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American Journal of Botany 97(5): 782–796. 2010.

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American Journal of Botany 97(5): 782–796, 2010; http://www.amjbot.org/ © 2010 Botanical Society of America

Understanding how reproductive traits evolved in the past leads to important insights into how organisms adapted. In sex-ually reproducing organisms, traits associated with outcrossing are thought to be under strong selection because of their direct effect on reproductive success; the radiation of fl oral morpholo-gies in angiosperms represents a great example of this (e.g., Darwin, 1871 ; Lloyd and Webb, 1992 ). Specifi cally in the case of animal-pollinated plant species, pollinator preference repre-sents a strong selective pressure to fl ower traits ( Darwin, 1862 ; F æ gri and van der Pijl, 1966 ; Stebbins, 1970 ; Schemske and Bradshaw, 1999 ). Attraction of pollinators and successful pol-len transfer represent the primary targets of selection during fl ower evolution, leading to repeated evolutionary shifts be-tween pollinators and, consequently, to the diversifi cation of fl oral forms ( Darwin, 1862 ; F æ gri and van der Pijl, 1966 ; Stebbins, 1970 , 1974 ; Harder and Barrett, 2006 ).

The association between particular pollinators and specifi c fl oral traits is thought to have led to the evolution of pollination syndromes, which correspond to suites of fl oral traits that are associated with the attraction of specifi c pollinators ( Vogel, 1954 ; F æ gri and van der Pijl, 1966 ; Stebbins, 1970 ; Fenster et al., 2004 ). In this context, homoplastic evolution of similar fl ower morphologies are thought to have resulted from strong selec-tion pressures by similar pollinators ( F æ gri and van der Pijl, 1966 ). Despite this, differing opinions exist regarding the

applicability of the syndrome concept (see Herrera, 1996 ; Strauss and Whittall, 2006 ; Waser, 2006 ). Nonetheless, the rec-ognition of pollinators as functional groups defi ned by their ecological similarities is generally considered a valuable con-cept for the study of fl ower specialization to pollinators ( Fenster et al., 2004 ). Evolutionary specialization leads to shifts in fl oral morphologies that are associated with the use of a subset of pol-linators compared to those visiting the ancestral morphology ( Armbruster et al., 2000 ; Fenster et al., 2004 ). The identifi ca-tion of specifi c shifts in fl oral morphologies allows us to estab-lish when signifi cant evolutionary changes took place, as well as to test specifi c hypotheses associated with the processes that may have led to such morphological shifts ( Grant and Grant, 1968 ; Armbruster and Webster, 1982 ; Armbruster et al., 1994 ; Johnson and Steiner, 1997 ; Hansen et al., 2000 ; Fenster et al., 2004 and references therein).

Shifts among fl oral morphologies resulting from the selec-tion exerted by specifi c pollinator groups can occur in three dif-ferent ways ( Armbruster, 1993 ): (1) gradual quantitative shifts that correspond to those shifts proposed by Darwin ’ s coevolu-tionary race model (1862); (2) gradual qualitative shifts with intermediate stages among fl owers pollinated by different pol-linators; and (3) qualitative shifts without intermediate mor-phologies ( Stebbins, 1970 , 1974 ; Armbruster, 1993 ). Despite the transient nature of the intermediate phases among fl oral morphologies, these morphologies may persist in plant popula-tions when the frequencies of the effective pollinators fl uctuate ( Stebbins, 1970 , 1974 ). This condition is thought to represent the rule, rather than the exception, for shifts between pollina-tion modes ( Stebbins, 1970 ). On the other hand, drastic varia-tion in the frequencies of pollinators might lead to rapid changes in traits that determine pollinator specifi city, leading to shifts without intermediate morphologies.

Little evidence is available as to how fl oral morphologies have shifted over time, making it diffi cult to evaluate the direction

1 Manuscript received 25 June 2009; revision accepted 25 February 2010. The authors thank B. Loeuille, M. Kaehler, R. Ree, S. Branco, R.

Olmstead, S. Graham, and an anonymous reviewer for comments that greatly improved this manuscript. This paper is part of the thesis of S.A., which was supported by FAPESP (Grant 06/59916-0) and MBG (Elizabeth E. Bascom Fellowship).

2 Author for correspondence (e-mail: [email protected])

doi:10.3732/ajb.0900182

EVOLUTION OF FLORAL MORPHOLOGY AND POLLINATION SYSTEM IN BIGNONIEAE (BIGNONIACEAE) 1

Suzana Alcantara 2 and L ú cia G. Lohmann

Universidade de S ã o Paulo, IB, Departamento de Bot â nica, Cidade Universit á ria, Rua do Mat ã o 277, S ã o Paulo, SP, CEP 05508-090, Brazil

The radiation of angiosperms is associated with shifts among pollination modes that are thought to have driven the diversifi ca-tion of fl oral forms. However, the exact sequence of evolutionary events that led to such great diversity in fl oral traits is unknown for most plant groups. Here, we characterize the patterns of evolution of individual fl oral traits and overall fl oral morphologies in the tribe Bignonieae (Bignoniaceae). We identifi ed 12 discrete traits that are associated with seven fl oral types previously de-scribed for the group and used a penalized likelihood tree of the tribe to reconstruct the ancestral states of those traits at all nodes of the phylogeny of Bignonieae. In addition, evolutionary correlations among traits were conducted using a maximum likelihood approach to test whether the evolution of individual fl oral traits followed the correlated patterns of evolution expected under the “ pollination syndrome ” concept. The ancestral Bignonieae fl ower presented an Anemopaegma -type morphology, which was fol-lowed by several parallel shifts in fl oral morphologies. Those shifts occurred through intermediate stages resulting in mixed fl oral morphologies as well as directly from the Anemopaegma -type morphology to other fl oral types. Positive and negative evolutionary correlations among traits fi t patterns expected under the pollination syndrome perspective, suggesting that interactions between Bignonieae fl owers and pollinators likely played important roles in the diversifi cation of the group as a whole.

Key words: Bignonieae; discrete traits; evolutionary correlations; evolutionary specialization; fl oral traits; morphological transitions; pollinator shifts.

783May 2010] Alcantara and Lohmann — Evolution of floral morphology in Bignonieae

Most fl oral types of Bignonieae are thought to represent modifi cations from the basic Anemopaegma -type, except the Amphilophium -type, which is thought to have evolved from Pithecoctenium -type fl owers ( Gentry, 1974a ). In a previous study, the fl ower types defi ned by Gentry (1974a) were used to infer pollination syndromes that were mapped onto the phylog-eny of Bignonieae as a single multistate trait, indicating sev-eral homoplastic origins for pollination systems in the group ( Lohmann, 2003 ). Because the various traits that determine the individual fl ower types can show different levels of evolution-ary correlation, a more detailed evaluation of the patterns of evolution of individual fl oral traits can contribute important in-formation on the evolution of fl ower morphologies and pollina-tion systems as a whole. In particular, an understanding of the evolutionary patterns of individual fl oral traits of Bignonieae allows for a test of the hypothesis that the ancestral fl ower of Bignonieae was a “ specialized ” fl ower, pollinated by a narrow group of pollinators (i.e., an Anemopaegma -type fl ower, as sug-gested by Gentry, 1974a ), from which successive shifts in pol-linators subsequently occurred.

In this study, we specifi cally address (1) the patterns of evo-lution of individual fl oral traits, (2) the pattern of evolution of the overall fl ower morphology, and (3) the patterns of evolu-tionary correlation among pairs of individual fl oral traits. First, we used a likelihood approach to reconstruct the ancestral states of individual fl ower traits in Bignonieae. Second, we identifi ed the overall fl oral morphology reconstructed at each ancestral node by compiling the ancestral states of each individual fl oral trait (as defi ned by Gentry, 1974a ); these data were then used as a basis for mapping the inferred fl oral types as a single multi-state character and to reconstruct the evolutionary pattern of the overall fl ower morphology in the group. The information gath-ered through ancestral-state reconstructions of individual fl oral traits and overall fl oral morphology permits a detailed evalua-tion of the sequence of evolution in reproductive traits of Bi-gnonieae over time, allowing us to test specifi c predictions associated with the pollination syndrome concept. In particular, we tested whether shifts in fl oral morphologies occurred through intermediate phases (i.e., mixed morphologies). Last, we test for correlated patterns of evolution among individual fl oral traits. The analyses of evolutionary correlation allows us to evaluate whether transitions in particular traits are associated with transitions in other traits, as expected under the pollination syndrome hypothesis.

MATERIALS AND METHODS

Taxon and trait sampling — Taxon sampling was identical to that used in the combined molecular phylogeny of Lohmann (2006) . Specifi cally, we in-cluded 104 Bignonieae species, representing 20 of the 21 genera recognized by Lohmann (in press) ; only the monotypic Callichlamys was not included in the analysis. Species were selected to include all the morphological diversity of the group. In particular, this sampling strategy included representatives of all major morphological shifts in the group (see Appendix S1 in the Supplemental Data with the online version of this article).

Specifi c fl oral traits used are described in detail in Lohmann (2003) and Lohmann et al. (in press) . Of the 95 discrete characters originally coded by Lohmann (2003) , 12 fl oral characters were selected for the present study, seven of which were coded as binary and fi ve as multistate (Appendix 1). These 12 discrete fl oral traits were chosen because of their association with Gentry ’ s fl oral types ( Table 1 ; see Evolution of overall fl oral morphology for further details).

