(Malpighiaceae), a Neotropical Species

Flora 211 (2015) 26–39

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Structure and secretion mechanisms of ﬂoral glands in Diplopterys

pubipetala (Malpighiaceae), a neotropical species

∗

Clivia Carolina Fiorilo Possobom, Elza Guimarães, Silvia Rodrigues Machado

São Paulo State University, UNESP, Institute of Biosciences of Botucatu, Department of Botany, 18618-970 Botucatu, São Paulo, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: Detailed studies on the distribution, structure, and secretion activity of ﬂoral glands are important to

Received 15 July 2014

understand the relationship of ﬂowers with oil-collecting bees in Malpighiaceae. Here, we characterised

Received in revised form

the ﬂoral biology and the glands in sepals, petals and connective tissues of Diplopterys pubipetala. The

10 December 2014

data on the floral biology were obtained under field conditions. The samples from functional flowers

Accepted 9 January 2015

were prepared for anatomical, histochemical and ultrastructural studies. The bees of the genera Monoeca

Edited by Shahin Zarre.

and Centris were the most frequent visitors. While both insects searched for oil, the former also collected

Available online 14 January 2015

pollen and connective tissue secretions. The conspicuous and subsessile sepal glands are arranged in pairs

Keywords: on the abaxial surface, presenting structural and cellular machinery typical of epithelial elaiophores. The

oil is accumulated in the subcuticular space and released when the bee scraps the cuticle, causing its rup-

Floral glands

Histochemistry ture. The petal glands, observed at the ﬁmbriate edges, are diminutive, comprising secretory epithelium

Oil-collecting bees surrounding a central core of parenchymal cells supplied with vascular tissues. The petal glands are typ-

Pollination ically osmophores, and secretion occurs via diffusion through the thin cuticle. The glandular connective

Ultrastructure

comprises large globular secretory epithelial cells, which produce a bright and viscous secretion, mim-

icking pollen grains. This predominantly hydrophilic secretion is released to the surface of the connective

tissue traversing the thin cell wall and intact cuticle in regions with protruding protoplasts. In addition,

the sticky secretion produced from the glandular connectives might also increase the efﬁciency of trans-

port and pollen transfer. Taken together, these results show that each gland has a peculiar mechanism

and type of secretion, suggesting additional levels of ﬂoral specialisation for interactions with pollinators.

Introduction 1967; Anderson, 2004; Judd et al., 2007), which are frequently

associated with extraﬂoral nectar secretion and ants that protect

Malpighiaceae is a pantropically distributed family, represented plants against herbivores (Lobreau-Callen, 1989; Possobom et al.,

by approximately 1300 species which mostly occur in neotropical 2010; Aguirre et al., 2013). Nevertheless, this ant-plant interaction

region (Davis and Anderson, 2010). It is one of the most impor- may take part in a multitrophic interaction involving pollinators

tant plant families that secrete ﬂoral oil as a reward to pollinators with consequences on plant reproductive success, as showed by

and amongst the oldest clades to have acquired elaiophores (Vogel, Assunc¸ ão et al. (2014).

1974; Renner and Schaefer, 2010). These oil glands occur in approx- Neotropical species of Malpighiaceae are pollinated principally

imately 90% of the neotropical species, and when they occur in by specialized oil-collecting bees belonging to the tribes Centridini,

the paleotropical species, they seems to be related with extranup- Tapinotaspidini and Tetrapediini that are restricted to American

tial nectar secretion and not with oil secretion (Vogel, 1974, continent and are especially diverse in the tropical region (Alves-

1990a). These glands are morphologically very similar to those dos-Santos et al., 2007). These bees gathered the oil produced

on the petiole, abaxial surface or margins of leaves (Hutchinson, by elaiophores and use it for larval provisioning (Vogel, 1974;

Vinson et al., 1997; Reis et al., 2007) and cell lining (Neff and

Simpson, 1981; Simpson and Neff, 1981; Buchmann, 1987; Vogel,

∗ 1990a). The majority of Malpighiaceae species are hermaphroditic,

Corresponding author at: São Paulo State University, UNESP, Institute of Bio-

sciences of Botucatu, Department of Botany, Rubião Jr District s/n, PO Box 510, predominantly allogamous and highly dependent on pollen vec-

18618-970, Botucatu, São Paulo, Brazil. Tel.: +55 14 3880 0122;

tors for reproduction (Teixeira and Machado, 2000; Sigrist and

fax: +55 14 3815 3744.

