Annals of Botany 107: 1323–1334, 2011 doi:10.1093/aob/mcr084, available online at www.aob.oxfordjournals.org

Pterandra pyroidea: a case of pollination shift within Neotropical

Simone C. Cappellari1,2,*, Muhammad A. Haleem3, Anita J. Marsaioli3, Rosana Tidon2 and Beryl B. Simpson1 1Integrative Biology, The University of Texas at Austin, One University Station A6700, Austin, TX 78712, USA, 2Laborato´rio de Biologia Evolutiva, Departamento de Gene´tica e Morfologia, Instituto de Cieˆncias Biolo´gicas, Universidade de Brası´lia, CP 04457, Brası´lia, DF, 70904-970, and 3Instituto de Quı´mica, Universidade Estadual de Campinas, CP 6154, Campinas, SP, 13083-862, Brazil

* For correspondence. E-mail [email protected] Downloaded from https://academic.oup.com/aob/article/107/8/1323/173604 by guest on 29 September 2021

Received: 2 December 2010 Returned for revision: 1 February 2011 Accepted: 2 March 2011

† Background and Aims Most Neotropical of Malpighiaceae produce floral fatty oils in calyx glands to attract pollinating oil-collecting bees, which depend on this resource for reproduction. This specialized type of pollination system tends to be lost in members of the that occur outside the geographic distribution (e.g. ) of Neotropical oil-collecting bees. This study focused on the pollination ecology, chemical ecology and reproductive biology of an oil flower species, pyroidea (Malpighiaceae) from the Brazilian Cerrado. Populations of this species consist of with oil-secreting (glandular) flowers, plants with non-oil-secreting flowers (eglandular) or a mix of both types. This study specifically aims to clarify the role of eglandular morphs in this species. † Methods Data on were recorded by in situ observations. Breeding system experiments were con- ducted by isolating inflorescences and by enzymatic reactions. Floral resources, and floral oils offered by this species were analysed by staining and a combination of various spectroscopic methods. † Key Results Eglandular flowers of P. pyroidea do not act as mimics of their oil-producing conspecifics to attract pollinators. Instead, both oil-producing and oil-free flowers depend on pollen-collecting bees for reproduction, and their main pollinators are bumble-bees. Floral oils produced by glandular flowers are less complex than those described in closely related genera. † Conclusions Eglandular flowers represent a shift in the pollination system in which oil is being lost and pollen is becoming the main reward of P. pyroidea flowers. Pollination shifts of this kind have hitherto not been demon- strated empirically within Neotropical Malpighiaceae and this species exhibits an unusual transition from a specialized towards a generalized pollination system in an area considered the hotspot of oil-collecting bee diver- sity in the Neotropics. Transitions of this type provide an opportunity to study ongoing evolutionary mechanisms that promote the persistence of species previously involved in specialized mutualistic relationships.

Key words: Cerrado, elaiophores, fatty acids, floral oils, floral rewards, gas chromatography, Malpighiaceae, oil-collecting bees, oil flowers, pollination shift, pollination syndromes, Pterandra pyroidea.

INTRODUCTION are rarely restricted to interactions within a single functional group (Waser et al., 1996; La´zaro et al., 2008). Instead, inter- Many aspects of floral morphology and physiology have long actions tend to show at least some degree of ecological gener- been regarded as the result of selective pressures imposed by alization as most plants are visited by several species of the animals that are the most effective pollinators of particular animals that vary in their contribution to plant reproduction plant species (Darwin, 1877; Grant, 1949; Stebbins, 1970; (Armbruster, 2006). In addition, associations between plants Faegri and van der Pijl, 1979; Baker and Baker, 1983; and their pollinators are subject to continuous temporal and Herrera, 1988; Fenster et al., 2004). This relationship is best spatial fluctuations in their local environment affecting their illustrated by the convergence of combinations of floral traits populations (Herrera, 1988; Horvitz and Schemske, 1990; (morphology, type of resources offered, colour, scents, etc.) Fenster and Dudash, 2001; La´zaro et al., 2010). Thus, ecologi- found in unrelated plant species that depend on the same func- cal generalization in plant– interactions aids in the tional groups of pollinators (bats, birds, large bees, moths, etc.) maintenance and persistence of natural communities (Waser for reproduction (Faegri and van der Pijl, 1979; Fenster et al., et al., 1996). Nonetheless, it has been shown that ecological 2004; Armbruster, 2006; Cronk and Ojeda, 2008). Such con- variation can occasionally select for floral traits, leading to vergence in floral traits is triggered by similarities in diet pre- switches in pollination mode (Schemske and Horvitz, 1984; ference, body size and shape, and foraging behaviour of Thomson and Wilson, 2008). animals that are naturally attracted to flowers which appeal Because pollinators play a critical role in plant reproduction to their physiological and sensory systems (Faegri and van by promoting genetic variation through cross-pollination and der Pijl, 1979; Fenster et al., 2004). Although associations reproductive isolation, shifts in pollination systems are con- between plants and their pollinators can become evolutionarily sidered of major importance for understanding diversification specialized (Armbruster, 2006) over time, mutualistic partners and extinction of flowering plants (Grant, 1949; Stebbins,

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1970; Campbell, 2008). Most studies of pollination shifts have white or pink coloured elaiophores (Fig. 1A, B), or are com- focused on understanding how such shifts happen and on iden- pletely eglandular (Fig. 1C). The corolla is approx. 1.5cmin tifying their role in reproductive isolation between sympatric diameter and consists of five scentless clawed ranging species of congeners (Grant, 1994; Kay and Schemske, from light to dark pink in colour (Fig. 1D). Flowers of this 2002; Bradshaw and Schemske, 2003). species have nearly actinomorphic corollas but, due to differ- The Malpighiaceae is a medium-sized family (1300 ences in the thickness and size of the flag , they are con- species) of herbs, shrubs, vines and trees (Anderson et al., sidered zygomorphic (Anderson, 1997). Corollas lack a 2006). The vast majority of species in the family are distribu- disc and persist on inflorescences after pollination until fruit ted in the Neotropical region (85 %), but some species are maturation, changing their colour to white or yellowish-beige found in the tropics (15 %), especially in before dehiscing. The androecium consists of ten free Madagascar (Anderson, 1979; Davis et al., 2001, 2002). stamens with anthers that dehisce longitudinally. The gynoe-