Phylogenies — A well-supported phylogeny of Bignonieae based on plastid ( ndhF ) and nuclear ( PepC ) markers including 104 species of the tribe

of evolution in individual fl oral traits and the number of shifts in pollination systems ( Armbruster, 1992 , 1993 ; Johnson et al., 1998 ; Armbruster and Baldwin, 1998 ; Wilson et al., 2004 ). In-formation on the evolution of individual fl oral traits might pro-vide particularly useful insights into the processes that may have driven the evolution of fl ower morphologies as a whole. Specifi cally, a better understanding of the order of evolution and the lability of individual traits and of the patterns of corre-lated evolution among those traits could greatly enhance our understanding of the processes that may have led to current variation in fl oral morphologies ( P é rez et al., 2006 ). Some stud-ies have addressed the patterns of evolution of individual fl oral traits that are associated with attractiveness to animals (re-viewed by Fenster et al., 2004 ; with subsequent studies by Ackermann and Weigend, 2006 ; P é rez et al., 2006 , 2007 , Whittall and Hodges, 2007 ; Okuyama et al., 2008 ; Smith et al., 2008 ; Tripp and Manos, 2008 ; Yamashiro et al., 2008 ). How-ever, no study has ever evaluated the exact order of evolution of individual traits in a temporal context.

In this study, we review the pollination systems of the tribe Bignonieae, a clade of neotropical Bignoniaceae and evaluate the evolution of fl oral traits in this group. Bignonieae includes approximately 380 widely distributed species, mostly lianas but with some shrubs ( Lohmann, 2003 , 2006 ). The ecological and morphological diversity of this clade makes it an appropriate model for studies on evolutionary and ecological diversifi cation ( Gentry, 1990 ; Lohmann, 2006 ). Specifi cally, their pollination strategies are thought to have played major roles in the diversi-fi cation of the group, with changes in fl oral structure being as-sociated with shifts in pollinator guilds and phenological restraints delimiting differential use of shared pollinators ( Gentry, 1974b , 1990 ). Flowers of Bignonieae were previously classi-fi ed into seven fl oral types ( Gentry, 1974a ; Fig. 1 ) that agree with currently recognized pollination syndromes (sensu F æ gri and van der Pijl, 1966 ). Specifi cally, the Anemopaegma , Am-philophium , and Pithecoctenium fl oral types ( Fig. 1 ) are polli-nated by large and medium-sized solitary bees, the prevalent pollinators of Bignonieae ( Gentry, 1974a ; references in Appen-dix 2). More specifi cally, the Anemopaegma -type fl oral mor-phology corresponds to the classical syndrome of open-mouthed fl owers pollinated by large to medium-sized bees, mainly eu-glossine bees and anthophorids (described by F æ gri and van der Pijl, 1966 ), while Pithecoctenium -type fl owers are described as xylocopid-specialized fl owers, with thick texture and curved corollas limiting nectar robbing by hummingbirds and xylo-copids ( Gentry, 1974a ). In addition, Amphilophium -type fl ow-ers represent an extreme specialization to avoid nectar robbing because the corolla lobes are closed at anthesis and can only be opened by strong, large-bodied pollinators, usually xylocopids ( Gentry, 1974a ). The Martinella -type fl owers were originally described as representing the typical hummingbird-fl owers, with red-orange to deep violet fl owers, exserted stamens, and long tubular corollas ( Gentry, 1974a ). Hawkmoth pollination is thought to be associated with Tanaecium -type fl owers ( Gentry, 1974a ), while the Cydista -type fl owers are considered morpho-logically and phenologically specialized to a “ mimetic ” polli-nation mode by large to medium-sized bees. Cydista -type fl owers lack a nectar disk, but have colorful corollas with con-spicuous nectar guides ( Gentry, 1974a ). Butterfl ies and small bees putatively pollinate the small Tynanthus -type fl owered-species ( Gentry, 1974a ), which are considered to be generalists given that the small bee syndrome includes pollination by a broad spectrum of small insects.

784 American Journal of Botany [Vol. 97

resolved. The shortest possible branch lengths were also assigned to the six branch lengths created to resolve the fi ve polytomies. These time-calibrated, fully resolved trees were used in all analyses. To assess the potential impact of branch lengths to the analyses, we modifi ed branch lengths of the time-calibrated tree (L. G. Lohmann et al., unpublished data) in Mesquite 1.12 so that nodes reached the maximum or minimum confi dence intervals associated with each node. That is, branch lengths were modifi ed so that the minimum age estimated for each node would represent the youngest or oldest age indicated by the con-fi dence intervals. For convenience, hereafter we refer to these trees as those presenting minimum possible branch lengths and maximum possible branch lengths, respectively. Overall, three sets of 500 trees were produced from the PL Bignonieae tree as follows: (1) trees including the original branch lengths assigned by the PL analyses, (2) trees with minimum possible branch lengths, and (3) trees with maximum possible branch lengths.

Evolution of individual fl oral traits — Ancestral character state reconstruc-tions were carried out in Mesquite 1.12 ( Maddison and Maddison, 2006 ) using

( Lohmann, 2006 ) reconstructed monophyletic groups that represent 21 genera in a new generic classifi cation of Bignonieae ( Lohmann, in press ). Two fossils identifi ed as Callichlamys ( Chaney and Sanborn, 1933 ) and Paragonia / Arrabidaea — currently Tanaecium / Fridericia — ( Graham, 1985 ) were placed at nodes 32 and 102 of the resulting phylogeny (see Appendix S2 in the online Supplemental Data), with estimated ages of 34.8 ± 0.22 Myr and 35.35 ± 1.65 Myr, respectively. Complementary analyses using these calibra-tion points and a penalized likelihood (PL) approach ( Sanderson, 2002 ) led to a maximum likelihood (ML) tree with branch lengths proportional to time (L. G. Lohmann; C. Bell [University of New Orleans], and R. C. Winkworth [South Pacifi c], unpublished results). Confi dence intervals associated with the age of representative nodes were obtained through bootstrapping ( Sanderson, 2002 ). Additionally, the fi ve polytomies encountered in the PL tree were randomly resolved using the treefarm Package implemented in the program Mesquite 1.12 ( Maddison and Maddison, 2006 ).

To evaluate how alternative tree resolutions affected ancestral-state recon-structions, we created an additional set of 500 trees with polytomies randomly

Fig. 1. Morphological types of Bignonieae as classifi ed by Gentry (1974a) . (A, B) Anemopaegma -type. (A) Anemopaegma chamberlaynii (Sims) Bureau & K. Schum., (B) Fridericia candicans (Rich.) L. G. Lohmann. (C – F) Martinella -type. (C) Dolichandra cynanchoides Cham., (D) Lundia cordata (Vell.) DC., (E) Adenocalymma dichilum A. H. Gentry, (F) Pyrostegia venusta (Ker Gawl.) Miers. (G) Amphilophium -type, Amphilophium paniculatum (L.) Kunth. (H) Pithecoctenium -type, Amphilophium crucigerum (L.) L. G. Lohmann. (I) Cydista -type, Bignonia corymbosa (Vent.) L. G. Lohmann. (J) Tynanthus -type, Tynanthus cognatus (Cham.) Miers. (K) Tanaecium -type, Tanaecium jaroba Sw.


“ mixed ” morphology type. Mixed morphologies were assigned to classify over-all fl ower morphologies whenever fl owers included traits that were associated with multiple fl oral types described by Gentry (1974a) . To assess the evolution of the overall fl oral morphology, we compiled the ancestral states of the indi-vidual traits reconstructed at each node and used the same procedure described above to classify each ancestral fl ower into one of Gentry ’ s fl ower types (1974b) or into a mixed morphology. For binary traits, the assignment of fl oral types was solely based on the unambiguous reconstructions. For multistate traits, several characters were reconstructed ambiguously. However, in those cases, the prob-abilities assigned to each character allowed us to rule out particular character states that were improbable for a particular node; this information was used to establish the morphological types associated with particular nodes.

A literature review indicated that the pollination system of 46 species of Bignonieae had been studied in the fi eld (Appendix 2). For these 46 species, we were able to establish a direct association between Gentry ’ s fl oral types (1974b) and their respective pollinator groups. This information was then used to pre-dict the most likely pollination mode for the remaining species for which the pollination system had not been studied in the fi eld. Furthermore, these studies, provided suffi cient information for us to categorize the individual fl oral mor-phologies into specialized or generalized fl owers (sensu Fenster et al., 2004 ). Whenever a fl oral morphology was associated with a single functional guild of pollinator, that particular species was classifi ed as a specialist. On the other hand, whenever a fl oral morphology was pollinated by two or more functional groups, the species was considered to be a generalist. Based on the association between fl oral types and the pollination systems described for the tribe (see Table 1 ), we were able to classify all 104 species of Bignonieae into specialists and generalists. Similarly, comparisons with current pollination systems were extrapolated to infer the most likely pollination mode for the ancestral fl owers.

To evaluate whether the evolution of fl oral morphologies included an inter-mediate phase (i.e., mixed morphology), we considered whether changes were concentrated within a single branch or whether transitions were spread out over multiple branches, resulting in branches with mixed fl oral types. Transitions according to the former category were considered as punctuated, while transi-tions according to the latter type were considered as sequential. To account for the potential effect of ambiguous reconstructions of some characters at some nodes, we considered all possible reconstructions while classifying transitions as punctuated and sequential. We then carried a Kolmogorov – Smirnov one-sample test (appropriate for small samples) to test whether the number of punc-tuated and sequential shifts differed signifi cantly ( Zar, 1999 ).