Sazima, 2004; Costa et al., 2006; Cappellari et al., 2011). The close

E-mail address: [email protected] (S.R. Machado).

http://dx.doi.org/10.1016/j.ﬂora.2015.01.002

C.C.F. Possobom et al. / Flora 211 (2015) 26–39 27

interactions between Malpighiaceae and their speciﬁc pollinators glandular tissues, a set of buds was isolated in voile bags throughout

have influenced the diversification rates in Malpighiaceae since its floral development. During anthesis, some flowers were exposed to

origin (Renner and Schaefer, 2010; Davis et al., 2014). bees (Monoeca sp.), and subsequently these bees and non-visited,

In addition to the sepal elaiophores, in more than 10 Malpighi- bagged ﬂowers were collected for analysis using a scanning elec-

aceae genera, at least one species possesses diminutive petal glands tron microscope (SEM).

and/or glandular connectives on the anthers (Anderson, 1975, 1982, Voucher samples were deposited in the “Irina Delanova

1987, 1995; Gates, 1982; Johnson, 1986; Simpson, 1989; Anderson Gemtchujnicov” Herbarium (BOTU) of the Biosciences Institute,

and Davis, 2007). According to Vogel (1974), the petal glands of São Paulo State University (UNESP) at Botucatu, São Paulo, Brazil

Heteropterys chrysophylla produce non-polar lipids, different from (numbers 25,306–25,310).

those observed in sepal gland secretion. Lobreau-Callen (1989) also

detected lipids in the secretion from the petal glands of Burdachia Floral biology

primatocarpa and Glandonia macrocarpa, but no ecological function

has been suggested. Concerning the glandular anther connectives, The ﬂoral anthesis, longevity, time of opening and percentage

Gates (1982) suggested that the location of this tissue on the ﬂower of ﬂowers in anthesis in 12 D. pubipetala individuals were observed

favours contact with the ventral portion of the visitor, and secre- daily. The ﬂoral scents were examined using organoleptic tests, and

tion could thereby increase the adhesion of the pollen grains onto the osmophores were macroscopically detected through neutral

the body of the visitor. red staining (Vogel, 1990b). We also assessed the secretory activity

Detailed studies correlating the structure and function of floral of the floral glands during floral development. Focal observations

glands in Malpighiaceae have been limited to only a few species, of the ﬂoral visitors were conducted between 06:00 and 20:00 h,

and these studies primarily focused on sepal elaiophores (Vogel, and the time, duration, and frequency of visits, behaviour, resource

1974; Subramanian et al., 1990; Cocucci et al., 1996; Castro et al., collected, and site of body contact with the anthers and stigmas

2001). Thus, our current knowledge of petal glands and glandular were recorded according to Dafni et al. (2005). The behaviour of the

connectives is scarce (Vogel, 1974; Lobreau-Callen, 1989; Cocucci visitors was described based on ﬁeld observations and photograph

et al., 1996), and many aspects remain unknown, such as the and ﬁlm analyses. The specimens collected from each visitor species

structural organisation and secretion mechanisms. A comparative were analysed under a stereomicroscope to assess the presence and

analysis of the different glandular tissues associated with data on location of the pollen grains and the oil on the body.