Neotropical Malpighiaceae are well known for their remark- cium consists of a superior, tricarpelate ovary with each Downloaded from https://academic.oup.com/aob/article/107/8/1323/173604 by guest on 29 September 2021 able ‘oil flower pollination syndrome’ a pollination mode locule bearing a single ovule. The three styles found in the characterized by the production of floral oils (lipids) which centre of the flower have minute, transparent stigmas. are harvested by female oil-collecting bees that use the oils Differences in height of styles (longer) and anthers (shorter) to feed their brood and/or to line their nest cells (Vogel, indicate herkogamy. Fruits are dry and composed of three indi- 1974; Neff and Simpson, 1981; Simpson, 1989). Like other vidually seeded units (i.e. cocci) (Anderson, 1997). oil flower taxa, Malpighiaceae flowers generally lack nectar At our field site in the Cerrado, considered a hotspot of and rely on floral oils and, sometimes, pollen, as their main oil-collecting bee diversity in Brazil (Pinheiro-Machado rewards for pollinators (Vogel, 1974). Interactions of et al., 2002; Raw et al., 2002), we discovered mixed Malpighiaceae with oil-collecting bees from two tribes, P. pyroidea populations consisting of individuals with either and Tapinotaspidini (Apinae), are thought to be exclusively glandular or eglandular flowers. To understand ancient and have influenced the diversification rates in the the pollination ecology role of eglandular individuals in this family since its origin (Anderson, 1990; Davis et al., 2001; species and their influence on the oil–bee mutualism, the fol- Davis and Anderson, 2010). The strength of this association lowing non-mutually exclusive questions were addressed. (1) is best illustrated by the consistent floral morphology through- Are eglandular flowers mimics of their glandular conspecifics? out Neotropical members of the family, which show adap- If eglandular flowers attract pollinators by deceit, as described tations to oil-collecting bee pollination (Vogel, 1974; in populations of other Malpighiaceae (Sazima and Sazima, Anderson, 1979; Zhang et al., 2010). Among characteristic 1989; Teixeira and Machado, 2000), it would be expected floral features of the family are a zygomorphic corolla with a that (a) the ratio between different floral morphs would be differentiated ‘flag’ petal – a dorsal, differentiated petal biased towards glandular individuals in mixed populations; which is used by oil-collecting bees for orientation and and (b) oil-collecting bees would visit both floral morphs in support while harvesting resources on the flower – as well search for oil. (2) Is the lack of elaiophores associated with as the presence of oil-secreting calyx glands, called ‘elaio- the evolution of autogamy in this species? Autogamy and phores’ (Vogel, 1974; Anderson, 1979). The idea of floral con- loss of elaiophores were shown to be innovations in other oil- servatism as a result of selection imposed by pollinators is producing plants, which have colonized habitats where oil- further supported by the absence of typical floral features in collecting bees are scarce or absent (Se´rsic, 2004). (3) Does some Paleotropical taxa of this family where the Neotropical the current coexistence of glandular and eglandular flowers Malpighiaceae-associated oil-collecting bee groups do not represent a pollination shift in P. pyroidea which was orig- occur (Anderson, 1979, 2001; Davis, 2002; Davis and inally pollinated by oil-collecting bees? In the case of a polli- Anderson, 2010; Zhang et al., 2010). nation shift, one would expect eglandular individuals to be The present study focused on the pollination ecology of oil- pollinated by bees seeking rewards different from oil, as has producing (glandular) and oil-less (eglandular) individuals of been demonstrated in Old World Malpighiaceae, which have Pterandra pyroidea A. Juss. in an area of the Brazilian lost their association with oil-collecting bees (Davis, 2002). Cerrado. Pterandra is a of shrubs and trees that occurs To test these hypotheses, data on pollinators, breeding in forests and savannas of South and Central America systems, phenology and floral rewards were gathered and a (Anderson, 1997, 2005). The most recent systematic treatment comparison of these data for both floral types was conducted. recognizes 15 species in the genus (Anderson, 1997; Anderson et al., 2006) and phylogenetic analyses indicate Pterandra as one of the most basal lineages within Malpighiaceae (Davis MATERIALS AND METHODS et al., 2001; Davis and Anderson, 2010). Although most Study site species of the genus bear calyx glands, at least one other species, P. sericea W. R. Anderson, from Venezuela, is The study was conducted from 2007 to 2010 in the Reserva known to have glandular and eglandular flowers (Anderson, Ecolo´gica of IBGE (RECOR), a protected area located 1997). 35 km south of Brası´lia, Central Brazil (1000 m a.s.l., Pterandra pyroidea is a perennial shrub of 1–2 m height, 15856′S, 47853′W). The area is part of the Cerrado biotic with opposed, elliptical , and inflorescences composed realm, a seasonal South American ecosystem characterized of 4–6 fascicles of pink, showy flowers, located under apical by different types of phytophysiognomies that range from stem leaves. The calyx of P. pyroidea consists of five humid, open grasslands to savannas and closed canopy pinkish, triangular sepals each of which bears one or two forests (Eiten, 2001; Gottsberger and Silberbauer- Cappellari et al. — Pollination shift in Neotropical Malpighiaceae 1325

A B C Downloaded from https://academic.oup.com/aob/article/107/8/1323/173604 by guest on 29 September 2021

D E

F IG. 1. Morphology and pollinator interaction of Pterandra pyroidea flowers. (A) Inflorescence of a glandular individual showing elaiophores on the sepals and differences in height between styles and anthers. (B) Wilted inflorescence of a glandular individual with reduced elaiophores. (C) Dorsal view of an eglandular flower and floral buds showing sepals lacking elaiophores. (D) Ventral view of a floral corolla; arrow indicates the flag petal. (E) A bumble-bee (Bombus cf. brevivillus) collecting pollen on a flower.