To assess whether the number of traits used to defi ne the fl oral types had any impact on the assessment of the punctuated and sequential shifts, we tested whether punctuated shifts were caused by a higher number of traits with

a ML approach. This methodology fi nds the ancestral states for each node that maximize the probability of the observed states in the terminal taxa under a stochastic model of evolution ( Pagel, 1999 ). The assignment of individual an-cestral states is made from the likelihood estimated for each character state ac-cording to a decision threshold value, with the states that present the lowest likelihood values being rejected ( Pagel, 1999 ). This method allows the incorpo-ration of additional parameters (i.e., phylogenetic branch lengths, varying rates of evolution and evolutionary models), which leads to increased accuracy on the ancestral state reconstructions ( Pagel, 1997 ). The assignment of probabili-ties to all possible states at each node is particularly interesting for traits with a high number of transitions.

All morphological characters were equally weighted and considered unor-dered in all analyses. For the binary traits, two evolutionary models were tested: the Markov chain (Mk) with 1 parameter and the Mk with 2 parameters, repre-senting forward and backward rates of changes between character states. Models were chosen using the likelihood ratio test (LRT) following the recom-mendation of Posada and Buckley (2004) . For multistate traits, reconstructions using the Mk model with 1 parameter were conducted. We used a decision threshold value 2.0 when ancestral states were assigned to a given node ( Mad-dison and Maddison, 2006 ). These ancestral character state reconstructions were used to assess the rates of transition of fl oral traits and the shifts among fl oral morphologies in Bignonieae.

To further assess the effect of alternative topologies and branch lengths to the reconstructed ancestral states, we used the three sets of trees obtained (i.e., dated trees, trees with minimum branch lengths possible, and trees with maximum branch lengths possible) as a basis for carrying out the ancestral-state reconstruc-tions for the 12 characters studied. One tree was randomly chosen from all trees analyzed ( Fig. 2 ). Results from this tree were compared to the results obtained from all other trees and the following information were recorded: (1) the percent-age of trees that presented a particular node and (2) the percentage of trees in which the ancestral states were reconstructed unambiguously for a given node (see Case et al., 2008 ). These results were reported for the fi ve nodes that were randomly resolved (one of these corresponding to a supra-generic node), for the 35 suprageneric nodes, and for the 20 infrageneric nodes associated with shifts in fl oral morphology, leading to a total of 59 nodes analyzed overall.

Evolution of overall fl oral morphology — The original description of fl oral types in Bignoniaceae ( Gentry, 1974a ) was contrasted with the morphological matrix of Lohmann (2003) and Lohmann et al. (in press) to identify characters that were associated with the fl oral types described by Gentry (1974a) . Twelve morphological characters fi t these criteria and were used to classify the overall fl ower morphology of each species into one of the Gentry ’ s fl oral types or into a

Table 1. Gentry ’ s fl oral types described for Bignonieae followed by individual traits associated with each fl oral type and their putative pollination syndrome; traits associated with each morphology were compiled from Gentry (1974a) and from published pollination studies (see Appendix 2).

Floral typesCalyx shape

Double calyx

Corolla color

Nectar guides

Corolla shape

Corolla curvature

Corolla texture

Corolla tubes

Corolla lobes

Corolla mouth*

Anther position

Nectar disk

Pollination syndrome

Amphilophium Cupular Present Magenta Absent Tubular Straight Coriac. Rounded Bilabiate Closed Included Present 1. Large to medium-sized bees 2. Bats

Anemopaegma Cupular, tubular

Absent Magenta, yellow, white

Absent Infund. Straight Memb., rarely coriac.

Rounded Not-bil. Opened Included Present; absent in Lundia

1. Large to medium-sized bees 2. Small bees and insects

Cydista Cupular Absent Magenta Present Infund. Straight Memb. Flattened abaxially

Not-bil. Opened Included Absent Large to medium-sized bees

Martinella Spath., tubular,

urceolate

Absent 1. Red, magenta 2. White

Absent Tubular, infund.,

urceolate

Straight Coriac., rarely memb.

Rounded Not-bil. Opened Exserted Present 1. Hummingbirds (long tube) 2. Bats (short tube, see text)

Pithecoctenium Cupular Absent Yellow, white

Absent Infund. Curved Coriac. Rounded Not-bil. Opened Included Present Large to medium-sized bees

Tanaecium Cupular, tubular

Absent White Absent Tubular Straight Coriac. Rounded Not-bil. Opened Exserted Present Hawkmoths

Tynanthus Cupular Absent Magenta,white

Present Infund. Straight Memb. Rounded Bilabiate Opened Exserted, rarely

inserted

Absent, reduced

Small bees and insects, butterfl ies

Notes: *, at anthesis; spath., spathaceous; infund., infundibuliform; coriac., coriaceous; memb., membranous; not-bil., not bilabiate.


in the estimation of branch lengths also had very little impact on the reconstruction of the ancestral character state reconstruc-tions (Appendix S4). For example, nectar guides, corolla curva-ture, corolla texture, corolla tube, corolla lobes, corolla mouth at anthesis, double calyx, and nectar disk presented identical and unambiguously reconstructed ancestral states in all three sets of trees evaluated (data not shown). The ancestral state re-construction of anther position in relation to corolla lobes was also not affected by branch length (Appendix S4). Corolla shape presented identical ancestral state reconstructions in two sets of trees: trees with the original branch lengths (estimated by the PL analysis) and trees with maximum branch lengths. In the trees with the minimum possible branch lengths, ambiguities were encountered in two nodes. Calyx shape had the root node unambiguously reconstructed as cupular in the analyses that considered the minimum and maximum branch lengths; how-ever, a cupular/tubular condition was assigned in the set of trees with the original branch lengths. As far as corolla color is con-cerned, alternative branch lengths affected the ancestral state reconstructions of three of the 59 nodes considered. In two of those three ambiguously reconstructed nodes, the ambiguities present in the trees with the original branch lengths were un-ambiguously assigned in the tree with minimum branch lengths. The third node was unambiguously reconstructed in the trees with the original branch lengths and in the tree with maximum branch lengths; this same node was ambiguously reconstructed in the tree with minimum branch lengths (see Appendix S4).

Evolution of the overall fl oral morphology in Bignon-ieae — The diagnostic combination of traits presented in Table 1 allowed us to classify species of Bignonieae as belonging to a specifi c fl oral type (e.g., online Appendix S1). Among the 104 species sampled in the phylogeny, the Anemopaegma -type fl ower represented the most common condition (53 species), followed by the Martinella -type fl ower (11 species); nine spe-cies were reported as presenting mixed fl oral morphologies ( Fig. 2 ).

In most cases, fi eld studies on the pollination biology of Bi-gnonieae (see Appendix 2) corroborated the pollination syn-dromes originally associated with fl oral types ( Table 1 ). The only two exceptions were the recent description of visitation by bats in Amphilophium -type fl owers (L. G. Lohmann, personal observation) and bat pollination in Adenocalymma dichilum , a species we classifi ed as Martinella -type fl ower ( Fig. 1 ). Despite this, all species studied that presented Amphilophium , Cydista , Martinella , Pithecoctenium , and Tanaecium fl ower morpholo-gies were associated with a single pollinator group and were hence categorized as specialized. On the other hand, the Tynan-thus -type morphology was associated with multiple pollinator groups and hence considered to represent a generalized mor-phology. The open-mouthed bee syndrome of the Anemopaegma -type was confi rmed by most studies; however, a few studies also reported visitation by multiple pollinator groups in species with Anemopaegma -type fl owers (Appendix 2). Generalist species

transitions being concentrated within a single branch of the phylogeny and whether sequential shifts (with mixed morphologies) occurred due to a sequen-tial change of a lower number of traits at each branch. We carried out a t test for two samples with unequal variances ( Zar, 1999 ) and estimated the probability values using 1000 replicates in a random resampling of the data (using the resa-mpling probability estimates; Lowry, 2009 ). We used this same procedure to test whether there was any relationship between branch length and the occur-rence of punctuated vs. sequential changes. Specifi cally, we tested whether punctuated shifts were associated with longer periods of time, which could have resulted from the accumulation of character transitions within a given lineage and/or from the extinction of lineages with intermediate stages given the in-creased time.

Evolutionary correlation among fl oral traits — We tested for correlated evolution among fl oral traits using Pagel ’ s likelihood approach for discrete characters (1994) implemented in the Correl Package ( Midford and Maddison, 2006 ) for Mesquite ( Maddison and Maddison, 2006 ). Three traits originally coded as multistate by Lohmann (2003) (i.e., calyx shape, corolla shape, and corolla color) were recoded as binary, so that each state was transformed into a new trait coded as presence/absence. Double calyx and nectar disk were also recoded as binary, with the states “ only reminiscent present ” and “ reduced ” coded as absent. This coding scheme was necessary to assess possible correla-tions among gains and losses of each character state of the individual multistate characters because Mesquite does not allow evolutionary correlations to be per-formed between multistate characters. In total, we evaluated potential evolu-tionary correlations among 20 binary traits. For all 190 pairwise correlations tested, 100 searches were carried out, with the P value being estimated from 10 000 repeated simulations. We applied a Bonferroni adjustment (corrected alpha: 0.00027) to account for the use of multiple tests ( Zar, 1999 ).

Paired correlations between the presence/absence states of the multistate traits were evaluated to verify the directionality of changes among the states of those characters. Hypotheses of character correlations were accepted whenever a model with eight-parameters presented a better fi t than a simpler model of evolution with four parameters ( Midford and Maddison, 2006 ). The eight-parameter model differed from the four-parameter model in the rates of transition among states of two binary traits. Under the eight-parameter model, asymmet-ric rates of transitions were associated with each state of a character, varying according to the state of the other trait that was being evaluated. Whenever an eight-parameter model presented a better fi t than a four-parameter model, the asymmetric rates of transition associated with the character transition were as-sociated with any directionality among the correlated traits.

