Colleters on the Inflorescence Axis of Croton Glandulosus (Euphorbiaceae): Structural and Functional Characterization Author(S): Silvia R

Colleters on the Inflorescence Axis of Croton glandulosus (Euphorbiaceae): Structural and Functional Characterization Author(s): Silvia R. Machado, Lúcia M. Paleari, Élder A. S. Paiva, Tatiane M. Rodrigues Source: International Journal of Plant Sciences, Vol. 176, No. 1 (January 2015), pp. 86-93 Published by: The University of Chicago Press Stable URL: http://www.jstor.org/stable/10.1086/678469 . Accessed: 20/01/2015 12:03

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This content downloaded from 200.145.134.143 on Tue, 20 Jan 2015 12:03:00 PM All use subject to JSTOR Terms and Conditions Int. J. Plant Sci. 176(1):86–93. 2015. q 2014 by The University of Chicago. All rights reserved. 1058-5893/2015/17601-0007$15.00 DOI: 10.1086/678469

COLLETERS ON THE INFLORESCENCE AXIS OF CROTON GLANDULOSUS (EUPHORBIACEAE): STRUCTURAL AND FUNCTIONAL CHARACTERIZATION

Silvia R. Machado,1,* Lúcia M. Paleari,* Élder A. S. Paiva,† and Tatiane M. Rodrigues*

*Universidade Estadual Paulista, Instituto de Biociências de Botucatu, Distrito de Rubião Júnior, 18618-970 Botucatu, São Paulo, Brazil; and †Universidade Federal de Minas Gerais, Instituto de Ciências Biológicas, 31270-901 Belo Horizonte, Minas Gerais, Brazil

Editor: Pamela K. Diggle

Premise of research. Croton glandulosus L. is a ruderal annual species of Euphorbiaceae native to the American tropics and widespread in urban and rural areas in Brazil. We investigated the structure, histochemistry, and ultrastructure of glands on the inflorescence axis of C. glandulosus to explore the relationship between their structure and function. Methodology. Reproductive apexes were prepared according to standard methods for plant anatomy, SEM, and TEM. Histochemical tests were performed using fresh material. Pivotal results. Mature glands are bowling pin shaped and are differentiated into a single-layered secretory epidermis arranged radially around a parenchymatous central axis supplied with xylem. In the neck region of the glands, the epidermal cells present structural components typical of lipophilic secretion, whereas the parenchyma cells are specialized in the production of hydrophilic compounds. The resinous secretion is released by cuticle pores occurring exclusively on the tip of the gland. Conclusions. This is the first report of the occurrence of colleters on the inflorescences of a Croton species. Our observations clearly indicate that glands on the inflorescence axis of C. glandulosus are resin-secreting colleters that may have important protective functions.

Keywords: anatomy, colleters, Croton glandulosus, histochemistry, ultrastructure.

Introduction standard-type colleters were found on the leaves of Astraea lobata, Brasiliocroton mamoninha, and Croton species. How- Croton L. is the second-largest and most diverse genus of ever, detailed structural studies and functional studies on these Euphorbiaceae and is typical of dry to moist vegetation glands in Croton are lacking. widespread in tropical and subtropical regions of the world Colleters have been defined as secretory structures present (Webster 1994; Govaerts et al. 2000; Van Ee et al. 2011). on the adaxial surface of vegetative organs and reproductive Species of Croton can usually be recognized in the field by the organs and are thought to protect young organs from desicca- monocline inflorescences (Berry et al. 2005), pungent odor, tion (Kronestedt-Robards and Robards 1991), herbivores, and stellate pubescence or lepidote pubescence, clear to colored pathogens (Thomas 1991). They produce a viscous fluid insol- latex, and leaves that turn orange before dehiscing (Van Ee uble in water, mucilage, or a mixture of mucilage and lipophilic et al. 2011). Several Croton species are used to treat hyper- substances (Fahn 1979; Evert 2006; Machado et al. 2012). tension, inflammation, malaria, virosis, and other diseases Structurally, colleters can appear as trichomes or emergences (Salatino et al. 2007). supplied (or not supplied) with vascular tissues (Evert 2006). A high morphological diversity of secretory structures with Croton glandulosus L. is one of the most common and varied distribution patterns has been ascribed to Croton spe- widespread species of the genus. It is a ruderal annual species cies, including extrafloral nectaries in the petiole and leaf mar- native to tropical America and is widespread in urban and gins, floral nectaries, idioblasts of lipophilic substances, and rural areas in Brazil (Kissmann and Groth 1999; Lorenzi laticifers (Sá-Haiad et al. 2009 and literature therein). Glands 2008). Personal observations in the field showed an unusual structurally resembling colleters were described by Sá-Haiad kind of external gland on the inflorescence axis of this species, et al. (2009) in leaves of some genera of Euphorbiaceae, includ- whose sticky exudate is gathered by different phytophagous ing Croton. According to Vitarelli (2013; Riina et al. 2014), insects, including Meliponinae bees (L. M. Paleari, Y. Messi, and S. R. Machado, unpublished manuscript). In this article, we report for the first time the occurrence of 1 Author for correspondence; e-mail: [email protected]. colleters on the inflorescences of a Croton species. We describe Manuscript received March 2014; revised manuscript received August 2014; the structure, histochemistry, and ultrastructure of these glands electronically published December 17, 2014. in C. glandulosus in order to explore a relationship between