the behaviour of visitors could provide insights into the role of ﬂoral

exudates in plant-pollinator interactions. Anatomical analysis

In the present study, we characterised the anatomy, histochem-

istry and ultrastructure of the sepal, petal and connective glands For general histology, the samples were ﬁxed in Karnovsky’s

of Diplopterys pubipetala (A. Juss.) W.R. Anderson & C. Davis, as it solution (Karnovsky, 1965) for 24 h, dehydrated in an ethanol

relates to interactions with pollinators. series, and embedded in 2-hydroxyethyl-methacrylate resin (His-

toresin, Leica, Heidelberg, Germany). Serial cross and longitudinal

sections (5–8 ␮m thickness) were obtained using a rotatory micro-

Materials and methods tome. Subsequently, the sections were stained with 0.05% toluidine

blue, pH 4.3 (O’Brien et al., 1964), and mounted in synthetic resin.

Study site and plant material The images were obtained using a light microscope equipped with

a camera. To examine the vascularisation of the petal glands, the

The cerrado, or the Brazilian savannah, covers nearly ﬁmbriate petal fragments were cleared according to Fuchs (1963).

2 million km , approximately 22% of the country’s land sur-

face. The cerrado is extremely variable in terms of physiognomy, Histochemical analysis

ranging from open grasslands to forests with discontinuous grass

layers. A continuum of savanna formations between these two Hand-cut sections of fresh material were subjected to the fol-

extremes spans the entire range of woody plant density. Con- lowing treatments for histochemical analysis: ferric trichloride for

comitant with the seasonal water deﬁcit, the cerrado environment phenolic compounds, Lugol reagent for starch grains, ruthenium

experiences a high irradiance load and elevated vapour pressure red for pectin/mucilage, Sudan IV for total lipids, 7% cupric acetate

deﬁcits (Oliveira Filho and Ratter, 2002). solution to detect resins (Johansen, 1940), mercuric bromophenol

This study was conducted using Diplopterys pubipetala from the blue for proteins (Mazia et al., 1953), periodic acid-Schiff’s reagent

◦ ◦

cerrado area (22 42 38 S and 48 18 35 W), located in Botucatu (PAS) for total polysaccharides (Jensen, 1962), Wagner’s reagent

municipality, São Paulo, Brazil, from 2006 to 2008. for alkaloids (Furr and Mahlberg, 1981), and naphtol + dimethyl-

Diplopterys pubipetala is a shrub with vining branches; petiolate paraphenylene-diamine (NADI) reagent for terpenes (David and

leaves bearing minute glands; axillary inﬂorescences comprising Carde, 1964). The control samples were tested according to the

2–3 condensed racemes 2–3(5) pairs of ﬂowers or cymes of 3–5 speciﬁcations for each analysis. For all tests, the sections were

condensed racemes; ﬂowers with a ﬁve-lobed calyx containing a mounted in glycerine under a coverslip. Micrographs were obtained

pair of glands on the four lateral sepals; a yellow corolla with ﬁve using a light microscope equipped with a digital camera.

clawed petals and four lateral petals reﬂexed between the sepals;

a posterior petal with a thick ﬂeshy claw and basal ﬁmbriae, occa- Ultrastructural analysis

sionally gland-tipped; stamens with glabrous ﬁlaments; basiﬁxed

anthers; introrse dehiscence; three carpels and a densely pilose For scanning electron microscopy (SEM), the samples were ﬁxed

−1

ovary with a single ovule in each locule; three styles with capitate in 2.5% glutaraldehyde in a 0.1 mol L phosphate buffer, pH 7.3, for

stigmas; and samara fruit (Gates, 1982). 24 h, dehydrated in a graduated acetone series, critical point-dried

Using a stereomicroscope, the sepals, petals and connective using CO2, mounted on aluminium stubs, coated with gold, and

samples bearing actively secretory glands were collected from examined at 15 kv.