Gottsberger, 2006). The annual precipitation in this area these populations had only eglandular flowers and two had (1400 mm) is concentrated during the rainy season only glandular flowers. One population was composed of (October–March/April). The dry season extends from glandular and eglandular individuals and it was used to deter- April/May to September/October and it is characterized by mine the ratio of glandular to eglandular individual plants per cooler temperatures, especially at night (Gottsberger and area (m2). Phenology data for five glandular and five eglandu- Silberbauer-Gottsberger, 2006). Observations and experiments lar individuals were recorded weekly over one flowering on populations of P. pyroidea were carried from July to season (August–October 2008). The data on flower production October during the second half of the dry season at our field for each flower type were analysed using a repeated measures site. Field work was conducted in a shrub savanna (Campo analysis of variance (ANOVA; a ¼ 0.05) with one between- Sujo) and an open-canopy scrub woodland (Cerrado; sensu subjects variable and one within-subjects variable using the Eiten, 2001). Vouchers of each Pterandra morphotype are statistical package R. Variation in the number of elaiophores deposited at the herbaria of RECOR (IBGE), Instituto de in glandular individuals was quantified by counting the Botaˆnica de Sa˜o Paulo (SP), Universidade de Brası´lia (UB) number of glands per flower for 30 randomly selected and The University of Texas at Austin (TEX). The identity flowers from five different individuals in this population. of plant vouchers was confirmed by M. C. Mamede from the Instituto de Botaˆnica de Sa˜o Paulo. Pollen quantification Preliminary observations on pollinator visitation frequency Population survey and phenology to each floral type indicated that glandular flowers received Preliminary observations carried out in 2007 indicated that fewer visits than eglandular flowers. Therefore, pollen pro- individuals of P. pyroidea have either glandular or eglandular duction was quantified to determine if differences in quantity flowers. Although glandular flowers may have reduced of pollen produced by glandular and eglandular flowers were numbers of glands, glandular and eglandular flowers are not significant. We collected 5–6 buds per plant from five individ- found on the same plant. Six populations were studied and uals of each morphological type and used individual anthers to each was examined to determine the proportion of individual produce a total of 26 pollen slides for each morph (glandular plants bearing glandular or eglandular flowers. Three of or eglandular). This was the appropriate sample size required 1326 Cappellari et al. — Pollination shift in Neotropical Malpighiaceae for determining large differences in the average pollen pro- TABLE 1. Fatty acids detected as methyl ester derivatives in duction between floral types (power level ¼ 0.80; a ¼ 0.05) Pterandra pyroidea floral oils according to a power analysis using the G-Power v.3 statistical program. To avoid differences due to intermediate stages of Peak RT RA elaiophore occurrence, only buds that had ≥8 elaiophores number Name of chemical compound (min) RI (%) were used to make slides of glandular flowers. Pollen grains 1 Docasanoic acid 25.45 2569 0.85 from individual anthers were suspended in liquid solution 2 Tetracosanoic acid 29.28 2760 0.89 and the number of pollen grains in known aliquots of the 3 5,7-Diacetoxydocosanoic acid 33.40 2974 85.80 liquid was counted (Protocol 3.2inDafni et al., 2005). 4 5,7-Diacetoxytetracosanoic acid 36.86 3146 7.25 Permanent slides were made for each sample using glycerin jelly containing fuchsin stain. The total pollen production Peak number refers to peaks detected in the chromatogram (Fig. 3); RT, retention time in minutes; RI, calculated retention index; RA, percentage of Downloaded from https://academic.oup.com/aob/article/107/8/1323/173604 by guest on 29 September 2021 per flower was calculated by multiplying the number of relative abundance in comparison with other compounds in the extract. grains in the aliquot by the number of aliquots in the liquid and then by the number of anthers per flower. The mean number of pollen grains for each floral morph were compared 3.5 kV, capillary voltage ¼ 43 V, capillary temperature ¼ using a two-sample, two-tailed Student’s t-test (a ¼ 0.05). 275 8C. Full scan experiments (range m/z 100–1300) were Additionally, a Kolmogorov–Smirnov test for independent performed in both the linear trap and the Orbitrap. Fragment samples (a ¼ 0.05) was performed to compare the distribution masses were acquired as profile data at a resolution of of data points between the two floral types. Both tests were 30 000 at m/z 400. The automatic gain control (AGC) ion performed using the statistical package R. population target in full scan MS was 50 000 for LTQ-MS and 500 000 for Orbitrap-MS and the ion population target Chemical analyses of floral oils for MSn was set to 10 000 for LTQ-MS. The maximum ion injection time was 10 ms. Mass data were analyzed with To characterize the floral oils produced by P. pyroidea,a Xcalibur software (Thermo Finnighan). total of 27 flowers (¼137 elaiophores) were extracted in The NMR spectral data were acquired with an 11 T Varian 3 mL of ethyl acetate for 10 min from which a total of 3 mg Inova spectrometer, operating at 499.88 MHz for 1H-NMR and of residue was obtained. Thin-layer chromatography (TLC) at 125.71 MHz for 13C-NMR. CDCl was used as solvent, and . 3 revealed one major spot (Rf 0 1, ethyl acetate/hexane 2/8) tetramethylsilane (TMS; 0.0 p.p.m.) as an internal reference compatible with the presence of carboxylic acid derivatives. (d 0.0 ppm). Chemical shifts (d) were recorded in ppm and Therefore, the floral oils were subjected to methylation using coupling constants J in Hz. diazomethane as the methylating agent. The methyl esters The major constituent of the floral oil was isolated from the were then analysed by gas chromatography with electron methylated crude floral oils using silica column chromato- impact mass spectrometry (GC/MS-EI) according to methods graphy eluted with hexane and increasing amounts of ethyl previously established (Reis et al., 2007). Another 67 flowers acetate. The column fractions were monitored by TLC eluted (¼554 elaiophores) were extracted in ethyl acetate, methylated with hexane and ethyl acetate (8:2) and developed with sulfu- and used to isolate compounds and determine their structure ric acid–anisaldehyde solution (Wagner et al., 1984). The iso- with nuclear magnetic resonance (NMR) techniques. lated pure compound (10 mg) was considered a standard and The GC-MS analyses were carried out using an Agilent- characterized by 1H- and 13C-NMR spectroscopy. The results 6890/5973 system equipped with an HP-5 fused silica capillary obtained from NMR analysis were in good agreement with column (30 m × 0.25 mm × 0.25 mm). The oven temperature the structure proposed by the mass fragmentation pathway. was programmed from 150 to 290 8Cat48Cm21. Injector temperature was 240 8C. Helium was used as carrier gas at a flow rate of 1 mL m21. Mass spectra were taken at 70 eV Breeding system with a mass scanning from m/z 40 to 700 amu (satomic mass units). The retention indices (RIs) were obtained by Floral phenology was examined in situ. To test if a temporal co-injecting the crude floral oil with a normal hydrocarbon separation of reproductive activity existed, we examined 17 mixture (C17,C19,C25 and C32; Aldrich) as an internal stan- flowers from five individuals of each morph on the first day dard. Plotting the n-hydrocarbon retention times vs. the hydro- the flowers