This content downloaded from 200.145.134.143 on Tue, 20 Jan 2015 12:03:00 PM All use subject to JSTOR Terms and Conditions MACHADO ET AL.—COLLETERS OF CROTON GLANDULOSUS 87 their structure and function. We hope that the results may were cut with a microtome and stained with 1% toluidine blue. contribute to understanding the role of these glands in the in- Ultrathin sections (80 nm) were cut with an ultramicrotome teractions of C. glandulosus with the associated insect fauna. and contrasted with uranyl acetate and lead citrate (Reynolds 1963). Observations were made with a Tecnai Spirit TEM (FEI) Material and Methods at 60 kV.

Plants Results Reproductive apexes and inflorescence axes of Croton glandulosus were collected from wild populations in weedy Pattern Distribution and Morphology of Colleters areas in the Botucatu municipality, São Paulo state, Brazil (lat. The colleters of Croton glandulosus occur along the inflo- 7 ′ 7 ′ – 22 55 S, long. 48 30 W), during 2010 2013. Vouchers were rescence axis from the floral bud stage (fig. 1A,1B) and are deposited at the herbarium at the Department of Botany, located on the edge of bracts and on the base of the floral re- Botucatu Campus of the Universidade Estadual Paulista ceptacle (fig. 1C). Mature glands are already observed on very (UNESP), Brazil. young reproductive axes and are characteristically dark yellow- amber colored (fig. 1B, inset). They can be solitary (on bract Stereomicroscopy and SEM edges) or in groups of three (on floral receptacle bases). When grouped, the colleters are arranged in a linear row (fig. 1D) with Detailed study of the distribution pattern of glands and the central gland less developed and colorless. The secretion is morphology of glands was performed using stereomicroscopes varnish-like translucent and appears as a thin film that covers and scanning electron microscopes. For SEM, samples were the surface of the gland (fig. 1B, inset) when freshly produced or fixed in 2.5% glutaraldehyde (0.1 M sodium phosphate buffer, resembles glassy globules when accumulated at the apical re- at pH 7.3, overnight at 47C). The material was then dehydrated gion of the colleter (fig. 1E). SEM observations revealed pores in a graded acetone series, critical-point dried, mounted on exclusively on the tip of the colleters through which secretion is aluminum stubs, and sputter-coated with gold. Observations released (fig. 1E, arrows). were carried out on a Quanta 200 SEM (FEI, Gräfelfing, Ger- Mature colleters exhibit a massive body consisting of a very many) at 20 kV. large bulbous base and a narrow neck with a shape that resembles a bowling pin (fig. 1B,1D) on a nonsecretory stalk. Light Microscopy The stalk can be very short in solitary colleters on the bracts For histological characterization of the glands, samples were (fig. 1B,1C) or elongated, as those associated with the re- fixed in 2.5% glutaraldehyde, dehydrated through an ethanol ceptacle base (fig. 1C,1D). series, and then processed according to the conventional methods of (2-hydroxyethyl)-methacrylate HistoResin embedding Structure and Histochemistry (Leica Microsystems, Nussloch/Heidelberg, Germany). Sections (3–5 mm thick) were cut using a Leica RM 2245 microtome, Colleters are emergences differentiated into a single-layered stained with 1% toluidine blue in 1% aqueous sodium tetra- secretory epidermis arranged radially around a parenchyma- fi borate solution (O’Brien et al. 1964), and mounted in syn- tous central axis supplied with xylem ( g. 2A). A thick, smooth thetic resin (Entellan). cuticle coats the gland and becomes thinner and porous on the fi The main classes of compounds in the gland tissues and ex- tip of the colleter ( g. 2A). In this region, the cuticle detaches udates were investigated in freehand sections of fresh material from the outer tangential wall, causing a small space where fi using the following histochemical tests: PAS reagent for total secretion is temporarily accumulated ( g. 2A). Structural anal- fi polysaccharides (Jensen 1962), ruthenium red for pectins and ysis con rmed that release of secretion is restricted to the dis- mucilages (Johansen 1940), Sudan IV for total lipids (Johansen tal portion of the colleter. The secretion in the subcuticular 1940), mercuric bromophenol blue for total proteins (Mazia space stained positively for mucilage, lipids, and proteins. et al. 1953), Nadi reagent for essential oils and resins (David Secretory epidermal cells are elongated in the anticlinal direc- fi and Carde 1964), and ferric trichloride for phenolic compounds tion, like a palisade when seen in longitudinal section ( g. 2A). (Johansen 1940). Control reactions were carried out simul- Mature colleters, active in secretion, exhibit juxtaposed epi- taneously according to the specifications for each test. Obser- dermal cells with prominent nuclei aligned in the midregion of fi vations were carried out on a Leica DMR light microscope, the cells, dense cytoplasm, small vacuoles, and cuticle rmly fi and images were recorded digitally with a Leica DFC 500 cam- adhered to the outer tangential wall ( g. 2B). At a posterior era (Leica Microsystems). stage, epidermal cells exhibit a loose aspect with the rise of intercellular spaces filled with secretion, along with a reduced cytoplasm, larger vacuoles, and detached cuticle (fig. 2C). TEM Parenchyma cells are axially elongated in the neck region of For TEM, samples were fixed in 2.5% glutaraldehyde (0.1 the colleter and have distinctly thinner cell walls and abundant M sodium phosphate buffer, at pH 7.3, for 6–8hat47C) and cytoplasm. In the bulbous region of the colleter, the paren- fi post xed with 1% osmium tetroxide (OsO4) in the same chyma cells exhibit irregular shapes and sizes, reduced cyto- buffer for 2 h at room temperature. After washing in distilled plasm, developed vacuole, and large druses (fig. 2A). Tracheary water, the material was dehydrated in a graded ethanol series elements, more frequently in numbers of three, occur in the and embedded in Araldite resin. Semithin sections (0.5 mm) central portion of the parenchyma axis (fig. 2B). The stalk is