ﬂowers at different developmental stages for anatomical, histo- For transmission electron microscopy (TEM), the samples were

−1

chemical, and ultrastructural studies. In addition, to evaluate the ﬁxed in 2.5% glutaraldehyde in a 0.1 mol L sodium phosphate

◦

effect of the visitor contact on the ﬂoral structures, particularly the buffer, pH 7.3, for 6–8 h at 4 C, post-ﬁxed with 1% osmium tetroxide

28 C.C.F. Possobom et al. / Flora 211 (2015) 26–39

Fig. 1. Floral visitors of Diplopterys pubipetala. (A) Centris aenea and (B–C) Monoeca sp. visiting ﬂowers. (A–B) Bees collecting sepal gland secretions using the two anterior

pairs of legs. Note the area of the body of the Monoeca bee in contact with the anthers (arrows). (C) Bees collecting pollen and the connective tissue secretions using the

anterior pair of legs (arrow). Scale bars A–C = 5 mm.

(OsO4) in the same buffer for 2 h at room temperature, dehy- begins to open. The ﬂowers released a mildly sweetish scent, and

drated in a graduated ethanol series, and embedded in epoxy positive reactions to neutral red were primarily observed at the

resin (Araldite). The ultrathin sections were contrasted using 5% ends of the ﬁmbriae and in the apical region of the connective tis-

uranyl acetate in 50% ethanol and lead citrate (Reynolds, 1963). sues. The glands in the ﬁmbriate edges are more numerous and

The sections were examined using TEM at 80 kv. In addition, the developed in the posterior petal especially in the basal portion of

samples treated using the zinc iodide–osmium tetroxide (ZIO) the limb, when compared to the lateral petals. Although visitors

technique (Reinecke and Walther, 1978) to improve the visuali- were not observed collecting secretions of these glands, the bees

sation of endomembranes and the osmium-imidazole technique stand out for the precision of their landing behaviour, always with

(Angermüller and Fahini, 1982) to enhance the staining and preser- their head turned towards the posterior petal (Fig. 1A and B).

vation of lipids. Diplopterys pubipetala ﬂowers were exclusively visited by bees

(Table 1). The peak of visitation occurred between 09:00 h and

Results 10:00 h, when the scent was noticeably stronger as compared to

other periods of the day. The most frequent visitors were Centris

(64%) and Monoeca (34%) bees, corresponding to 98% of the total

Floral biology

recorded visits (Table 1). Sporadic and brief visits from bees belong-

ing to the genera Bombus, Epicharis and Xylocopa were recorded, but

All D. pubipetala ﬂowers are hermaphrodites with dehiscent

it was not possible to observe the visiting behaviour and the type

anthers and longevity of two days. Approximately 5% of the buds

of resource collected by these species. The visits from Centris bees

opened each day at approximately 06:00 h. A strong zygomorphy

(Fig. 1A) were short, lasting less than 2 s on each ﬂower. The visits

characterises D. pubipetala ﬂowers. Clusters of white pollen grains

from Monoeca bees (Fig. 1B and C) lasted approximately 3 s on each

were exposed at approximately 08:00 h, two hours after the ﬂower

Table 1

Floral visitors of Diplopterys pubipetala in Botucatu, São Paulo, Brazil.

Tribe Bees Number of visits (frequency %) Foraged resource

Centridini Centris (Centris) aenea (Erichson, 1848) 37 (23.3) O

Centris (Centris) varia (Erichson, 1848) 37 (23.3) O

Centris (Heterocentris) analis (Fabricius, 1804) 4 (2.5) O

Centris sp. 1 1 (0.6) O

Centris sp. 2 22 (13.8) O

Centris sp. 3 1 (0.6) O

Epicharis ﬂava (Friese, 1900) 1 (0.6) NI

Tapinotaspidini Monoeca cf brasiliensis (Lepeletier, 1825) 24 (15.1) O

Monoeca sp. 30 (19) O/P

Bombini Bombus sp. 1 (0.6) NI

Xylocopini Xylocopa sp. 1 (0.6) NI

Total 159 (100)

O: oil, P: pollen, NI: not identiﬁed.

