flowers and bractlets (based on comparisons with candidate to selection generated by one function (pollination or defense) outgroups) was diffuse deployment of bractlets around the and later being coopted for the other, often several times staminate flowers arranged in 4 to 5 fertile branches (Fig. 3). In independently. This underscores the historical contingency of this arrangement, male floral buds were protected by a layer of resin. After resin became a reward for pollinators, the resin- secreting bractlets became rearranged into a gland-like structure. The staminate flowers were then located below the gland, near the pistillate flowers, creating bilateral symmetry, enhancing the consistent positioning of the pollinators and thus floral precision and accuracy (Fig. 3; refs. 40–42). However, this resulted in resin no longer protecting the staminate flowers from pollen-feeding insects (ϭ ‘‘transfer exaptation,’’ Table 1; refs. 28, 33). Resin was later replaced by pollen or fragrance as the pollinator reward 5 to 6 times; in 3 to 4 of these, the resiniferous bractlets were ‘‘immediately’’ (intermediates not extant) rede- ployed in the diffuse fashion that protects the staminate flowers. Fig. 3. Repeated reorganization of staminate flowers and resiniferous These reversals suggest that florivores that consume male ga- bractlets in the male subinflorescence (cymule) of Dalechampia (4-armed metes generate strong selection and that the easiest line of clade depicted), associated with (i) the inferred shift from defense of male evolutionary response was to reactivate preexisting developmen- flowers with resin to attraction of pollinators with resin (left arrow), and (ii) tal information for the arrangement of the bractlets, information the shift from pollination by resin-collecting bees to pollination by other kinds that was still in the genome but suppressed by regulatory genes. of insects and redeployment of bractlets in a defensive arrangement (right Support for this hypothesis derives from the fact that these arrow). The redeployment of flowers in the putative ancestral arrangement apparent atavisms have occurred in identical detail indepen- argues for the role of regulatory-gene changes in both transitions. Circles represent staminate flowers and curved lines the resiniferous bractlets. The dently 3 to 4 times on both deep and shallow branches in both orientation of the diagram is the same as the orientation of the inflorescence South America and Madagascar. Additionally, the reversal in- in nature (i.e., the top of the diagram is the top the inflorescence) (Fig. 1). cludes rearrangement of the male flowers to the inferred an-

18088 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0907051106 Armbruster et al. Downloaded by guest on September 27, 2021 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 SPECIAL FEATURE Parameter Mean ML estimate (Ϯ SE) Mean likelihood ratio (Ϯ SE) P (mean) Interpretation

␭ 1.003 Ϯ 0.004 85.94 Ϯ 2.55 (against ␭ ϭ 0) Ͻ0.001 Strong phylogenetic signal in data; trait variance explained signiﬁcantly by tree topology ␣ 2.834 Ϯ 0.586 — ␤ Ϫ2.801 Ϯ 0.579 — ␬ 0.317 Ϯ 0.024 5.608 Ϯ 0.586 (against ␬ ϭ 0) Ͻ0.05 Stasis on longer branches, some punctuated divergence ␦ 0.300 Ϯ 0.019 4.838 Ϯ 0.411 (against ␦ ϭ 1) Ͻ0.05 Early adaptation important (adaptive radiation), evolution of defense is not purely gradualistic Evolutionary trend, 0.329 Ϯ 0.055 (against model A) NS No detected overall directional trend for gradual model increase in number of lines of defense Evolutionary trend, 1.359 Ϯ 0.105 (against model A) NS No detected overall directional trend for speciation model increase in lines of defense