RESULTS

Evolution of individual fl oral traits — The ancestral condi-tion of most traits analyzed was unambiguously reconstructed on the completely resolved, penalized likelihood tree used in the current study (data available upon request). Even for corolla color, the most homoplastic trait analyzed, the ancestral condi-tion was unambiguously assigned to most nodes (Appendix S3 in the online Supplemental Data). The 12 binary traits consid-ered presented a better fi t to the Mk1 model of evolution (Ap-pendix 1). Rates of character change on the time-calibrated phylogeny varied. For example, corolla curvature and double calyx showed lower rates of evolution than anther position in relation to corolla lobes, corolla shape, and corolla color (Ap-pendix 1).

The alternative resolutions of the fi ve polytomies encoun-tered on the penalized likelihood tree did not affect the ances-tral state reconstructions (see online Appendix S4). Variations

Fig. 2. The completely resolved penalized likelihood tree of Bignonieae used for the maximum likelihood (ML) ancestral state reconstructions, show-ing the evolution of fl oral morphologies in the tribe. Floral morphologies mapped in this phylogeny were inferred from the ancestral state reconstructions of the 12 discrete traits showed in Table 1 . Branch lengths are proportional to time (Myr). Black branches marked with an asterisk (*) represent branches in which the reconstructed morphologies could not be assigned due to uncertainties in the reconstruction of individual character states (see Table 2 ). Rep-resentations of the extant species as well as the ancestral morphology of the tribe reconstructed by ML are shown (see Fig. 1 for further information on the species represented).

®


among the 20 selected Bignonieae traits, 74 were signifi cant (38.95%; Table 3 ). Most correlations were negative, suggesting that particular fl ower traits never occur together within the same fl ower. For example, the negative correlation between yellow corolla and exserted position of the anther indicates that yellow corollas generally present inserted anthers. Simi-larly, a negative correlation between red and curved corollas indicates that red-fl owered corollas generally present straight tubes. Nectar guides were also negatively correlated with tubu-lar corollas, suggesting that whenever nectar guides are pres-ent, the state tubular corolla is not. On the other hand, strong positive correlations were observed between urceolate and red corollas, tubular and white corollas, presence of nectar guides and infundibuliform corollas, and presence of double-calyx and tubular corollas, indicating that these traits usually occur together. Other positive correlations were found between dou-ble-calyx and magenta corollas, closed corolla mouth at anthe-sis and magenta corollas, spathaceous calyx and red corollas, tubular and red corollas.

Among the three characters originally coded as multistate by Lohmann (2003 , in press ) but recoded as presence/absence for the analyses of correlated evolution (i.e., calyx shape, corolla shape and color), negative correlations among the states of the same character were prevalent because a single species cannot present multiple states of the same character. For example, the corolla of a species cannot be tubular and urceolate simultane-ously. Overall, the assigned rates of change between the states of these three traits and the reconstructed ancestral states (Ap-pendix S5 in the online Supplemental Data) indicate the follow-ing transitions: (1) Calyx shape: cupular shape often precedes the evolution of all other calyx shapes, with tubular and spatha-ceous calyces always evolving from ancestors with cupular ca-lyces (see Appendix S5-A). Furthermore, spathaceous and urceolate calyces are negatively correlated and occur in dis-tantly related lineages, with tubular calyces having evolved multiple times. (2) Corolla shape: tubular and urceolate corollas most often evolved from ancestors with infundibuliform corol-las (see Appendix S5-B). (3) Corolla color: yellow, white, and red corollas often evolved from ancestors with magenta fl ow-ers, with white and red corollas being negatively associated with yellow corollas (see Appendix S3). Moreover, the assigned rates of transition in other characters that are correlated with fl ower color are generally affected by the color of fl owers and not the other way around.

DISCUSSION

The radiation of angiosperms is greatly associated with the diversifi cation of fl ower forms, which is thought to have re-sulted from natural selection, leading to adaptation to different pollination strategies ( Darwin, 1862 ; F æ gri and van der Pijl, 1966 ; Stebbins, 1970 ). Despite the prevalent concept that shifts in fl oral morphology are driven by selective processes (e.g., Stebbins, 1970 , 1974 ; Harder and Barrett, 2006 ), we still lack evidence of how fl oral morphologies have shifted over time (see introduction). Thus, we assessed in this paper the evolu-tionary pattern of (1) transitions in the individual fl oral traits, (2) shifts in the overall fl oral morphology, and (3) correlations between paired fl oral traits in the neotropical tribe Bignonieae (Bignoniaceae). We discuss below our major results attempting for an association between fl oral shifts and putative pollinator groups in this clade.

usually presented smaller and magenta corollas, with infl orescences displaying a large number of fl owers (common in Fridericia ). On the other hand, species in which a specialized pollination by large to medium-sized bees were reported, usually presented larger and yellow corollas (common in Adenocalymma and Anemopaegma ). Even though Anemopaegma -type fl owers (as described by Gentry, 1974a ) fi t better a specialized pollination system, the wide variation in fl ower morphologies associated with the Anemopaegma -type fl owers indicates that some Ane-mopaegma -type fl owers might be generalists. Hence, both pos-sibilities (generalists and specialists) were considered for this morphological type.

Ancestral character state reconstructions indicated that the ancestral fl ower of Bignonieae presented an Anemopaegma -type fl ower ( Fig. 2 ). This ancestral fl ower was likely magenta (53% probability); however, the exact condition was ambigu-ously reconstructed (magenta/yellow/white). Among the 12 individual fl oral traits associated with fl oral types ( Table 1 ), seven did not present ambiguities in any of the 59 nodes consid-ered ( Table 2 ). Calyx shape, corolla color, nectar guides, co-rolla shape, and anther position in relation to corolla lobes led to some ambiguously reconstructed nodes. Such uncertainty makes the identifi cation of the ancestral morphology of six in-ternal nodes ambiguous; these nodes could be assigned as Ane-mopaegma -type fl owers or other alternative type morphologies depending on the assigned state of the ambiguously recon-structed characters ( Table 2 ). It was still possible to identify seven internal nodes that were not affected by ambiguous re-constructions and in which the most likely morphology was characterized by a mixed pattern ( Table 2 ).

Overall, we identifi ed 29 shifts in fl oral morphologies, most of which were concentrated next to terminal branches in the phylogeny ( Fig. 2 ). Generally, shifts from the basic Anemo-paegma -type into one of Gentry ’ s fl ower types were encoun-tered; less commonly, shifts into mixed fl oral traits, not associated with any of Gentry ’ s types, were encountered. The transition from an Anemopaegma -type to a Martinella -type fl ower was the most common, having occurred at least eight times. The repeated evolution of a Tanaecium -type fl ower from an ancestor presenting an Anemopaegma -type fl ower was also common. Amphilophium -type fl owers, on the other hand, are always inferred to have evolved from ancestors with Pithecoctenium -type fl owers, while Tynanthus -type fl owers evolved a single time from an ancestor presenting an Anemo-paegma -type fl ower and once from an ancestor with a Cydista -type fl ower.

Among the 29 shifts encountered in fl oral morphologies, 10 showed transitions through mixed morphologies (sequential), while 19 showed a punctuated pattern (i.e., without intermedi-ate stages). Even though the ancestral morphology of six of these 19 shifts were ambiguous, all most likely resolutions of these ambiguous assignments resulted in punctuated shifts ( Ta-ble 2 ). Overall, the number of sequential and punctuated shifts did not differ (Kolmogorov – Smirnov test: D max = 0.155, D 0,05 = 0.27), and no difference was detected between the length of branches associated with punctuated or sequential shifts in fl o-ral morphologies ( t test = 0.953, df = 27, P = 1). Similarly, no difference was encountered between the numbers of traits in-volved in shifts with or without intermediate fl oral types ( t test = 0.1567, df = 27, P = 0.88).

Evolutionary correlations among fl oral traits in Bignon-ieae — Among the 190 pairwise correlation tests carried out


Table 2. Ancestral states of traits that determine Gentry ’ s (1974a) morphologies in Bignonieae reconstructed using a maximum likelihood (ML) approach. Node numbers are indicated in Appendix S2 (see the online version of this manuscript) and correspond to the 35 suprageneric nodes and 24 infrageneric nodes considered here (see Material and Methods, Evolution of individual fl oral traits for further information). The six nodes created to resolve the fi ve polytomies encountered in the penalized likelihood (PL) tree are indicated by (*).