C.C.F. Possobom et al. / Flora 211 (2015) 26–39 29

Fig. 2. Calyx glands of Diplopterys pubipetala. (A) Treatment with Sudan IV. Note the stained secretions in the subcuticular space (*). (B–C) Scanning electron micrographs. (B)

Sepal glands of non-visited ﬂowers, showing intact surfaces. Note the protuberance (*) ﬁlled with secretions. (C) Sepal glands after visits from bees. Note the ruptured cuticle

(arrows). (D) Light micrographs showing a longitudinal resin section. Note the secretory epithelial cells (ep), sub-epithelial parenchyma (sp) and parenchyma supplied by

phloem (ph) and xylem (xy). (E–J) Transmission electron micrographs. (E) Epithelial cells at the secretory phase, showing dense cytoplasm, nucleus (nu), vacuoles (va), plastids

(pl) and oil droplets (ol). Note the subcuticular space (ss) with accumulated secretions, a proper cuticle (ct) and channels between anticlinal cell walls (*). (F) Dense epithelial

cell cytoplasm showing part of the nucleus (nu), ribosomes and polyribosomes, rough endoplasmic reticulum (rer), Golgi bodies (Gb) with vesicles, plastids (pl), mitochondria

(mi), multi-vesicular bodies (mb) and oil droplets (ol). (G) Plastids (pl) surrounded with rough endoplasmic reticulum (rer). (H) Plasmodesmata (arrow) connecting epithelial

(ep) and sub-epithelial (se) cells. (I) The epithelial outer periclinal cell wall at the pre-secretory stage, showing an intact cuticular layer. (J) Cuticular layer with small gaps (*)

formed from the dissolution of the pectin strata. Scale bars: A, B and C = 500 ␮m; D = 100 ␮m; E, H = 1 ␮m; F and G = 500 nm; I and J = 2 ␮m.

30 C.C.F. Possobom et al. / Flora 211 (2015) 26–39

ﬂower. Bees of both genera landed directly upon the ﬂower with gradually dissolves, forming small gaps (Fig. 2J) that coalesce

their legs positioned precisely between the lateral petals. During oil and give rise to the large subcuticular space (Fig. 2E). Concur-

collection, the bees did not touch the sepals opposite the posterior rently, the middle lamella between the anticlinal walls dissolves,

petal, where there were typically no glands. forming channels between the epithelial cells where secretions

The entire ventral portion of each bee was in contact with the accumulate (Fig. 2E). The epithelial cells present a dense cyto-

reproductive structures of the ﬂowers, including the glandular por- plasm rich in polyribosomes, mitochondria, rough and smooth

tion of the anther connective. These bees reached the glands of the endoplasmic reticulum, plastids, and oil droplets (Fig. 2E–J). The

calyx, scraping this structure with the ﬁrst pair of legs and col- Golgi bodies are scarce, and few of these organelles are developed

lecting the secreted oil stored in the subcuticular space (Fig. 2A), (Fig. 2F). The mitochondria were globular with well-developed

which was stored on the last pair of legs upon leaving the ﬂower. cristae (Fig. 2F–G), and either dispersed throughout the cytoplasm

The non-visited ﬂowers exhibited sepal glands with intact cuticles or aggregated near the plasma membrane. The plastids were large

(Fig. 2B), while ﬂowers visited by oil-collecting bees of the Monoeca and spherical, with a dense stroma devoid of inner membranes and

sp. exhibited cuticle rupture (Fig. 2C). ﬁlled with oil droplets (Table 2, Fig. 2E–G). The endoplasmic retic-

In addition, the two Monoeca bee species also pressed their ulum cisternae were dispersed throughout the cytoplasm (Fig. 2F)

abdomens against the reproductive organs repeatedly during oil or closely juxtaposed with plastids and mitochondria (Fig. 2G). Oil

collection. These bees were also observed collecting pollen with droplets (whose contrast was improved with osmium-imidazole,

the ﬁrst pair of legs, immediately after collecting oil (Fig. 1C). To Table 2) were also dispersed throughout the cytoplasm (Fig. 2E).