This content downloaded from 200.145.134.143 on Tue, 20 Jan 2015 12:03:00 PM All use subject to JSTOR Terms and Conditions Fig. 1 Stereomicroscope images (A, B) and scanning electron microscope images (C–E) showing the distribution and morphology of colleters on the inflorescence axis of Croton glandulosus. A, Colleters on apex of the inflorescence axis. B, Portion of an inflorescence axis showing colleters on the edge and base of the bracts. The inset shows a mature colleter with bowling pin shape and dark yellow-amber color. C, Colleters grouped at the bract (br) base around the floral receptacle (rc) and solitary colleter on the bract edge. The flower was removed. D, Three-grouped colleters at the bract base. Note the small and misshaped central colleter. E, Resin globules on the tip of a colleter. Arrows indicate cuticle pores. Scale bars p 1cm(A), 0.25 cm (B), 200 mm(C), 150 mm(D), 25 mm(E).

This content downloaded from 200.145.134.143 on Tue, 20 Jan 2015 12:03:00 PM All use subject to JSTOR Terms and Conditions MACHADO ET AL.—COLLETERS OF CROTON GLANDULOSUS 89

Fig. 2 Photomicrographs of colleters from a Croton glandulosus inflorescence axis. A, Longitudinal section showing secretory elongate epidermal cells (se) arranged in palisade around central axis of parenchyma cells (pa). Note subcuticular space (ss) and pore in cuticle (arrow) at tip of colleters. B, C, Cross sections. B, Colleter active in secretion showing juxtaposed epidermal cells with dense cytoplasm and tracheary elements (te) immersed in the parenchyma core. C, Colleter in a later developmental stage showing epidermal cells with a loose aspect, reduced cytoplasm, and enlarged vacuome. Observe intercellular spaces and subcuticular spaces filled with secretion. Scale bars p 50 mm. constituted by common epidermal and parenchyma cells with throughout the cytosol (fig. 3C) and near the plasma mem- no features of secretory activity (fig. 2A). brane. The Golgi bodies are poorly developed and randomly distributed in the cytoplasm (fig. 3D). Plastids surrounded by endoplasmic reticulum are polymorphic, lacking inner mem- TEM branes, and contain dense stroma and lipid droplets (fig. 3D). The ultrastructural organization of the cells of the epider- The vacuolar compartment is poorly developed and consists mis and cells of the parenchyma in the neck region differs of small vacuoles containing flocculated material or mem- from that in the bulbous region of the colleter. In the tip re- brane debris (fig. 3D). In the bulbous region of the colleter, gion, the epidermal cells are collenchymatous, showing thick- the epidermal cells show thinner walls, thin cuticle, prominent ened walls with an irregular contour (fig. 3A). The outer tan- nucleus, less dense cytoplasm, scattered mitochondria, and gential wall is covered with a thick cuticle, showing a wide plastids with thylakoids and electron-dense globules (fig. 3E). cuticular layer that is lamellar and traversed by a reticulated The parenchyma cells in the neck region of the colleter have network of pectins (fig. 3B). As the secretion process continues, thin walls, prominent nucleus, abundant cytoplasm, and vac- the cuticle detaches, causing a small space (fig. 3B); the mid- uoles of different sizes with membranous and flocculate ma- dle lamella along the radial walls becomes loose with signs of terial (fig. 4A). Plasmodesmata connect the protoplasts of con- dissolution (fig. 3C). Accumulations of amorphous electron- tiguous parenchyma cells (fig. 4A,4B,4D,4E). The organelle translucent material (previously identified as polysaccharides population is represented by numerous mitochondria (fig. 4A, and lipids) mixed with black granulations (previously identi- 4E), abundance of hyperactive Golgi bodies, abundance of fied as proteins) are seen inside the intercellular and subcu- secretory vesicles (fig. 4B,4C,4G), rough endoplasmic retic- ticular spaces (fig. 3B,3C). ulum profiles (fig. 4B,4D), polyribosomes (fig. 4C), peroxi- The cytoplasm of epidermal secretory cells is very dense and somes (fig. 4C), and scarce plastids (fig. 4A). The mitochon- contains free ribosomes, polyribosomes, mitochondria, Golgi dria have well-developed cristae (fig. 4C,4D). The plastids are bodies, plastids, and abundance of endoplasmic reticulum oval and present thylakoids, small starch grains, and dense (fig. 3C,3D). Smooth endoplasmic reticulum profiles are ran- globules (fig. 4F). Multivesicular bodies are seen attached to domly distributed in the cytoplasm and are predominant in the plasma membrane (fig. 4E). Periplasmic spaces containing these cells (fig. 3C). Large oil drops were seen scattered fibrilar material and membrane debris are commonly observed

This content downloaded from 200.145.134.143 on Tue, 20 Jan 2015 12:03:00 PM All use subject to JSTOR Terms and Conditions Fig. 3 Transmission electron microscopy of secretory epidermal cells of colleters on Croton glandulosus inﬂorescence axis. A–D, Neck region. A, Collenchymatous epidermal cells showing thick cuticle (ct), prominent nucleus, and dense cytoplasm. B, Detail showing irregular contour of cell wall (cw) and thick lamellar cuticular layer traversed by a reticulate network. Note accumulation of secretion in the subcuticular space (ss). C, Distal portion of cell exhibiting proliferated smooth endoplasmic reticulum (ser), mitochondria (mi), and oil drops (ol). Black granulations and amorphous secretion is accumulated in the intercellular space (asterisk). D, Polymorphic plastids (pl) surrounded by endoplasmic reticulum (pr), scarce Golgi bodies (Gb), and extensive rough endoplasmic reticulum (rer). E, Cell of the bulbous region with thinner walls, thin cuticle, prominent nucleus, less dense cytoplasm, and plastid with thylakoids and electron-dense globules. va p vacuole. Scale bars p 10 mm(A), 5 mm(B), 1.5 mm(C), 1 mm(D), 2 mm(E).