NodeCalyx shape

Double calyx

Corolla color

Nectar guides

Corolla shape

Corolla curvature

Corolla texture

Corolla tubes

Corolla lobes

Corolla mouth*

Anther position

Nectar disk Floral type

2 Absent Absent Infund. Straight Memb. Rounded Not-bil. Opened Included Present Anemopaegma 3 Cupular Absent M/Y Absent Infund. Straight Memb. Rounded Not-bil. Opened Included Present Anemopaegma 4 Cupular Absent Y Absent Infund. Straight Memb. Rounded Not-bil. Opened Included Present Anemopaegma 5 Cupular Absent Y Absent Infund. Straight Memb. Rounded Not-bil. Opened Included Present Anemopaegma 6* Cupular Absent Y Absent Infund. Straight Memb. Rounded Not-bil. Opened Included Present Anemopaegma 10 Cupular Absent Absent Infund. Straight Memb. Rounded Not-bil. Opened Included Present Anemop ./ Martinella 26 Absent Y Absent Infund. Straight Memb. Rounded Not-bil. Opened Included Present Anemopaegma 29 Cupular Absent M/Y Absent Infund. Straight Memb. Rounded Not-bil. Opened Included Present Anemopaegma 30 Cupular Absent M/Y Absent Infund. Straight Memb. Rounded Not-bil. Opened Included Present Anemopaegma 31 Cupular Absent M/Y Absent Infund. Straight Memb. Rounded Not-bil. Opened Included Present Anemopaegma 32 Cupular Absent M/Y Absent Infund. Straight Memb. Rounded Not-bil. Opened Included Present Anemopaegma 33 Cupular Absent M Absent Infund. Straight Memb. Rounded Not-bil. Opened Included Present Anemopaegma 34 Cupular Absent M Absent Infund. Straight Memb. Rounded Not-bil. Opened Included Present Anemopaegma 35 Cupular Absent M Absent Infund. Curved Coriac. Rounded Not-bil. Opened Included Present Pithecoctenium 38* Cupular Absent M Absent Infund. Curved Coriac. Rounded Not-bil. Opened Included Present Pithecoctenium 39 Cupular Present M Absent Infund. Curved Coriac. Rounded Bilabiate Opened Included Present Pithecoc .- Amphilop .41 Cupular Present M Absent Infund. Curved Coriac. Rounded Bilabiate Opened Included Present Pithecoc .- Amphilop .42 Cupular Present M Absent Tubular Curved Coriac. Rounded Bilabiate Closed Included Present Amphilophium 56 Cupular Absent M Absent Infund. Straight Memb. Rounded Not-bil. Opened Included Present Anemopaegma 57 Cupular Absent M/Y Absent Infund. Straight Memb. Rounded Not-bil. Opened Included Present Anemopaegma 58 Cupular Absent Y Absent Infund. Straight Memb. Rounded Not-bil. Opened Included Present Anemopaegma 66 Cupular Absent M Absent Infund. Straight Memb. Rounded Not-bil. Opened Included Present Anemopaegma 75 Cupular Absent M Absent Infund. Straight Memb. Rounded Not-bil. Opened Included Present Anemopaegma 76 Cupular Absent M Absent Infund. Straight Memb. Rounded Not-bil. Opened Included Present Anemopaegma 79 Cupular Absent M Infund. Straight Memb. Flattened Not-bil. Opened Included Absent Cydista 87 Cupular Absent M Present Infund. Straight Memb. Flattened Not-bil. Opened Included Absent Cydista 88 Cupular Absent M Present Infund. Straight Memb. Rounded Not-bil. Opened Included Absent Anemop. - Cydista 94 Cupular Absent R/M/Y Absent Infund. Straight Memb. Rounded Not-bil. Opened Included Present Anemop ./ Martinella 97 Cupular Absent R/Y Absent Infund. Straight Memb. Rounded Not-bil. Opened Present Anemop .- Martinella 101 Cupular Absent M/Y Absent Infund. Straight Memb. Rounded Not-bil. Opened Included Present Anemopaegma 102 Cupular Absent M/Y Absent Infund. Straight Memb. Rounded Not-bil. Opened Included Present Anemopaegma 103 Cupular Absent M/W Absent Infund. Straight Memb. Rounded Not-bil. Opened Included Present Anemopaegma 104 Cupular Absent M/W Absent Infund. Straight Memb. Rounded Not-bil. Opened Included Present Anemopaegma 107 Cupular Absent Absent Infund. Straight Memb. Rounded Not-bil. Opened Included Present Anemopaegma 113 Cupular Absent M/W Absent Infund. Straight Memb. Rounded Not-bil. Opened Included Present Anemopaegma 116 Cupular Absent M/W Absent Straight Memb. Rounded Not-bil. Opened Included Present Anemop ./ Tanaecium 117 Cupular Absent M/W Absent Straight Memb Rounded Not-bil. Opened Included Present Anemop ./ Tanaecium 121 Cupular Absent M/W Absent Infund. Straight Memb. Rounded Not-bil. Opened Included Present Anemopaegma 122 Cupular Absent M/W Absent Infund. Straight Memb. Rounded Not-bil. Opened Included Present Anemopaegma 123 Cupular Absent M/W Absent Infund. Straight Memb. Rounded Not-bil. Opened Included Present Anemopaegma 124 Tubular Absent M/W Absent Infund. Straight Memb. Rounded Not-bil. Opened Included Present Anemopaegma 127 Cupular Absent M/W Absent Infund. Straight Memb. Rounded Not-bil. Opened Included Present Anemopaegma 129* Cupular Absent M/W Absent Infund. Straight Memb. Rounded Not-bil. Opened Included Present Anemopaegma 140* Cupular Absent M Absent Infund. Straight Memb. Rounded Not-bil. Opened Included Present Anemopaegma 144* Cupular Absent M Absent Infund. Straight Memb. Rounded Not-bil. Opened Included Present Anemopaegma 150 Absent R/M Absent Straight Coriac. Rounded Not-bil. Opened Included Present Anemop .- Martinella 154 Tubular Absent M/W Absent Straight Memb. Rounded Not-bil. Opened Included Present Anemop ./ Tanaecium 160 Cupular Absent M Absent Infund. Straight Memb. Rounded Not-bil. Opened Included Present Anemopaegma 163* Cupular Absent M/W Absent Infund. Straight Memb. Rounded Not-bil. Opened Included Present Anemopaegma 164 Cupular Absent M/W Absent Infund. Straight Memb. Rounded Not-bil. Opened Included Present Anemopaegma 165 Cupular Absent M Absent Infund. Straight Memb. Rounded Not-bil. Opened Included Present Anemopaegma 169 Cupular Absent M Absent Infund. Straight Memb. Rounded Not-bil. Opened Included Present Anemopaegma 178 Cupular Absent W Absent Infund. Straight Memb. Rounded Bilabiate Opened Exserted Reduced Tynanthus 185 Cupular Absent W Present Infund. Straight Memb. Rounded Not-bil. Opened Included Absent Anemopaegma 188 Cupular Absent W Present Infund. Straight Memb. Rounded Not-bil. Opened Included Absent Anemopaegma 195 Cupular Absent M/Y Absent Straight Memb. Rounded Not-bil. Opened Included Present Anemop ./ Tan ./ Martin 197 Cupular Absent Y Absent Tubular Straight Memb. Rounded Not-bil. Opened Included Present Anemop .- Tan .- Martin 202 Tubular Absent M Absent Infund. Straight Memb. Rounded Not-bil. Opened Exserted Present Martinella 205 Urceolate Absent M Absent Infund. Straight Memb. Rounded Not-bil. Opened Included Present Anemop .- Martinella

Note: M, magenta; R, red; Y, yellow; W, white; infund., infundibuliforme; memb., membranous; coriac., coriaceous; not-bil., not bilabiate; blank cells correspond to ambiguous character state reconstructions by ML. Despite the ambiguity for the reconstructions of the character corolla color, states are reported whenever only two states were assigned to a given node. A slash (/) between fl oral types means that different fl oral types could be assigned to the node depending on the alternative resolutions of one or more ambiguous reconstructed traits; a dash (-) means that morphologies were reconstructed with mixed character states.


ated by these variables did not affect the reconstructions reported here.

Four of the fi ve lowest rates of evolution were encountered in the following traits: corolla curvature, corolla tube, corolla mouth at anthesis, and double calyx. The character corolla mouth at anthesis and double calyx are associated with the Amphilophium -type fl owers. On the other hand, curved corolla and corolla tube are associated with the fl oral types Pithecoctenium and Cydista , respectively. Despite the utility of these characters for the identifi cation of clades within genera, these traits were homoplasious and did not represent synapomorphies of any genera ( Lohmann, 2006 ). Nectar guides, corolla texture, corolla lobes, anther position in relation to corolla lobes, nectar disk, corolla shape, and calyx shape were also homoplastic (online Appendix S5). Yet, the state transitions were in general concen-trated in terminal branches that were sparsely distributed along the phylogeny (e.g., anther position in relation to corolla lobes, see online Appendix S3).

Evolution of individual fl oral traits — The unambiguous as-signment of most ancestral states of Bignonieae was surprising given that previous studies found high levels of ambiguity while reconstructing ancestral states of fl oral traits (see Case et al., 2008 ). Furthermore, given the high levels of homoplasy in fl o-ral traits of Bignonieae ( Lohmann et al., in press ), diffi culties in the reconstruction of the ancestral states of those traits had been anticipated. However, the homoplasy in fl oral traits of Bignon-ieae was shown to be restricted to specifi c lineages and/or character types ( Lohmann et al., in press ). Specifi cally, most binary-coded traits were unambiguously reconstructed, while multistate characters were shown to be more homoplasious, with the ambiguities being concentrated within particular gen-era (Appendix S5-A with the online version of this article). Alternative resolutions of polytomies did not affect the ancestral-state reconstructions. Similarly, the alternative branch lengths evaluated also did not impact the ancestral-state reconstructions. Thus, we are confi dent that the phylogenetic uncertainty gener-

Table 3. Evolutionary correlations between fl oral traits of Bignonieae. Above diagonal: differences among – logL of the four-parameter models (independent evolution) and eight-parameter models (correlated evolution among traits). Below diagonal: P values calculated by 10 000 simulations. Signifi cant differences among the – logL are marked with an asterisk (*), indicating a better fi t of the data to the model of correlated evolution between two traits.