opened. Pollen availability was determined directly carbon carbon number × 100 generated the equation RI ¼ by observation of the anthers. Pollen viability was tested using 50.52 X + 1284.04 by linear regression with a correlation pollen from five fresh flowers from five different individuals of coefficient R2 ¼ 0.999. The RI of each constituent (Table 1) each floral morph collected on the first day of floral opening. was obtained by inserting the retention time (X ) into the Pollen grains were stained with aceto-carmine solution above equation. (Radford et al., 1974) and mounted on slides with colourless Electrospray ionization mass spectrometry (ESI-MS) was glycerin jelly. The percentage of viable pollen was evaluated performed using a LTQ-Orbitrap hybrid mass spectrometer by counting the number of robust stained grains among 300 (Thermo Fischer Scientific, Bremen, Germany). Nitrogen gas grains per slide. was used for the nebulization, desolvation and the It has been shown that several species of Malpighiaceae collision-induced dissociation (CID). The sample was diluted have a stigmatic cuticle that becomes filled with liquid when in methanol, infused at a flux of 10 mLmin21, and detected receptive (Sigrist and Sazima, 2004). Thus, stigmatic receptiv- in the positive ion mode; sheath gas ¼ 8, spray voltage ¼ ity of P. pyroidea was determined by observing the stigmas Cappellari et al. — Pollination shift in Neotropical Malpighiaceae 1327 under high magnification (×40) using a field microscope and Entomological Collection at the Universidade de Brası´lia. by conducting a hydrogen peroxide test (Zeisler, 1938). The Bees were identified by A. J. C. Aguiar (University of number of ovules per flower was quantified for the same 26 Brası´lia), S. C. Cappellari, G. A. R. Melo (Federal University flowers of each floral type that were used for pollen of Parana´) and J. L. Neff (Central Texas Melittological quantification. Institute) based on reference collections and the taxonomic To determine P. pyroidea’s dependence on external vectors literature (Silveira et al., 2002; Moure et al., 2007). for reproduction, breeding system studies were conducted on glandular and eglandular morphs (2009–2010). Inflorescences (which varied in the number of flowers they RESULTS bore) were marked and isolated with net bags in the bud Populations stage. The following tests were applied: (a) no manipulation to test for spontaneous selfing; (b) hand-pollination of Pterandra pyroidea is the only species in the genus occurring Downloaded from https://academic.oup.com/aob/article/107/8/1323/173604 by guest on 29 September 2021 flowers with pollen of the same plant to test for potential gei- in the Central Brazilian Cerrado (Gavilanes and Ferreira, tonogamy; (c) manual cross-pollination to test for outbreeding 1974–1976; Anderson, 1997; Pereira et al., 1998; Mendonc¸a and the effect of manual pollination; and (d) open, non-bagged et al., 2008) and populations of P. pyroidea were common inflorescences served as controls testing for the success rate of throughout the different types of Cerrado vegetation (Campo natural pollination. For geitonogamy and cross-pollination Sujo, Campo Cerrado, Cerrado and Cerrada˜o sensu Eiten, experiments, flowers were emasculated prior to pollination. 2001) at our field site. The population with plants of both For hand-pollination, pollen was applied onto a glass slide floral morphs had 212 individuals distributed in an area of and rubbed against a stigma. All flowers were pollinated on 1850 m2, of which 32 were glandular and 180 eglandular the first day of flowering and remained bagged until fruits (ratio of 1:6), with densities of 0.017 individuals/m2 and were fully developed or dropping of the corolla unit was 0.11 individuals m22, respectively. In glandular individuals, observed. Mature fruits were collected, counted, opened and the number of elaiophores per flower was variable (mean ¼ the number of seeds produced counted in the laboratory as a 8, s.d. ¼ 2.12, min. ¼ 2, max. ¼ 10, n ¼ 30). In addition, measure of reproductive success. Fruit set results were ana- glandular flowers of some individuals showed a strong lysed with the SAS statistical package by a binomial logistic reduction in elaiophore size (Fig. 1B). regression with the Firth method to control for quasi- separation of data in logistic models (Firth, 1993). The P-value obtained for the interaction between floral type and Phenology treatment approached significance (P ¼ 0.12). Therefore, Flowers of both floral morphs opened throughout the day at exploratory post-hoc analyses were additionally run with x2 our field site. Initial opening occurred between 0800 and (a ¼ 0.05) tests for independence in fruit set of floral 0900 h when temperatures increased to .20 8C. Glandular morphs within each treatment as well as between treatments and eglandular P. pyroidea plants both bloomed for about 10 within each floral morph. weeks during the second half of the dry season. Temporal vari- ation in the beginning but not in the duration of the flowering Pollinators period was observed in populations of both floral morphs studied in 2008 (August–October), 2009 (July–September) Preliminary observations of floral visitors were carried out in and 2010 (June–August). During the collection of phenologi- 2007 and the results of these observations are reported in the list cal data in 2008, flower production peaked in the second week of pollinator species observed on flowers. Preliminary obser- of September. During this period, eglandular individuals pro- vations were conducted to determine a time window that duced, on average, more flowers per plant (mean ¼ 137 would cover most of the pollinator activity period since flowers per plant, s.d. ¼ 96.4) than their glandular conspecifics drastic temperature decreases at night were observed to delay (mean ¼ 95 flowers per plant, s.d. ¼ 151) (Fig. 2A). However, pollinator activity in the morning hours during the dry a repeated measures ANOVA of the weekly flower production season. During the 2008 and 2009 flowering seasons, randomly by the two floral morphs throughout the entire flowering chosen individuals of Pterandra with either glandular or season indicates that the difference was not significant eglandular flowers were observed individually for 30 min (F ¼ 0.48, P ¼ 0.54, d.f. ¼ 1). several times per week, totalling 16.5 h of observation time per floral morph. Overall, 66 observations of 30 min each were carried out, 40 of which were in the morning hours Pollen production (0730–1200 h). Floral visitors were collected and notes on their behaviour on flowers (touching/not touching reproductive The difference in the total number of pollen grains per flower parts) as well as the resource collected (pollen/oil) were produced by glandular (mean ¼ 115 642, s.d. ¼ 33 896, recorded. Visitation frequency (VF) was calculated by dividing median ¼ 116 200, n ¼ 26) vs. eglandular flowers (mean ¼ the total number of individuals per bee species recorded on 112 161, s.d. ¼ 65 069, median ¼ 88 150, n ¼ 26) was not sig- flowers by the total observation time for each floral morph nificant (t ¼ 0.24, P ¼ 0.81, d.f. ¼ 37.63, a ¼ 0.05). However, (16.5 h, 2008–2009 seasons) yielding a rate of bees per hour. it is noteworthy that the maximum count of pollen per flower was The number of visitors between morphs was compared by a higher in eglandular than glandular flowers (maxeglandular ¼ two-factor negative binomial distribution in the statistical 287 600 vs. maxglandular ¼ 182 500; Fig. 2B), and a non- package R. Bee vouchers were deposited in the parametric comparison of the variation in the data distribution 1328 Cappellari et al. — Pollination shift in Neotropical Malpighiaceae