This content downloaded from 200.145.134.143 on Tue, 20 Jan 2015 12:03:00 PM All use subject to JSTOR Terms and Conditions Fig. 4 Transmission electron microscopy of secretory parenchyma cells of colleters on Croton glandulosus inflorescence axis. A–F, Neck region. A, Parenchyma cells connected by plasmodesmata (arrows) exhibiting prominent nucleus (nu), abundance of mitochondria, and vacuoles (va) with different sizes containing membranous and flocculate material. B, Hyperactive Golgi bodies (Gb), mitochondria (mi), polysomes, and rough endoplasmic reticulum (rer). Arrow indicates plasmodesmata. C, Mitochondria with dilated cristae, peroxisome (pe), polysomes, and Golgi bodies with adjacent vesicles. D, Extensive rough endoplasmic reticulum and voluminous mitochondria near periplasmic space (ps). E, Multivesicular body (mb) connected to plasma membrane. Arrows indicate plasmodesmata. cw p cell wall. F, Plastids (pl) with thylakoids and starch grains (st). Note wide intercellular space (asterisk) filled with secretion. G, Parenchyma cell subjacent to the secretory epidermis (se) in the bulbous region of colleter showing nucleus with irregular shape and plastids with thylakoids and electron-dense globules. dr p druse. Scale bars p 5 mm(A, G), 0.7 mm(B), 1 mm(C, E, F), 0.5 mm(D).