Trait 01 02 03 04 05 06 07 08 09 10 11 12 13 14 15 16 17 18 19 20

01. Nectar guides

0.357 0.302 0.0001 0.002 0.109 0.065 0.393 0.0002 0.0003 0.548 0.0 0.002 0.538 0.003 0.548 0.082 0.0001 0.164 0.054

02. Corolla curvature

0.4 0.0 0.283 0.0 0.0002 0.154 0.0 0.001 0.141 0.046 0.0 0.0 0.0 0.021 0.012 0.0002 0.0 0.359 0.01

03. Corolla texture

0.91 10.67* 0.17 0.0 0.0002 0.0 0.018 0.486 0.026 0.172 0.0 0.271 0.0 0.005 0.072 0.0002 0.0 0.434 0.099

04. Corolla tube

8.97* 0.32 0.92 0.65 0.083 0.28 0.31 0.034 0.78 0.76 0.0 0.02 0.04 0.002 0.31 0.03 0.0 0.002 0.005

05. Corolla lobes

0.44 21.25* 1.43* 0.25 0.0001 0.0 0.0 0.0002 0.16 0.049 0.03 0.0004 0.0 0.0 0.63 0.28 0.0 0.055 0.0

06. Corolla mouth at anthesis

1.6 4.92* 4.58* 2.09 5.94* 0.24 0.0001 0.145 0.32 0.191 0.17 0.109 0.112 0.126 0.079 0.28 0.0001 0.0002 0.0001

07. Anther position

5.72 1.85 6.7* 1.36 1.29* 1.14 0.249 0.0002 0.0001 0.007 0.0 0.091 0.0 0.003 0.011 0.0 0.0 0.0002 0.062

08. Double calyx

0.38914.6* 6.5 0.21 5.32* 6.88* 1.2 0.577 0.919 0.075 0.88 0.041 0.36 0.045 0.012 0.103 0.0 0.19 0.159

09. Corolla tubular

5.43* 3.73 1.31 1.97 2.95* 1.63 8.12* 0.64 0.0 0.545 0.14 0.0002 0.0 0.011 0.301 0.0 0.0 0.0 0.0002

10. Corolla infundibuliform

1.08* 0.75 1.71 2.02 1.13 1.54 12.62* 0.44 50.34* 0.0002 0.36 0.0007 0.015 0.03 0.026 0.0 0.0 0.0 0.0002

11. Corolla urceolate

0.3 0.48 0.15 0.48 2.32 0.25 3.04 1.56 0.63 2.57* 0.07 0.206 0.148 0.231 0.137 0.002 0.199 0.059 0.25

12. Nectar disk10.8* 0.55* 0.95* 7.27* 0.84 1.56 2.25* 0.46 2.51 2.61 0.58 0.23 0.0 0.34 0.01 0.88 0.23 0.2 0.013. Calyx

cupular1.05 2.04* 2.01 0.001 2.13* 1.4 5.14 1.42 9.2* 3.83 0.73 1.46 0.0 0.0 0.0 0.006 0.053 0.179 0.237

14. Calyx tubular

0.86 8.71* 9.21* 0.88 5.79* 1.09 9.49* 1.14 5.59* 2.93* 0.73 0.77* 30.17* 0.0 0.382 0.0001 0.005 0.521 0.0001

15. Calyx spathaceous

1.08 2.63 3.2 0.88 2.78* 0.72 4.76 0.95 3.47 1.58 0.07 1.47 11.07* 2.55* 0.007 0.0002 0.201 0.0 0.41

16. Calyx urceolate

0.3 2.23 2.96 0.25 0.54 0.61 3.07 2.75 1.59 50.34 0.44 0.36 5.15* 1.25 3.19 0.015 0.0 0.381 0.061

17. Corolla red

2.35 1.93* 4.07* 0.91 1.28 0.69 7.23* 2.08 9.36* 10.96* 2.14 0.43 4.43 2.9* 1.95* 2.45 0.0 0.0 0.0

18. Corolla magenta

5.8* 0.61* 21.48* 5.03* 18.37* 3.22* 4.39* 11.6* 7.31* 7.06* 0.63 3.65 1.86 0.94 1.67 1.5* 8.74* 0.0 0.0001

19. Corolla yellow

2.4 0.69 1.15 1.86 0.62 3.47* 6.82* 1.96 5.28* 6.12* 3.04 2.96 3.54 1.72 3.86* 1.07 8.15* 9.76* 0.0

20. Corolla white

4.06 2.5 2.31 3.13 3.67* 3.9* 2.52 2.86 4.89* 5.06* 1.72 3.63* 2.51 4.74* 1.32 1.29 5.51* 9.76* 13.22*


and generalist pollination modes. As far as the Anemopaegma -type fl owers are concerned, specifi c fl oral traits (e.g., fl ower size, number of fl owers per infl orescence, and corolla color) al-low us to identify which of the present species are associated with a specialized or a generalized pollination mode (see Re-sults). Unfortunately, however, these features did not allow us to identify the pollination mode associated with the ancestral mor-phologies here reconstructed as Anemopaegma -type.

The Anemopaegma -type fl ower represents the predominant condition among current species of Bignonieae and was here reconstructed as the ancestral fl ower of the tribe. This pattern suggests conservatism in fl oral shape, despite recurrent evolu-tions of different morphologies in the tribe. Given that the an-cestral Anemopaegma -type fl owers could not be associated with a generalized or specialized morphology, at least two evo-lutionary scenarios can be hypothesized. First, a specialized ancestral Bignonieae fl ower may have been followed by several shifts among pollinator groups, several losses of specialization in some Anemopaegma -type species and generalization through morphological shifts from Anemopaegma to Tynanthus -type fl owers. Alternatively, a generalized ancestral morphology may have been followed by several specializations along the history of Bignonieae with at least two shifts to Tynanthus -type fl ow-ers, maintaining a generalist pollination system. These alterna-tive scenarios assume two extremes of a continuum concerning the specialization – generalization of fl owers to pollinators; how-ever, we cannot ignore all possible intermediate levels of pol-linator association ( Waser, 2006 ). In particular, intermediate stages may have occurred several times during the evolutionary history of Bignonieae, even among groups that maintained the Anemopaegma -type fl owers, as exemplifi ed by the present spe-cialized and generalized species sharing this morphology.

Most comparative studies have encountered great lability in the degree of specialization or generalization of fl owers in dif-ferent plant groups ( Armbruster and Baldwin, 1998 ; Fenster et al., 2004 ; Tripp and Manos, 2008 ). While some evolutionary changes may narrow the spectrum of pollinators, e.g., Calo-chortus lilies ( Dilley et al., 2000 ; Patterson and Givnish, 2004 ), and some clades of Ruellia ( Tripp and Manos, 2008 ), others seem to broaden the spectrum, e.g., Aphelandra ( McDade, 1992 ) and Dalechampia ( Armbruster and Baldwin, 1998 ). Nev-ertheless, most studies refl ect shifts from one functional group of pollinators to another ( Goldblatt and Manning, 1996 ; Johnson et al., 1998 ; Steiner, 1998 ; Wilson et al., 2004 ; P é rez et al., 2006 ; Okuyama et al., 2008 ; Smith et al., 2008 ; Tripp and Manos, 2008 ). Similar patterns of change are also observed during the diversifi cation of Bignonieae ( Fig. 2 ). Below we discuss shifts in morphologies associated with specifi c pollination modes in Bignonieae, but highlight that this pattern might represent an underestimation due to our incomplete sampling. Clades within which a more comprehensive taxon sampling might lead to a signifi cant impact on the results here encountered are discussed in further detail.

It is frequently suggested that the relationship between pollinators and fl owers mediated by fl oral traits is prone to parallelisms and reversals ( Armbruster, 1993 ; Goldblatt and Manning, 1996 ; Bruneau, 1997 ; Johnson et al., 1998 ; Patterson and Givnish, 2004 ; P é rez et al., 2006 ; Thomson and Wilson, 2008 ). In Bignonieae, we observe that the ancestral morphol-ogy (i.e., Anemopaegma -type) led to multiple evolutions of more derived fl ower types ( Fig. 2 ), corroborating the hypothesis of parallel evolution. A careful observation of the fl ower mor-phology of species of Bignonieae that were not included in

The exception to the unequivocal reconstructions of ances-tral states is the character corolla color. This trait had several ambiguous reconstructed suprageneric nodes and was also used in the defi nition of fl ower types in Bignonieae ( Gentry, 1974a ). Despite the ambiguous reconstruction of fl ower color of the an-cestral Bignonieae, there is a higher probability of a magenta ancestral fl ower. Yet, evolutionary correlations indicated higher rates of change from magenta fl owers to any other fl ower color. In addition, the patterns of color diversity illustrate an asym-metry in color transitions in this group. Specifi cally, most ex-tant species of Bignonieae are magenta or yellow, with these colors rarely co-occurring within the same genus. On the other hand, red and white corollas have evolved multiple times within different lineages, even within those characterized by yellow or magenta fl owers (Appendix S3). Seven of eight studies of fl ower color shifts compiled by Rausher (2008) indicate that changes in color determined by anthocyanins (i.e., deep purple, magenta, and red fl owers) were caused by loss of function (LOF) mutations in genes associated with the synthesis of those pigments. This pattern implies an unequal rate of color transi-tions from blue to red and from pigmented to white fl owers in a great number of taxa ( Rausher, 2008 ). Multiple LOF mutations on anthocyanin pathways (associated with magenta fl owers) may have led to the various origins of white fl owers in Bignon-ieae. In turn, LOF mutations in genes associated with the pro-duction of anthocyanins combined with the activation of fl avonoids/carotenoid production (e.g., Ono et al., 2006 ) may have been associated with the evolution of yellow and orange fl owers. However, to test hypotheses concerning the causes of extreme color lability in Bignonieae, we would need a better understanding of the biosyntheses of fl ower pigments in this group, combined with fi eld studies on the impact of color poly-morphism on the variation of pollinator attractiveness.