140 A 100 90 120 80 3 70 100 60 80 50 40

60 (%) Abundance 30 40 20 10 4 1 2 Downloaded from https://academic.oup.com/aob/article/107/8/1323/173604 by guest on 29 September 2021 20 0 Mean number of flowers per plant of flowers Mean number 20 30 40 0 Retention time (min)

13-Aug 29-Aug 12-Sep 25-Sep 10-Oct F IG. 3. Total ion chromatogram of Pterandra pyroidea floral oils. Normalized Date percentage of the relative abundance (RA) of each component in floral oil indi- cated by the vertical axis, and retention time (RT) of each compound shown in B minutes in the horizontal axis. Numbers refer to compounds in Table 1.

250 m/z 43 is indicative of an acetyl group, while intense peaks at m/z 131, m/z 173, m/z 259 and m/z 157 suggest a 200 5,7-diacetoxy substitution. Moreover, the ESI-LTQ-Orbitrap mass spectra suggest a molecular mass of 493.3510 amu . 150 (at 30 000 resolution) for component #3 and 521 3821 amu (at 30 000 resolution) for component #4, both consistent for pseudo molecular ions Na C27H50O6 and Na C29H54O6, 100 respectively. Therefore, combined spectroscopic data indicate that these fatty acids detected are: (a) 5,7-diacetoxydocosanoic

Pollen grains per flower (× 1000) per flower grains Pollen acid (Fig. 3, peak #3) and (b) 5,7-diacetoxytetracosanoic acid 50 (Fig. 3, peak #4), comprising the major and minor non- methylated components of the floral oil, respectively (Table 1). Glandular Eglandular Floral morph

F IG. 2. (A) Flowering phenology of Pterandra pyroidea during the dry Breeding systems season of 2008. Glandular phenology is indicated by a solid line (n ¼ 5) and eglandular (n ¼ 5) by a dotted line. (B) Pollen production by glandular Styles of freshly opened flowers are initially bent and (n ¼ 26) and eglandular (n ¼ 26) flowers of P. pyroidea. Grey squares indicate become fully erect 30–60 min after the flower opens. the respective averages for each morph: glandular (average ¼ 115 642; s.d. ¼ Flowers are protogynous (n ¼ 17) as the stigmatic region 33 896; median ¼ 116 200); eglandular (average ¼ 112 161; s.d. ¼ 65 069; becomes receptive and reacts to hydrogen peroxide before ¼ median 88 150); black dots indicate outliers. anthers are fully open. Anthers dehisced 2–3 h after the flowers opened, but passive pollen deposition on stigmas is of pollen production between glandular and eglandular samples prevented by spatial separation of floral organs (herkogamy) approached significance (D ¼ 0.35, P ¼ 0.08). (Fig. 1A). Most anthers are empty by the second day of flower- ing. In glandular flowers, 94 % of pollen grains stained with aceto-carmine solution (mean ¼ 283 stained grains, N ¼ 5) and in eglandular flowers 92 % of the pollen stained Chemistry of floral oils (mean ¼ 274 stained grains, n ¼ 5), indicating full fertility. Glandular individuals of P. pyroidea produce, on average, There was virtually no variation in the number of ovules 0.12 mg of oils per flower (s.d. ¼ 0.06 mg), and individual per flower in each morph (meanglandular ¼ 3, n ¼ 26; elaiophores contain, on average, 0.02 mg of oils (s.d. ¼ meaneglandular ¼ 2.96, n ¼ 26). 0.004 mg). The total ion chromatogram of the methylated The results of the breeding system experiments (Table 2) floral oil indicated that the chemical composition of refer to the total number of flowers tested (n). Neither morph P. pyroidea floral oil is rather simple, composed of one produced fruits by spontaneous selfing (autogamy) (nglandular ¼ major and three minor free fatty acids (Fig. 3). Mass fragmen- 45, neglandular ¼ 92). As a result of the geitonogamy experiments, tation, and 1H- and 13C-NMR chemical shift assignment indi- 40 % of the tested glandular flowers (n ¼ 5) and 25 % of the cated that the first two components (Fig. 3, peaks #1 and #2) eglandular flowers (n ¼ 64) produced fruits, indicating that are free fatty acids with no carboxyl group substitutions. The both forms are self-compatible. Cross-pollination experiments same techniques revealed that the other free fatty acids yielded fruit sets in 2 % (n ¼ 37) of the glandular and 12.3% detected (Fig. 3, peaks #3 and 4) have substitutions with two (n ¼ 97) of the eglandular flowers. Interestingly, the open, acetoxy groups each. In the mass spectrum the base peak at unbagged flowers of glandular individuals (n ¼ 28) did not Cappellari et al. — Pollination shift in Neotropical Malpighiaceae 1329

TABLE 2. Results of breeding systems experiments of Pterandra pyroidea at the Reserva Ecolo´gica do IBGE, Brasilia, Brazil

Glandular Eglandular

Treatment n Flowers Fruits (%) Seed set n Flowers Fruits (%) Seed set x2 P-value d.f.

Spontaneous selfing (autogamy) 4 45 0 (0.0) 0 4 92 0 (0.0) 0 0 1 1 Manual selfing (geitonogamy) 1 5 2 (40.0) 2 6 64 16 (25.0) 27 0.042 0.83 1 Cross-pollination 2 37 1 (2.0) 1 8 97 12 (12.3) 15 1.860 0.17 1 Control (open pollination) 3 28 0 (0.0) 0 8 340 92 (27.0) 134 1.021 ,0.05 1

n refers to the number of individual plants used for experiments; flowers refer to the total number of flowers tested per flower morph and treatment; fruits (%) report counts of fruits produced as a results of each experiment and the proportion of fruit set in parentheses; seed set reports counts of total seeds produced in each experiment; x2 indicates the post-hoc x2 test statistic comparison of test results between floral morphs; P-value is the obtained by x2. Downloaded from https://academic.oup.com/aob/article/107/8/1323/173604 by guest on 29 September 2021

TABLE 3. Binomial logistic regression and maximum likelihood estimates results for comparisons in fruit set of Pterandra pyroidea flowers

Comparison Parameter d.f. Estimate s.e. Wald’s x2 Pr . x2 Exp (Est.)

Flower type Glandular vs. eglandular 1 –0.3591 0.3521 1.0404 0.3077 0.698 Between treatments Self-pollination vs. control 1 –2.2041 0.7978 7.6335 0.0057 0.110 Manual pollination vs. control 1 1.9540 0.4870 16.1017 , .0001 7.057 Cross-pollination vs. control 1 0.1042 0.4740 0.0483 0.8260 1.110 Interaction between Glandular vs. eglandular and self-pollination vs. control 1 0.7139 0.7978 0.8008 0.3709 2.042 treatments and flower type Glandular vs. eglandular and manual pollination vs. control 1 0.7298 0.4870 2.2462 0.1339 2.075 Glandular vs. eglandular and cross-pollination vs. control 1 –0.2754 0.4740 0.3377 0.5612 0.759