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

cestral decussate position, even though there is no obvious than 70% of the species, those sampled represented all major subgeneric adaptive reason for this to occur. groups and all geographic regions in the global distribution, and captured The 2 exceptions to this trend can be explained by the fact that critical diversity in pollinator reward, blossom morphology, and defense the bractlets lost their ability to secrete resin in the process of within Dalechampia (30–42, 44, 45). We expect that the effects of incomplete sampling are minimal, perhaps resulting in lower power but not bias or type EVOLUTION adapting to new pollinators, and as such were not influenced by 1 error. DNA extractions, PCR, and sequencing followed standard procedures selection for greater protection of the staminate flowers. In the (46–49), with design of an internal primer for ETS amplification and sequenc- lineage containing D. spathulata, the bractlets instead secrete ing (5Ј-caa ctg ctc tta ggg gtt gct gtt-3Ј). Sequences were aligned in SeaView monoterpene fragrances and remain asymmetrically clustered, 4.0 (50) using MUSCLE (51) and adjusted manually. Four independent Bayesian orienting the pollinating bees as does the resin gland (38, 39). Markov chain Monte Carlo (MCMC) analyses were conducted using MrBayes Hence, these species have not been ‘‘released’’ from pollinator- 3.1 (52), each using 3 ‘‘heated’’ and 1 ‘‘cold’’ chain(s) and 3 data partitions (for mediated selection against diffuse deployment. In the other ITS, ETS, and cpDNA sequences). Best-fit evolutionary models for Bayesian exceptional lineage (D. brownsbergensis), the resin gland is phylogenetic analysis based on the Akaike information criterion in MrMod- eltest 2.3 (53) were GTRϩIϩ⌫ for the ITS region; HKYϩ⌫ for the ETS, and vestigial and nonsecretory (37). GTRϩG for cpDNA sequences. Each MCMC analysis was terminated at 10 ϫ 106 Defensive escalation in Dalechampia has occurred through generations (1 tree saved every 1,000 generations), when the average stan- increases in the number of lines of defense rather than refine- dard deviation of split frequencies (SDSF) was Ͻ0.004 across runs. The first ment of a single line. The 4-armed clade exhibits a pattern of 25% of generations (including all with SDSF Ն0.01) were discarded as burn-in. limited late escalation with few reversals. In contrast, the Posterior probabilities for each clade were obtained from a 50% majority-rule 5-armed clade exhibits dramatic early escalation followed by consensus of the approximately 40,000 retained trees. Nonparametric boot- strapping (10,000 replicates) of the full dataset using maximum parsimony numerous minor losses or gains of defense systems much later ϭ ϭ (Fig. 2). One small clade, D. stipulacea and relatives, exhibits all (MP) was also implemented, using PAUP* (54), with addseq simple, swap TBR, and maxtrees ϭ 1 (see ref. 55). Although the entire dataset was used to but 1 line of defense ‘‘invented’’ by Dalechampia over its entire obtain the best estimate of phylogeny, only the ITS partition was used for evolutionary history, illustrating the tendency for additive ac- comparative analyses (all dependent on branch lengths), based on missing ETS cumulation rather than substitution of lines of defense. and trnK data for some taxa in the dataset and evidence for relatively Anther difference between the 4-armed and 5-armed clades is clock-like evolution of ITS sequences in angiosperms (56). Unconstrained that the latter species generally have higher levels of defense branch lengths were used for analyses involving Pagel’s Continuous program (Fig. 2) and tend to occupy secondary habitats. This would seem because model B of Continuous cannot be used with ultrametric trees (57–59). at first to contradict classic ideas (e.g., ref. 43) about fugitive Ultrametric trees were used for analysis of binary traits under the assumption species being less defended. However, it is possible that the that molecular branch lengths involving neutral (or nearly neutral) substitu- tions in gene spacers or introns are most meaningful for studies of phenotypic leaves of late-succession species in the 4-armed clade are de- evolution if they reflect relative time (i.e., are ultrametric). To obtain ultra- fended by structural carbohydrates and tannins or other pheno- metric trees, ITS data were analyzed using BEAST v1.4 (60) under a relaxed lics (not measured), and a survey of these compounds in the clock (uncorrelated lognormal; ref. 61), Yule process of speciation, and best-fit future would be valuable. model of sequence evolution for ITS (GTRϩIϩ⌫; see above), with 4 ␥ categories In conclusion, a broad overview of this group’s evolutionary and constrained monophyly of Dalechampia. Each of 4 independent MCMC history yields evidence of historically contingent evolution, analyses was terminated at 30 ϫ 106 generations (saving 1 tree per 4,000 generations), when TRACER v1.4 (62) indicated that the effective sample size including exaptation and the recurrence of developmental ata- Ͼ visms. It also suggests that exaptation and atavisms have played of the posterior distribution was 1,000 (1,183.8 to 1,876.5) across runs, with a burn-in of 25%. The 4 post–burn-in posterior distributions were combined important roles in morphologic and chemical evolution of both (22,500 trees total) and resampled every 100,000 generations using LogCom- defense and attraction systems. 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- Materials and Methods acters in BayesTraits, ITS data alone were analyzed using 4 independent runs Phylogenetic Analysis. A molecular tree based on concatenated cpDNA (3Ј trnK of MrBayes, as in the combined-data analyses, with termination at 15 ϫ 106 introns/partial matK) and 18S–26S nuclear ribosomal ETS and ITS sequences generations (SDSF Ͻ0.006) and a burn-in of 32.5%. was generated from single individuals of 81 taxa (88 populations) of Da- lechampia and 2 closely related outgroup species in Plukenetia and Tragia Analyses of Trait Evolution. The sequence of evolutionary change in binary using Bayesian inference (Table S2). Although taxon sampling involved less defensive and pollination traits was estimated by optimizing traits using ML

Armbruster et al. PNAS ͉ October 27, 2009 ͉ vol. 106 ͉ no. 43 ͉ 18089 Downloaded by guest on September 27, 2021 optimization in Mesquite (63). MP optimization was used for multistate traits parameters with BiSSE (64) as implemented in Mesquite (63). However, we (Fig. 2) and graphic display of binary-trait change (Fig. S2) but not for analysis found no significant effects of character state on these parameter estimates. or interpretation. Statistical analyses of correlated trait evolution, order of This nonsignificant result indicates that our BayesTraits analyses were unlikely trait change, and the influence of the state of one trait on the probability of to have been compromised by biased sampling caused by diversification or evolutionary change in the other were based on the Ominibus, Order, and extinction rates being strongly influenced by character state (see ref. 65). Contingency tests, respectively, in BayesTraits-Discrete (18, 58). BayesTraits- Continuous (57, 59) was used to assess patterns of total defense evolution, ACKNOWLEDGMENTS. We thank Bridget Wessa for help in DNA sequencing; whereby number of lines of defense was treated as a continuous trait (al- Michael Park and Wayne Pfeiffer for analytical assistance; numerous graduate though actually ordinal, with 9 states). See Agrawal et al. (24) for a clear and postdoctoral students for help in the field, greenhouse, and laboratory; explanation of analyses and interpretation of parameters. and David Ackerly and 3 anonymous reviewers for comments on earlier drafts. To assess whether any defense or pollination innovations had effects on Funding was provided by National Science Foundation Grants DEB-9020265, rates of speciation, extinction, or net diversification, we estimated these -9318640, -9596019, and -0444745).

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