Corolla color, corolla lobes, corolla shape, and calyx shape are important features in the attractiveness of fl owers to pollina-tors ( Vogel, 1954 ; F æ gri and van der Pijl, 1966 ). In a review of comparative studies on the shifts in pollination syndromes, Fenster et al. (2004) showed that changes in fl oral traits are as-sociated with pollinator functional groups in ways that are eas-ily predicted by traditional pollination syndromes (F æ gri and van der Pijl, 1966 ). Different traits seem to show different lev-els of association with different pollinator groups, with fl ower morphology responding consistently to all functional groups. In Bignonieae, corolla color, corolla shape, and anther position provide excellent examples of high evolutionary lability. In ad-dition, pollination studies indicate that those traits are also as-sociated with particular pollinator groups (e.g., references in Appendix 2). Despite this, no empirical evidence is available to support the hypothesis that the lability in those traits has been caused by pollinator pressures. Indirect selection coupled with pleiotropy resulting in fl oral exaptations to several pollinator groups represents an alternative explanation to this pattern. It seems unlikely, however, that the reappearance of major fl oral features associated with specifi c pollinator groups represents a series of exaptations.

Evolution of the overall fl oral morphology in Bignon-ieae — Particular fl oral traits of Bignonieae have been tradition-ally interpreted as specializations to specifi c pollinators ( Gentry, 1974a ). The general trends encountered allowed us to formulate reasonable hypotheses about the pollinator groups and strategies associated with unstudied species. Exceptions are the Anemo-paegma -type fl owers, which are associated with both specialist


Tecomeae, Bignoniaceae.). Thus, the morphological shift to a Tynanthus -type fl ower might represent an example of an “ adap-tive wandering ” ( Thomson and Wilson, 2008 ) in which differ-ent morphologies related to generalized pollination systems may have emerged from diverse evolutionary responses to dif-ferent environments. As a result, this adaptive wandering may increase the morphological diversity in fl owers of a particular group, yet maintain a broad pollinator use range. This situation might apply to Bignonieae if the ancestral of the tribe presented a generalist fl ower. At least one generalization to the Tynan-thus -type fl ower, however, must have occurred from the spe-cialized Cydista -type.

The patterns of evolution of the traits that determine fl oral morphologies in Bignonieae suggest that shifts among fl oral types took place both with and without intermediate stages. As-suming an association between fl oral types and pollination modes, this pattern of change implies a combination of gradual (i.e., sequential evolution of correlated traits) and punctuated (i.e., concurrent evolution of correlated traits along a branch) shifts in pollination systems in Bignonieae. Shifts with interme-diate stages are thought to have been prevalent in most plant groups, with the intermediate stages being pollinated by two pollen vectors ( Stebbins, 1970 ). Two species of Bignonieae characterized by fl owers with mixed morphologies, Stizophyl-lum perforatum and Tanaecium pyramidatum , were visited by two groups of putative pollinators ( Gentry, 1974a ; Amaral, 1992 ; Carvalho et al., 2003 ). Additional fi eld studies conducted within a phylogenetic context are needed to assess whether these intermediate stages among Gentry ’ s fl oral types could be considered transient between different adaptive peaks. On the other hand, the dissociation between the mode of transition and branch length suggests that the reconstructed punctuated shifts do not represent an artifact created by putative extinction of lineages over time. In addition, the dissociation between the number of traits and the mode of transitions among morpholo-gies indicates that some traits must be more important in the determination of shifts among morphologies than others. This observation is in agreement with the qualitative nature of the pollination syndrome defi nition and with criticism concerning the unclear boundaries of the individual syndromes (e.g., F æ gri and van der Pijl, 1966 ; Fenster et al., 2004 ; Waser, 2006 ). Un-der a quantitative perspective, one should associate punctuated changes to changes in several traits simultaneously, while grad-ual changes should be associated with changes in a lower num-ber of traits. However, we did not fi nd any correlation between the number of traits and mode of change. Further analyses on the phenotypic space occupied by fl owers of Bignonieae and quantitative approaches to assess the association between mor-phologies and pollinators should provide important insights in this direction (S. Alcantara, F. B. de Oliveira [Universidade de S ã o Paulo], and L. G. Lohmann, unpublished data). The disso-ciation between branch lengths and number of traits needed to characterize the mode of morphological shifts (i.e., sequential or punctuated) provide interesting insights into the evolution of fl oral morphologies in Bignonieae. These patterns suggest that punctuated and sequential shifts in fl oral morphologies could both have led to the evolution of current fl oral types in this group. It further suggests that the shifts in fl oral morphologies are determined by complex evolutionary dynamics.

Evolutionary correlations among fl oral traits in Bignon-ieae — Negative correlations among discrete traits suggest that some combinations of characters are developmentally prohibited

the current study, associated with a careful evaluation of their putative position within the phylogeny (online Appendix S1), suggest that an even higher number of transitions in fl oral mor-phologies must have occurred during the diversifi cation of Bi-gnonieae. In addition, this observation also indicated that the Anemopaegma -type morphology could subsequently have re-evolved from a Tanaecium -type morphology, a hawkmoth-pol-linated fl ower. Because the sampling for this study was designed to encompass maximum diversity, many Tanaecium -type spe-cies within Tanaecium were not sampled. Unfortunately, the lack of a complete sampling in Tanaecium may have biased ancestral state reconstructions in this clade. Despite the uncer-tain reconstructions in some internal nodes of Tanaecium and the assignment of an Anemopaegma -type fl ower as the ances-tral condition for this genus, it seems unlikely that the Anemo-paegma -type fl ower would represent the ancestral condition in those internal nodes or the ancestral condition for Tanaecium . It has been previously suggested that the evolution of hawkmoth pollination might represent an evolutionary dead end ( Tripp and Manos, 2008 ). In Ruellia (Acanthaceae), another neotropi-cal and ecologically diverse group in terms of pollination sys-tems, the evolution of hawkmoth and bat pollination never led to the evolution of different pollination systems ( Tripp and Manos, 2008 ). Reversions of Anemopaegma -type fl owers from Tanaecium -type ancestors would imply an absence of con-straints in Tanaecium , contrasting with the results of Tripp and Manos (2008) ; the occurrence of reversions would not corrobo-rate their being an evolutionary dead end.

In contrast to hawkmoth-pollinated fl owers, bee-, insect-, and hummingbird-pollinated species of Ruellia have been shown to represent very labile fl oral morphologies ( Tripp and Manos, 2008 ). This lability was not observed in the humming-bird-pollinated fl owers here (i.e., Martinella -type fl owers). Even though Martinella -type fl owers appear repeatedly on the phylogeny of Bignonieae, this fl ower type never led to reversals into other fl oral types. Variants of the Martinella -type morphol-ogy that are associated with bat-pollination are restricted to three species of Bignonieae (i.e., Adenocalymma dichilum , Fridericia tynanthoides , and Pachyptera ventricosa ). Even though these species were not included in the current study, they occur in unrelated genera, also implying a homoplastic evolution. Amphilophium -type fl owers represent another fl ower type putatively pollinated by bats. Interestingly, Amphilophi-um -type fl owers also never led to the evolution of different fl ower types, suggesting that hummingbird and bat pollination might, instead, represent evolutionary dead ends in Bignonieae. In Penstemon ( Wilson et al., 2007 ) and Costus ( Kay and Schemske, 2003 ), two genera with many hummingbird-pollinated species, multiple origins of ornithophily never led to subsequent evolu-tions of any other fl ower types. However, transitions from hum-mingbird to bee pollination and from hummingbird to hawkmoth pollination ( Whittall and Hodges, 2007 ; Kulbaba and Worley, 2008 ; Tripp and Manos, 2008 ) have been documented in other plant groups (see references cited in Tripp and Manos, 2008 ). The transition of hummingbird to hawkmoth pollination, associ-ated with the directional increase in tube length in Aquilegia pro-vides an excellent example of the latter ( Whittall and Hodges, 2007 ). This pattern was not observed in Bignonieae, a group where hummingbird- and hawkmoth-pollinated fl owers seem to have both evolved independently from bee-pollinated ancestors.

The Tynanthus -type fl ower, pollinated by a great variety of insect groups ( Gentry, 1974a ), evolved at least twice in Bignon-ieae (Gentry also reported this fl ower type in Godmania , tribe


An evaluation of the phylogenetic uncertainty in the tree used for the current study indicates that our results are robust, regardless of the phylogenetic uncertainties. Similarly, the balanced sampling of fl oral morphologies of Bignonieae in the phylogeny of Lohmann (2006) suggests that the incom-plete taxon sampling would have a minor impact on the gen-eral shifts in fl oral morphologies inferred here. The incomplete taxon sampling does, however, likely contribute to an overall underestimation of the number of shifts in fl oral morpholo-gies. Positive and negative evolutionary correlations among fl ower traits are in agreement with the evolutionary patterns expected under the pollination syndrome concept. These fea-tures suggest that a functional relationship between Bignonieae fl owers and pollinator groups must have played a fundamental role in the morphological diversifi cation of the tribe. Studies on the multivariate aspects of evolution of fl oral phenotypes and a characterization of the ecological context under which the fl owers of Bignonieae evolve will provide further evidence on the processes that may have driven the evolution of fl oral morphologies in this group (S. Alcantara et al., unpublished data). Given the diversity of fl oral types and plant – pollinator interactions represented in Bignonieae, these analyses should greatly improve our knowledge on the processes that drive morphological evolution of fl owers and plant – pollinator inter-actions in the tropics as a whole.