This content downloaded from 200.145.134.143 on Tue, 20 Jan 2015 12:03:00 PM All use subject to JSTOR Terms and Conditions 92 INTERNATIONAL JOURNAL OF PLANT SCIENCES along the anticlinal walls (fig. 4D). Dissolution of middle la- The ultrastructural characteristics observed in the epider- mellae occurs along the anticlinal cell walls (fig. 4F). mal cells and parenchyma cells of C. glandulosus colleters are In the bulbous region, the parenchyma cells are characterized typical of cells that are involved in secretion. The richness of by reduced cytoplasm with numerous plastids and one well- membranous organelles, such as smooth endoplasmic reticu- developed vacuole frequently containing big druses (fig. 4G). lum, rough endoplasmic reticulum, and Golgi bodies, is com- Plastids contain thylakoids and abundant osmiophilic inclu- mon in glands that produce a mixed secretion (Evert 2006). sions (fig. 4G). Plasmodesmata connect the protoplasts of the Abundance of mitochondria reflects the high metabolism of epidermal cells and those with parenchyma cells (fig. 4G). these cells and characterizes several cell types involved in synthesis processes and transport of substances (Lüttge 1971). Pres- Discussion ence of plastids devoid of internal lamellae and surrounded by endoplasmic reticulum in the epidermal cells are indica- Our anatomical, histochemical, and ultrastructural data tions of terpene synthesis (Fahn 1979; Machado et al. 2006). clearly indicate that glands on the inflorescence axis of Croton Oil droplets in the stroma of plastids inside the vacuoles, dis- glandulosus are resin-secreting colleters. Typically, our obser- persed in the cytoplasm or outside protoplast, as observed in vations showed that colleters are active in secretion at very C. glandulosus colleters, are ultrastructural evidence of syn- early stages of flower development, as reported for different thesis of terpenoids (Turner et al. 1999; Rodrigues and Ma- plant species (Coelho et al. 2013 and references therein). To our chado 2012). Abundance of well-developed Golgi bodies with knowledge, this is the first report of the occurrence of colleters dilated cisternae and vesicles attached to these marginal regions on the inflorescences of a Croton species and the first histo- or situated close to these marginal regions, in addition to ex- chemical analysis and ultrastructural analysis of colleters in the tensive rough endoplasmic reticulum in C. glandulosus paren- genus. Although in their article Sá-Haiad et al. (2009) have chyma cells, is clear evidence of protein/carbohydrate-based discussed the morphological similarity of the glands in leaf mucilage synthesis (Fahn 1979; Evert 2006). Secretory vesicles margin to colleters, the authors concluded that these glands in the peripheral cytoplasm or their fusion with plasma mem- are extrafloral nectaries. brane indicate that secretion is granulocrine (Evert 2006). This The histochemical analysis of the secretion of colleters of C. is consistent with the presence of polysaccharides and proteins glandulosus indicates the presence of hydrophilic substances in the apoplast of the colleters. In addition, the presence of oil and lipophilic substances, and these data are consistent with drops scattered in the cytoplasm and outside the protoplast also the ultrastructural machinery of the secretory cells. Colleter se- suggests the occurrence of an eccrine mechanism where the cretion of C. glandulosus is insoluble in water as well as in or- molecules cross the plasma membrane by active transport (Nepi ganic solvents such as acetone and ethanol, as reported for 2007; Vassilyev 2010). Our data suggest that the epidermal resin-secretory colleters of Alibertia sessilis (Barreiro and Ma- cells appear to be more involved in the synthesis of lipophilic chado 2007). According to Langenheim (2003), resins on the substances, whereas the parenchyma cells are involved in the surface of young organs generally consist of a complex mixture synthesis of hydrophilic substances. The ultrastructural features of lipophilic flavonoids and terpenes that solidify at room of the epidermal cells and parenchyma cells located