collect the pollen, the bees pressed against the two posterior sta- Multi-vesicular bodies and vesicles are commonly observed in the

mens in an upward movement, thus also pressuring the glandular peripheral cytoplasm near the plasma membrane (Fig. 2F). The vac-

connective tissue. So, such secretion was also collected by Monoeca uoles are of different sizes and contain dense inclusions mixed

bees, in addition to oil and pollen. with ﬂocculate materials and membrane debris (Fig. 2E). Single,

An analysis of Centris bees revealed pollen grains scattered over large vacuoles derived from the fusion of smaller vacuoles were

the body and a dense, pale-coloured mass, likely a mixture of pollen observed in some epithelial cells (Fig. 2E). Plasmodesmata con-

and oil, stored on the third pair of legs. In contrast, the Monoeca necting epithelial cells with sub-epithelial cells were frequently

bees showed pollen grains concentrated in the ventral region of observed (Fig. 2H).

the abdomen, corresponding to the location of the central anterior The gland parenchymal cells have a central, well-developed vac-

stigma, and on the sides of the head, corresponding to the precise uole, and the cytoplasm is denser in sub-epithelial cells. In contrast

location of the two posterior stigmas. Pale yellow masses, compris- with the epithelial cells, the plastids are ellipsoids, showing inner

ing a mixture of pollen and oil, were observed on the hind legs of membranes, oil droplets and starch grains.

these bees.

Corolla glands

Morphological, histochemical and ultrastructural analyses of The yellow corolla has one posterior, two posterolateral, and

the ﬂoral glands two anterolateral petals (Fig. 3A). In contrast with the other petals,

the posterior petal has a smaller limb and thicker claw. The petals

Calyx glands exhibited ﬁmbriate edges with dilated terminations, corresponding

The calyx had glands arranged in pairs on the abaxial surface of with diminutive glands (Fig. 3A). These digitiform glands (Fig. 3B

the ﬁve sepals, varying from seven to ten (8.63 ± 0.93). The three and C) are more numerous and developed in the posterior petals,

sepals located opposite to the posterior petal lacked glands. The particularly in the basal portion of the limb, near the claw. The petal

glands are green, spherical to ovoid, subsessile, and inserted in glands and some sparse areas of the petal limb reacted positively to

the proximal region of the sepals. The accumulation of secretion NADI reagent (Fig. 3B), indicating the presence of terpenes (Table 2).

beneath the cuticle is observed just prior to anthesis. The secretion These same areas were also positively stained with neutral red,

reacted positively to Sudan IV staining, indicating the presence of indicating odour-producing areas.

lipids (Fig. 2A). The petal glands comprise a central core of homogeneous

Mature calyx glands comprised a uniseriate secretory epithe- parenchymal cells covered with a uniseriate secretory epithelium

lium and a central core of parenchymal cells supplied with and a thick cuticle (Fig. 3D–F). None of the examined samples

vascular tissues, primarily phloem, reaching the subepithelial lay- showed cuticle rupture. While the vascular terminations (compris-

ers (Fig. 2D). The epithelial tissue comprises narrow palisade cells, ing tracheal elements and phloem) enter the ﬁmbriae and reach

characterised by prominent nuclei, a densely stained cytoplasm and the cells close to the secretory epithelium in the posterior petal

large vacuoles predominantly localized to the distal pole of the cell (Fig. 3D), the vascularisation in the lateral petals passes through the