Comparisons between breeding system treatments applied to flowers, flower type (glandular/eglandular) and the interaction between treatment and flower type are reported. Pr.x2 indicates P-values for null model comparisons; Exp (Est.) indicate the odds ratio. Overall treatment and flower type interactions are indicated in the Results. produce fruits whereas eglandular flowers developed fruits in indicated significant differences between treatments (x2 ¼ 27 % of the marked flowers (n ¼ 340). The results of the bino- 37.76, P , 0.05). The residuals demonstrated that the fruit mial logistic regression with Firth’s correction indicate that the set by open pollination (Rs ¼ 2.79) exceeded the expected differences in fruit production depended on the different treat- values for this treatment, while fruit set by spontaneous ments applied (Wald x2 ¼ 17.38, P , 0.05, d.f. ¼ 3) and no selfing was lower than that expected (Rs ¼ –4.31). interaction between floral type and treatment applied could be detected (Wald x2 ¼ 5.78, P ¼ 0.12, d.f. ¼ 3). Pairwise Pollinators comparisons indicated that differences in fruit production could not be explained by the identity of the tested floral A total of 38 individuals representing 13 bee species were morph (Wald x2 ¼ 1.04, P ¼ 0.31; Table 3). Overall, differ- recorded on flowers of P. pyroidea (Table 4). During VF ences in the number of fruits produced by spontaneous observation periods (seasons 2008–2009), seven and 27 bees selfing (Wald x2 ¼ 7.63, P ¼ 0.05) as well as manual selfing were collected on glandular and eglandular flowers, respect- (Wald x2 ¼ 16.10, P , 0.05) within both floral types com- ively. The negative binomial distribution analysis indicated bined were significantly different from those obtained by the that the VF of pollinators to eglandular flowers was signifi- control treatment (Table 3). cantly higher than the VF to glandular flowers (u ¼ 2.25, Post-hoc tests of independence of fruit set results between s.d. ¼ 2.42, P , 0.05). For both morphs VFs were higher in floral morphs within each treatment showed a significant the morning than in the afternoon hours, especially between difference for the number of fruits produced in openly polli- 0900 and 1200 h. In glandular flowers, 86 % (n ¼ 6) of the nated flowers (x2 ¼ 8.71, P , 0.05; Table 2). An analysis of visitors were recorded in the morning, while in eglandular the residuals (Rs) of these data indicated that glandular flowers the percentage of visitors observed in the morning flowers produced fewer fruits than expected (Rs ¼ –2.64) in was 67 % (n ¼ 18). With the exception of a single patrolling this category. These differences were not found in other treat- male of Temnosoma sp., all visitors recorded on either floral ments. The post-hoc analyses of fruit sets for each treatment morph collected pollen (Table 4). Three species of bumble- within glandular flowers were indicative of deviations from bees, Bombus brevivillus, B. morio and Bombus sp., were the the expected values (x2 ¼ 29.47, P , 0.05). An analysis of most frequent visitors of eglandular flowers (VF ¼ 0.91 bees the residuals for each of the four treatments indicated that h21), two of which (B. brevivillus and B. morio) also visited the number of fruits formed by manual selfing was larger glandular flowers (VF ¼ 0.18 bees h21). When foraging on than expected (Rs ¼ 5.17). The post-hoc comparisons of Pterandra, bumble-bees use their middle and hind legs to fruit sets between treatments within eglandular flowers also hold on to petals while using their forelegs to scrape anthers 1330 Cappellari et al. — Pollination shift in Neotropical Malpighiaceae