below the temperature. Solidification of the secretion, initially sticky and neck region of the colleter indicate a gradual decrease in the then glassy when dry, as observed in C. glandulosus, is evidence secretory activity toward the base of the gland. Crystals of of its resinous nature (Langenheim 2003). Discharge of abun- calcium oxalate are restricted to the parenchyma cells located dant secretion in C. glandulosus occurs through the cuticular in the bulging portion of colleters of C. glandulosus and may pores that are present exclusively in the tip of the colleter; how- be merely excretory products, which can discourage herbivores, ever, it is probable that hydrophilic secretion also traverses the as discussed for other secretory structures (Roshchina and uninterrupted cuticle via the microchannels existing in the cu- Roshchina 1993). ticular layer, as already discussed in other glands (Miguel Resin produced by C. glandulosus colleters is waterproof et al. 2010; Stpiczyńska et al. 2012). and produced regularly and predictably. Concerning the prob- To our knowledge, the collenchymatous nature of the secre- able roles of C. glandulosus colleters, the protection afforded tory epidermal cells, as observed in the tip of the colleter, has by the resinous secretion is especially important if we con- not been described for colleters of other plant species. Thick sider that this species is a quite common ruderal species in the walls are probably involved with the apoplastic movement of Cerrado, an environment that is characterized by having a the substances, especially in the absence of plasmodesmata, as seasonal climate with a distinct dry season with high temper- observed in this colleter region. Moreover, the collenchyma- atures and high light intensity (Sano et al. 2008). Considering tous nature of the epidermal cells suggests that it may also have the ecological roles of resins (Langenheim 2003), we hypoth- a mechanical and/or protective function, preventing damage to esized that the production of resinous secretion throughout the secretory tissue by the visitors, as observed in the nectary of the period of reproductive development is particularly signif- Maxillaria coccinea by Stpiczyńska et al. (2003). Schyzogenous icant and probably plays an important role in protection of spaces resulting from the dissolution of middle lamella along the flower tissues from desiccation, herbivores, and patho- the anticlinal walls, as observed in C. glandulosus, are wall gens. In addition, resins contain volatile terpenes that may changes found on colleters of different species (Appezzato-da- also attract predators or parasitoids of herbivores attacking Glória and Estelita 2000). Such alterations are mediated by the C. glandulosus plants, providing an indirect defense (Paleari action of pectinases and cellulases (Ben-aire et al. 1979) and are et al., in prep.). consistent with the organelles population of the secretory cells As mentioned, C. glandulosus plants are visited by Meli- in C. glandulosus colleters. poninae female bees that collect gland secretions (Paleari et al.,

This content downloaded from 200.145.134.143 on Tue, 20 Jan 2015 12:03:00 PM All use subject to JSTOR Terms and Conditions MACHADO ET AL.—COLLETERS OF CROTON GLANDULOSUS 93 in prep.). Floral resins are a particularly valuable resource Acknowledgments for female bees in nest building (Armbruster 1984). Simulta- neously, volatile terpenes can attract bees that use such sub- We thank the Fundação de Amparo à Pesquisa do Estado stances as precursors for sex pheromones (Armbruster 1993). de São Paulo (process nos. 2008/52134-2 and 2008/55434-7) In summary, our observations clearly indicate that glands on for ﬁnancial support, the staff of the Electron Microscopy the inﬂorescence axis of C. glandulosus are resin-secreting col- Center of UNESP for helping in the sample preparation, and leters, and their importance to plant-insect interactions de- Dr. Bárbara de Sá-Haiad for helpful comments on the manu- serves further research. script. S. R. Machado receives grants from the CNPq council.

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