(Fig. 2D). Histochemical analyses indicated that epithelial cells in ﬁmbriae but does not reach the glandular region. The epithelial cells

the secretory phase contain phenolic substances, polysaccharides are thin-walled and vary in size and shape, depending on whether

and proteins (Table 2). The glandular parenchyma comprises two the glands are located on the lateral or posterior petal. The cross

well-deﬁned regions: (1) the subepithelial parenchyma, formed section revealed that the glands in the posterior petals had narrow

by 1–3 layers of packed, thin-walled parenchymal cells with a columnar epithelial cells, and few parenchymal, xylem and phloem

dense cytoplasm and vacuoles ﬁlled with phenolic substances; cells in the central region (Fig. 3E). The lateral petal glands showed

and (2) the innermost region of the gland, comprising larger, cuneiform epithelial cells and the central parenchyma, which is not

isodiametric parenchymal cells with a less dense cytoplasm, well- vascularised, comprised fewer cells. Histochemical analyses indi-

developed vacuoles, and small intercellular spaces. Starch grains cate the presence of polysaccharides, proteins and terpenes in the

were detected in the parenchymal cells (Table 2). glandular epithelium at the secretory phase (Table 2).

The TEM observations revealed a wide subcuticular space ﬁlled TEM observations revealed a smooth cuticle comprising a

with heterogeneous material, delimited by thin anticlinal walls of prominent cuticular layer and a thin cuticle proper (Fig. 3G).

epithelial cell and a thin cuticle (Fig. 2E). The subcuticular space The polysaccharide stratum of the cuticular layer disintegrates

results from the degradation of the cuticular layer during gland during gland development, forming small gaps (Fig. 3G) where

development. Immature glands present a thick cuticle compris- the protoplast secretions accumulate. The epithelial cells contain

ing an amorphous pectin layer, a loose ﬁbrous stratum, and the abundant cytoplasm rich in polyribosomes (Fig. 3H–M), mitochon-

cuticle proper (Fig. 2I). As the gland develops, the pectin stratum dria with well-developed cristae (Fig. 3H, L and M), rough and

C.C.F. Possobom et al. / Flora 211 (2015) 26–39 31

Fig. 3. Corolla glands of Diplopterys pubipetala. (A) Fimbriate and glandular edges (rectangles) of the posterior (pp), posterolateral (plp), and anterolateral (alp) petals. Note

the claw of the posterior petal (*). (B) The edges of posterior petals treated with NADI reagent. (C) Scanning electron micrographs of the posterior petal glands. (D–F) Light

micrographs showing longitudinal (D) and transverse (E–F) resin sections. (D–E) Posterior petal glands, showing epithelial cells and parenchyma supplied with phloem

(ph) and xylem (xy). (F) Lateral petal glands, showing quadrangular or cuneiform epithelial cells and a small number of parenchymal cells. (G–M) Transmission electron

micrographs. (G) Outer periclinal cell wall with small gaps (*) in the cuticular layer. (H) Cytoplasm with polyribosomes, Golgi bodies (Gb) and mitochondria (mi). (I) Abundant

rough endoplasmic reticulum (rer). (J) Plastids (pl) ﬁlled with oil droplets, marked with imidazole, near proliferated endoplasmic reticulum (er). (K) Plastids (pl) ﬁlled with

oil droplets (ol) surrounded with rough endoplasmic reticulum (rer). Observe Golgi body marked with ZIO technique. (L) Oil droplets (ol), plastids (pl), mitochondria (mi)

and plasmodesmata (arrows) in the anticlinal cell wall. (M) Epithelial and sub-epithelial cells connected by plasmodesmata (arrows). Note the reduced cytoplasm and large

vacuole of the sub-epithelial cells. Scale bars: A = 5 mm; B = 500 ␮m; C = 100 ␮m D and E = 50 ␮m; F = 25 ␮m; G, H, I, K and L = 500 nm; J, M = 1 ␮m. (For interpretation of the

references to colour in the text, the reader is referred to the web version of this article.)

32 C.C.F. Possobom et al. / Flora 211 (2015) 26–39

Table 2

Histochemical analysis of the ﬂoral glands of Diplopterys pubipetala.