TABLE 4. Floral visitors of glandular and eglandular flowers of strategy because oil-collecting bees would more frequently Pterandra pyroidea encounter the mimic (eglandular) than the putative model (glandular flowers) while foraging for oils. Moreover, oil- Total individual bees collecting bees were rarely observed on Pterandra flowers. The single individual oil-collecting bee (Paratetrapedia sp.) Glandular Eglandular Resource Bee species flowers flowers collected caught during our study was foraging for pollen and not for oil on glandular flowers. Bees of the oil-collecting bee genus Apis mellifera Linnaeus 1 C 2 C Pollen Paratetrapedia are thought to be ‘oil thieves’ of Augochlora sp. – 1 C Pollen Malpighiaceae flowers and are considered to be primarily Bombus brevivillus 2 C 10 C Pollen associated with oil flowers from other plant families (Vogel, Franklin Bombus morio 1 C 4 C Pollen 1974). Thus, it is unlikely that the only oil-collecting bee Swederus caught on glandular flowers is a significant pollinator. In Downloaded from https://academic.oup.com/aob/article/107/8/1323/173604 by guest on 29 September 2021 Bombus sp. – 2 C Pollen addition, the lack of any oil-collecting bee visits to eglandular Exomalopsis auropilosa –1C Pollen flowers is not consistent with a hypothesis of auto-mimicry in Spinola this species. Therefore, our data suggest that eglandular indi- Exomalopsis campestris –1C Pollen Silveira viduals of P. pyroidea do not seem to attract pollinators by Paratetrapedia sp. 1 C – Pollen deceptive auto-mimicry. Paratrigona lineata 2 C 4 C Pollen The results of the breeding system experiments provide evi- Lepeletier dence for dependence on biotic pollination by glandular as Temnosoma sp. – 1 F Patrolling Tetragona sp. 1 C 1 C Pollen well as eglandular individuals of P. pyroidea. Although Trigona spinipes –2C Pollen P. pyroidea is capable of self-fertilization, self-pollination is Fabricius prevented by herkogamy and dichogamy. A change in repro- Trigonisca intermedia –1C Pollen ductive strategy from mainly outcrossing to complete autoga- Moure mic has been suggested for some Calceolaria Total 8 30 (Calceolariaceae) species that have lost oil production after ‘Total individual bees’ refers to data collected from 2007 to 2010. colonizing Andean high altitude habitats where oil-collecting bees are rare or absent (Molau, 1988; Se´rsic, 2004; Cosacov et al., 2009). However, our results indicate that autogamy is and harvest pollen which is later transferred to the corbicula not part of the reproductive system of P. pyroidea. (Fig. 1E). The ventral part of their body comes in contact Several lines of evidence suggest that P. pyroidea is under- with the stigmas and anthers during the visit as the bees going a transition in pollination syndrome in which oil is no rotate on the flower in order to reach all anthers that encircle longer the principal resource sought by pollinators on either the gynoecium. Two species of stingless bees, Paratrigona glandular or eglandular flowers. This hypothesis is supported lineata (VFglandular ¼ 0.12; VFeglandular ¼ 0.24) and by the observation that bumble-bees were the most common Tetragona sp. (VFglandular ¼ 0.06; VFeglandular ¼ 0.06), and visitors of both floral types and, given their high visitation fre- the Africanized honey bee, Apis mellifera (VFglandular ¼ quencies, bumble-bees are likely to be the effective pollinators 0.06; VFeglandular ¼ 0.12), visited both floral morphs as well. of Pterandra. The similar body sizes and foraging behaviours Some stingless bees collected pollen from anthers without of bumble-bees and oil-collecting centridine and Monoeca coming into contact with the stigma, whereas honey bees con- bees also indicate that bumble-bees could potentially replace tacted the reproductive parts and had similar foraging habits to oil-collecting bees as sternotribic pollinators of Pterandra bumble-bees. A single female oil-collecting bee, flowers. Furthermore, eglandular flowers attracted more visi- Paratetrapedia sp. (Tapinotaspidini), was observed on tors and produced more fruits under natural conditions than flowers during the observation period foraging for pollen on their glandular conspecifics, suggesting that the eglandular, glandular flowers of P. pyroidea (VFglandular ¼ 0.06; oil-less phenotype is currently favoured by the local environ- VFeglandular ¼ 0). No centridine or other oil-collecting bees mental conditions. In addition, the presence of intermediate were observed to visit or approach either flower type. stages (flowers with reduced elaiophores) and predominance of large populations of eglandular individuals in the area suggest an increase in frequency of the eglandular phenotype. DISCUSSION Evolutionary scenario for a pollination shift in Pterandra This study focused on clarifying the role of eglandular individ- uals in populations of the oil flower P. pyroidea. The discus- Evolutionary models of pollination shifts propose that tran- sion is therefore concentrated on answering the questions sitions in pollination systems occur in populations of plants initially stated. We showed that, in our study site, individuals and pollinators that have experienced ecological (e.g. lack of with eglandular flowers occur at a higher frequency than pollinator visits and lack of flowering individuals of a given their glandular conspecifics in mixed populations of floral floral host) and evolutionary changes (e.g. mutations) which morphs, and oil-collecting bees were rarely observed on set them apart from their parent populations (Horvitz et al., Pterandra flowers. The bias in floral morph ratio towards 1990; Se´rsic and Cocucci, 1996; Bradshaw et al., 2003; eglandular individuals recorded in mixed populations of this Cronk and Ojeda, 2008; Thomson and Wilson, 2008). Based species indicates that cheating (mimicry) would be an unstable on our observations, glandular individuals of P. pyroidea are Cappellari et al. — Pollination shift in Neotropical Malpighiaceae 1331 experiencing a reduction in visitation frequency by the pollina- Species-specific associations have not been demonstrated in tors originally associated with this species (i.e. oil-collecting the oil flower pollination syndrome involving Malpighiaceae. bees), and the occurrence of either glandular or eglandular Instead, a lack of specificity has been proposed based on the flowers in individual plants indicates that this trait has a use of multiple species for oil acquisition among the main pol- genetic basis. Although the presence of elaiophores and the linators of this group (centridine and Monoeca bees) in areas need for biotic vectors for reproduction both indicate that with a high diversity of Malpighiaceae (Gottsberger, 1986; this species interacted with oil-collecting bees in the past, it Vogel, 1988; Sigrist and Sazima, 2004). Thus, it is probable is not clear which factors led to the loss of the association. that instead of losing the opportunity to interact with oil- Natural variation in pollen production among individuals of collecting bees, populations of P. pyroidea became rather unat- P. pyroidea could have attracted more bumble-bees, leading tractive to the local representatives of this pollinator group. to a switch from ‘occasional’ to ‘frequent’ pollen-gathering Both quality and quantity of floral oils may be potential visitors of its flowers. Such an increase in visitation by reasons for the low attractiveness of this species as an oil Downloaded from https://academic.oup.com/aob/article/107/8/1323/173604 by guest on 29 September 2021 bumble-bees could have led to a higher contribution to fruit source to oil-collecting bees in the area. Our results indicate production by this pollinator group. These conditions could that P. pyroidea flowers produce low quantities of oil have set the stage for a gradual switch from oil to pollen (mean ¼ 0.12 mg per flower) when compared with other flowers followed by morphological changes as a result of pol- Malpighiaceae species at our study site, which produce an linator selection, mutations or both. During the blooming average of 1 mg of oils per flower (M. A. Haleem and period of P. pyroidea bumble-bees occasionally visit and S. C. Cappellari, unpubl. res.). In addition, the oils produced collect pollen from two other oil-producing Malpighiaceae by P. pyroidea have a simple composition with four major species anisandra (A. Juss.) Gates and lipid compounds, two of which in low quantity (, 1% of pachyphylla A. Juss. (S. C. Cappellari, unpubl. the total composition, Table 1), while other Malpighiaceae at res.). However, the visitation rate of bumble-bees per hour to the field site have complex floral oils formed by several these species is 3–50 times lower than that to glandular and types of fatty acids (Haleem et al., 2010; M. A. Haleem and eglandular P. pyroidea flowers combined. A comparison in S. C. Cappellari, unpubl. res.). Although no correlation pollen production between flowers of these species and between floral oil quantity, chemical complexity and increased P. pyroidea would be necessary to determine if differences visitation of oil-collecting bees has been demonstrated, the fact in the production of this resource can explain differences in that other Malpighiaceae species are frequently visited by oil- visitation by bumble-bees and other non-oil-collecting bees. collecting bees indicates that there may be an important differ- ence between the resources provided by other Malpighiaceae and those of P. pyroidea flowers. Oil loss in an oil-collecting bee hotspot Examples of disassociations in the oil flower pollination Presumed shifts from oil to pollen flowers associated with syndrome within the range of oil flowers and oil-collecting elaiophore loss have been reported for Old World species of bees are not restricted to the plant side of the mutualism. Malpighiaceae as a result of dispersal events to habitats For instance, loss of