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or maladaptive. This fi nding agrees with the hypothesis that “ non-adaptive ” phenotypes would be excluded under a syn-drome scenario. For example, lack of exserted anthers in fl ow-ers with yellow corollas and curved corollas in red fl owers are in agreement with the described syndromes of melittophily by large to medium-sized bees, and ornithophily by humming-birds, respectively ( Thomson and Wilson, 2008 ). The absence of fl owers with exserted anthers and curved corollas might rep-resent a functionally maladaptive combination because tubular, curved fl owers might prevent hummingbirds from accessing the nectar disk, while exserted anthers might favor pollen trans-fer by hummingbirds ( Grant and Grant, 1968 ).

Among the positive correlations observed, some correspond directly to expectations under the pollination syndrome con-cept. In particular, nectar guides are positively correlated with infundibuliform corollas (and negatively correlated with tubu-lar corollas), as expected by melittophily. Tubular corollas are negatively correlated with nectar guides and positively corre-lated with red and white corollas, as expected by hummingbird and bat-hawkmoth pollination, respectively. In Aquilegia , the switch to white tubular corollas pollinated by hawkmoths tends to occur from red tubular corollas pollinated by hummingbirds, which in turn evolved from fl owers pollinated by bees ( Whittall and Hodges, 2007 ). However, we did not fi nd any indication of this directionality in Bignonieae (see above).

Interestingly, rates of transition in fl ower characters that are cor-related are generally affected by the color of fl owers and not the other way around, suggesting that fl ower color generally guides the transition in other morphological changes. This pattern is in agree-ment with the proposition that reward and signaling characters might drive shifts between syndromes ( Wilson et al., 2006 ; Thomson and Wilson, 2008 ). Changes in morphology and pollen presentation would follow to improve the effectiveness of the new pollinators ( Thomson and Wilson, 2008 ). In this case, detailed studies on the fl ower color transitions in Bignonieae would greatly contribute to a better understanding of the evolution of pollination syndromes in the tribe. These studies, with a historic context pro-vided by upcoming species-level phylogenies (L. G. Lohmann, unpublished data), would be a fruitful approach to understanding pollination shifts at lower taxonomic levels in this group, as well as the ecological processes involved in those shifts.

Despite these interesting results, we highlight that several correlations were not signifi cant after the application of the Bonferroni ’ s adjustment. This correction is recognized to be greatly conservative ( Zar, 1999 ). Hence, putatively weak but signifi cant correlations may have been ignored. The phyloge-netic patterns of correlation among fl oral traits in Bignonieae are being carefully evaluated with a quantitative, multivariate perspective (S. Alcantara et al., unpublished data). We expect that these further analyses on the evolution of fl owers of Bi-gnonieae will give us further insights on the correlated nature of the evolution of fl oral traits.

Conclusions and perspectives — Our results indicate an in-creasing diversifi cation of pollinator systems in Bignonieae, de-parting from an entomophilic ancestral state and passing through several and repeated shifts in fl oral morphology. The overall pat-tern observed indicates an evolution of more derived fl ower types from an ancestor with Anemopaegma -type fl owers. It further suggests repeated specializations, in combination with shifts to generalized Tynanthus -type fl owers. These shifts in fl oral mor-phologies followed both gradual and punctuated modes, suggest-ing that shifts occurred under variable evolutionary dynamics.


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Appendix 1. Characters and respective character states from Lohmann (2003) and Lohmann et al. (in press) . N : number of states, Rate of transition: per Myr assigned by maximum likelihood (ML) reconstructions in the ML-completely resolved and dated tree.

Character States N Rate of transition

01. Nectar guides 1. Absent 2 0.001622. Present

02. Corolla curvature 1. Straight 2 0.000512. Curved

03. Corolla texture 1. Membranous 2 0.001572. Coriaceous

04. Corolla tube 1. Rounded 2 0.001072. Flattened abaxially

05. Corolla lobes 1. Bilabiate 2 0.001032. Not bilabiate

06. Corolla mouth at anthesis 1. Opened 2 0.001032. Closed

07. Anther position in relation to corolla lobes

1. Included 2 0.007732. Exserted

08. Double calyx 1. Absent 3 0.000512. Present3. Only reminiscent present

09. Corolla shape 1. Tubular 3 0.005662. Infundibuliform3. Urceolate

10. Nectar disc 1. Absent 3 0.001052. Reduced3. Present

11. Calyx shape 1. Cupular 4 0.003032. Tubular3. Spathaceous4. Urceolate

12. Corolla color 1. Red 4 0.007982. Magenta3. Yellow4. White

796 American Journal of Botany

Appendix 2. Pollination systems of 45 species of Bignonieae. Species names follow Lohmann (in press) . Flower types were classifi ed according to Gentry (1974b) . Legitimate visitors include all functional groups reported as pollinators in the reference cited herein. Other visitors include illegitimate visitors such as nectar and pollen robbers or visitors that were not observed as resource-thieves.

Species References Flower type Legitimate visitors Other visitors

Adenocalymma bracteatum Amaral 1992 Anemopaegma Large to medium-sized bees Adenocalymma comosum Porsch 1929 in Machado & Vogel 2004 Anemopaegma Hummingbirds Adenocalymma dichilum Machado & Vogel 2004 Martinella Bats Large to medium-sized

bees Adenocalymma marginatum Amaral 1992 Anemopaegma Large to medium-sized bees Amphilophium askersonii Lohmann 2003 Amphilophium Bats, large to medium-sized bees Amphilophium buccinatorium Baker et al. 1998 Martinella Hummingbirds Amphilophium crucigerum Gentry 1974b ; Amaral 1992 Pithecoctenium Large to medium-sized bees Amphilophium paniculatum Amaral 1992 ; L.G. Lohmann pers. obs. Amphilophium Bats, large to medium-sized bees Amphilophium pannosum Gentry 1974b Amphilophium Large to medium-sized bees Anemopaegma chamberlaynii Amaral 1992 ; Correia et al., 2006 Anemopaegma Large to medium-sized bees Anemopaegma orbiculatum Gentry 1974b Anemopaegma Large to medium-sized bees Bignonia aequinoctialis Gentry 1974a , b Cydista Large to medium-sized bees Bignonia corymbosa Gentry 1974a Cydista Large to medium-sized bees Bignonia diversifolia Gentry 1974a Cydista Not observed Beetles (mainly), small

bees Bignonia hyacinthina Gentry 1974b Tynanthus Small bees Small insects Callichlamys latifolia Gentry 1974b Anemopaegma Large to medium-sized bees Dolichadra cynanchoides Galetto 1995 ; Bianchi et al. 2005 Martinella Hummingbirds Dolichandra unguis-cati Gentry 1974b Anemopaegma Large to medium-sized bees Fridericia candicans Gentry 1974a , b Anemopaegma Large to medium-sized bees Small bees Fridericia caudigera Carvalho et al. 2003 Anemopaegma Large to medium-sized bees Fridericia chica Gentry 1974a , b Anemopaegma Large to medium-sized bees Fridericia conjugata Gentry 1974a , b; Correia et al. 2005 Anemopaegma Large to medium-sized bees Small bees, butterfl ies and

hummingbirds Fridericia corallina Gentry 1974a , b Anemopaegma Large to medium-sized bees Fridericia fl orida Gentry 1974a , b Tynanthus Small bees Butterfl ies Fridericia japurensis Gentry 1974b Cydista/Tanaecium Large to medium-sized bees Small bees, butterfl ies Fridericia mollissima Gentry 1974a , b Anemopaegma Large to medium-sized bees Fridericia patellifera Gentry 1974a , b Anemopaegma Bees Hummingbirds, bees,

butterfl ies, wasps, fl ies Fridericia pubescens Araujo & Sazima 2003; Carvalho et al.

2003 Anemopaegma Large to small bees Hummingbirds

Fridericia samydoides Amaral 1992 Anemopaegma Large to medium-sized bees Fridericia speciosa Abreu & Vieira 2004 Martinella Hummingbirds Fridericia triplinervia Amaral 1992 Anemopaegma Large to medium-sized bees Lundia cordata Lopes et al., 2002 Martinella Hummingbirds Small bees, fl ies, wasps,

hummingbird Lundia corymbifera Gentry 1974b Anemopaegma Small hawkmoths Lundia obliqua Amaral 1992 Anemopaegma Large to medium-sized bees Mansoa hymenaea Barrows 1977 Anemopaegma Large to medium-sized bees Wasps, ants, small bees Martinella obovata Gentry 1974b Martinella Hummingbirds Pachyptera kerere Gentry 1974b Tanecium Large to medium-sized bees Pyrostegia venusta Gobatto-Rodrigues & Stort 1992; Gusman

& Gottsberger 1996 Martinella Hummingbirds Large to medium-sized

bees Stizophyllum perforatum Amaral 1992; Carvalho et al. 2003 Anemopaegma/

Martinella Butterfl ies; large to medium-sized bees

Stizophyllum riparium Gentry 1974b Anemopaegma/Martinella

Large to medium-sized bees

Tanaecium jaroba Gentry 1990 Tanaecium Hawkmoth Tanaecium pyramidatum Gentry 1974b; Carvalho et al. 2003 Anemopaegma/

Pithecoctenium Large to medium-sized bee, wasps

Tanaecium selloi Amaral 1992 Anemopaegma Bees Small insects, butterfl ies Tynanthus croatianus Gentry 1974b Tynanthus Small bees Small insects, large to

medium-sized bees Xylophragma mirianthum Carvalho et al. 2003 Anemopaegma Large to medium-sized bees Xylophragma seemanianum Gentry 1974b Anemopaegma Large to medium-sized bees

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