oil-collecting behaviour has been docu- where oil-collecting bees do not exist, such as Madagascar mented in the Neotropical genus Centris (Apidae: Centridini) (Anderson, 2001; Davis, 2002). Among Neotropical (Neff and Simpson, 1981; Renner and Schaefer, 2010). members of the family, about nine lineages are known to be Morphological and behavioural studies indicate that some partially or completely eglandular (Anderson, 1979; species in the subgenera Paracentris, Penthemisia and Anderson et al., 2006; Anderson and Corso, 2007). Wagenknechtia do not collect oils, have reduced oil-collecting However, it is not clear which factors may have triggered apparatuses, or lack them completely (Snelling, 1956; Neff the loss of elaiophores in the Neotropical region because and Simpson, 1981; Zanella, 2002). Although some of these most eglandular lineages occur within the geographic distri- Centris species occur in areas outside the distribution of oil bution range of oil-collecting bees. The Cerrado, in particular, flowers, field observations indicate that oil foraging was also is a centre of oil-collecting bee diversity, and the local lost in species which have current distributions that overlap apifauna at our field site is one of the richest in terms of with oil flowers in the families Krameriaceae and oil-collecting taxa, especially those associated with Malpighiaceae (Snelling, 1956; Neff and Simpson, 1981). Malpighiaceae flowers (Raw et al., 1998, 2002). In the Because non-oil-collecting Centris species seem to be Cerrado near Brası´lia, a total of 61 oil-collecting bee species restricted to xeric habitats such as the Andean deserts of have been recorded (Boaventura, 1998; Raw et al., 2002; or the Sonoran desert in North America, it S. C. Cappellari, unpubl. res.), most of which do forage on has been proposed that floral oil collection may have been other oil-producing species of Malpighiaceae (Cappellari lost in species that nest in habitats where brood and larval pro- et al., 2009). In addition, the blooming season of visions are not threatened by the risk of excessive humidity P. pyroidea overlaps with that of ten other Malpighiaceae and microbial infections (Neff and Simpson, 1981). species, all of which are visited by oil-collecting bees (Cappellari et al., 2009; S. C. Cappellari, unpubl. res.). The Maintenance of floral dimorphism in Pterandra visitation rates of oil-collecting bees to these species are 7–79 times that of P. pyroidea. Therefore, we conclude that It is important to point out that the observed floral dimorph- the ongoing shift in pollination system observed in ism in P. pyroidea does not seem to be a local phenomenon P. pyroidea was most probably not caused by the absence of restricted to our field site, or a recent phenomenon. In the taxo- oil-collecting bees. nomic description of the genus, A. de Jussieu (1858) reported 1332 Cappellari et al. — Pollination shift in Neotropical Malpighiaceae the existence of glandular and eglandular flowers in evolution of new morphological structures in addition to the P. pyroidea, an observation that was later confirmed loss or reduction of elaiophores. For example, in some (Anderson, 1997). In fact, P. pyroidea is the best-sampled Calceolaria species, that occur in areas where visitation by oil- species in the genus and has an extensive geographic distri- collecting bees is scarce, pollen or food bodies replaced oils as bution. It occurs throughout the Cerrado in Brazil (Distrito the major floral reward, attracting bumble-bees and birds as Federal, and states of Goia´s and Minas Gerais) and eglandular alternative pollinators (Se´rsic and Cocucci, 1996; Se´rsic, flowers are present in collections from multiple localities 2004). In Neotropical Malpighiaceae, pollination of eglandular (Anderson, 1997; S. C. Cappellari, pers. obs.). The geographi- lineages has not been investigated. Generally, the loss of oil pro- cally widespread existence of eglandular individuals is consist- duction in Neotropical species of the family is associated with ent with the hypothesis that populations of P. pyroidea may be changes in floral traits, such as an increase in anther size (e.g. converging on a pollination system in which pollen is offered ) or pollen production (e.g. automimetic as the main floral reward. Similarly, the lack of visitation by Byrsonima), a switch to poricidal anthers (e.g. Coleostachys), Downloaded from https://academic.oup.com/aob/article/107/8/1323/173604 by guest on 29 September 2021 oil-collecting bees is consistent with the results of a 2 year production of floral fragrance (e.g. Psychopterys) and fewer long plant–pollinator community study conducted in a zygomorphic corollas; all attributes of melittophilous flowers Cerrado area within 20 km distance of our field site in which that have pollen as the major reward (Anderson, 1979; P. pyroidea occurred as well (Boaventura, 1998). Teixeira and Machado, 2000; Anderson, 2007; Anderson and Variation in the environmental conditions (e.g. plant com- Corso, 2007). We suggest that the nearly radial corollas of munity composition, density of oil-collecting bee populations) P. pyroidea flowers with well exposed, large anthers may outside our study site and study period may explain the persist- have provided the basis for a switch in pollination system as ence of glandular flowers in P. pyroidea throughout its geo- simplified floral symmetry may decrease flower handling time graphic range. For instance, fruit production by glandular for generalist pollinators, such as bumble-bees, that visit large individuals was observed but not quantified at our field site arrays of flowers to collect pollen. in 2008. In addition, gene flow between populations of differ- Our observations along with other studies on pollination ent floral morphs could contribute to the reintroduction of the shifts (Se´rsic and Cocucci, 1996; Armbruster and Baldwin, glandular phenotype into populations. Although glandular 1998; Se´rsic, 2004; Thomson and Wilson, 2008) corroborate flowers received fewer visits than their eglandular conspecifics, the idea that plant and pollinator species, even though involved both are visited by the same bumble-bee species, which could in evolutionarily specialized associations like the oil-flower promote pollen transfer between morphs. Furthermore, lateral syndrome, are potentially able to cope with changes in local growth and re-sprouting, both common features in environmental conditions and form novel mutualistic bonds. P. pyroidea and other Cerrado plants that are subject to fire, If differences in floral oil characteristics of P. pyroidea may also contribute to the persistence of glandular individuals indeed affect the foraging behaviour of oil-collecting bees in these areas. then perhaps competition with other oil hosts at our study site may have mirrored those experienced by species of the family that have dispersed to areas outside of the oil-collecting Shifts from specialized to generalized pollination syndromes bees’ distributional range (e.g. Africa) in terms of bee visita- Specialized pollination systems are characterized by pheno- tion. Although the causes for the absence of oil bee visits typic traits that enhance pollination by restricting the type of are distinct in each of these scenarios (competition vs. disper- visitors and reducing gamete loss (Armbruster, 2006). sal), the evolutionary outcome, a pollinator shift to pollen- Pollinator shifts from generalized to specialized pollination collecting bees, might be analogous, resulting in a similar systems have been observed in several instances involving strategy to cope with competition or lack of original pollinators shifts from bee to bird pollination and bird to moth pollination in a newly colonized environment. However, further research (reviewed in Thomson and Wilson, 2008). Shifts in the oppo- is needed to arrive at general conclusions about the ecological site direction, i.e. from specialized to generalized pollination and evolutionary mechanisms associated with loss of oil pro- systems, are less common and tend to be associated with the duction in P. pyroidea and other eglandular lineages of absence of specialized pollinators in habitats colonized by Malpighiaceae. plants (Armbruster and Baldwin, 1998; Davis, 2002; Davis et al., 2002; Se´rsic, 2004). Our observations indicate that the ACKNOWLEDGEMENTS switch in the pollination system of P. pyroidea represents a transition from an evolutionarily specialized pollination We thank the staff of the Reserva Ecolo´gica do IBGE for field system (sensu Armbruster, 2006) in which floral adaptations work support; Maria C. Mamede for plant voucher identifi- (e.g. elaiophores or flag petals) are being lost in a plant cation; Antonio J. C. Aguiar (UnB), Gabriel A. R. de Melo species that occurs sympatrically with the group of pollinators (UFPR) and John L. Neff (CTMI) for help with taxonomic originally associated with it. This transition is furthermore identification of bee specimens; Dianne Souza, Gabriela illustrated by the rotating movements performed by bumble- Correa and Paulo Henrique Pinheiro for help with field data bees on flowers, indicating that the flag petal is not needed collection; Erika Hale (UT Austin) for statistical support; for orientation by these pollinators as well as by the lack of oil- John L. Neff and Christian Rabeling for critical input on the collecting bees harvesting oils from the elaiophores in the manuscript. A.J.M. and M.A.H. are grateful to CAPES/ glandular form of this species. Petrobra´s-ANP for financial support and TWAS-CNPq for Shifts in the pollination systems of other oil flower lineages providing a fellowship to M.A.H. This research was funded have happened several times and often have included the by a NSF Doctoral Dissertation Improvement Grant Cappellari et al. — Pollination shift in Neotropical Malpighiaceae 1333

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