Staining procedure Target Compounds Calyx glands Corolla glands Stamen glands

Reactivity Site Reactivity Site Reactivity Site

Sudan IV Total lipids ++ Cuticle ++ Cuticle ++ Cuticle

++ Subcuticular space

NADI Terpenes − ++ Epithelium ++ Epithelium

Ferric Trichloride Phenols + Epithelium − + Epithelium (globular cells)

++ Parenchyma ++ Epithelium (ﬂat cells)

Cupric acetate Resins − − −

Schiff (PAS) Neutral Polysaccharides ++ Cell wall ++ Cell wall ++ Cell wall

+ Epithelium + Epithelium + Secretory epithelium

Ruthenium red Pectin/ Mucilage ++ Cell wall ++ Cell wall ++ Cell wall

+ Epithelium + Epithelium + Secretory epithelium

Mercuric bromophenol blue Proteins ++ Epithelium ++ Epithelium ++ Epithelium

Wagner’s reagent Alkaloids − − −

Lugol Starch + Parenchyma − −

−: negative, +: slightly positive, ++: strongly positive

smooth endoplasmic reticulum (Fig. 3H, I and K), Golgi bodies with and ﬁlled with a ﬁne granular stroma containing electron-dense

associated vesicles (Fig. 3H and K), large plastids (Fig. 3J–M), and granules and tubules/vesicles (Fig. 5A, D and E). The juxtaposition

oil droplets (Fig. 3L). The large, unusually shaped plastids are ﬁlled of mitochondria and plastids is commonly observed (Fig. 5A, D and

with oil droplets of various sizes (Fig. 3J–M). Elements of both E). Large secretory vesicles are ﬁlled with a homogenous granular

smooth and rough endoplasmic reticulum occur throughout the material (Fig. 5E and F), preliminarily identiﬁed as polysaccharides.

cytoplasm (Fig. 3H and I) but are more abundant near plastids Interestingly, these vesicles are axially oriented towards protrud-

(Fig. 3K). We also observed plasmodesmata connecting epithelial ing areas (Fig. 5E and F) and juxtaposed with mitochondria (Fig. 5E).

cells with parenchymal cells (Fig. 3M). The parenchymal cells have a SER proliferation, RER and hyperactive Golgi bodies are observed

reduced cytoplasm and a central well-developed vacuole (Fig. 3M). subjacent to the wide spaces between the plasma membrane and

the cell wall (Fig. 5B and C). The periplasmic spaces are translu-

cent and ﬁlled with ﬁbrillar material (Fig. 5G and H). Vesicles are

Androecium glandular tissue commonly observed in the peripheral cytoplasm near the plasma

The ten stamens (Fig. 3A) have pale-yellow glandular connective membrane (Fig. 5A, B, G and H).

tissue comprising highly developed globular cells (Fig. 4A), which A summary of the general, anatomical, histochemical and ultra-

reacted positively to NADI reagent (Table 2, Fig. 4B), and ﬂat cells structural characteristics of the ﬂoral glands observed in Diplopterys

on the basal and lateral region, which reacted positively to ferric pubipetala is presented in Table 3.

chloride reagent (Table 2). Translucent, slightly viscous exudates,

identiﬁed as mucilage (Table 2), were observed over the surface of

the connective tissue (Fig. 4A). 4. Discussion

SEM revealed globular cells located on the apical and median

region of the connective tissue (Fig. 4C), displaying a reticulated In the present study, we described the ﬂoral biology and the

surface in the pre-secretory phase (Fig. 4D and E) and “bubbled” glands on the sepals, petals and stamen connectives of Diplopterys

surface in the secretory phase (Fig. 4F–H). Turgid globular cells were pubipetala, comparing the anatomy, histochemistry and ultrastruc-

observed next to depleted cells in the secretory phase. Flocculate ture of the ﬂoral glandular tissues. Each gland has a particular

material, previously identiﬁed as polysaccharides, is also observed anatomical structure, histochemistry and secretion mechanism,

on the glandular surface. suggesting additional levels of specialization associated with bee

The cross sections showed that the connective tissue surface attraction. In addition, we provide the ﬁrst description of the

is lined with a layer of globular bulky secretory cells (Fig. 4I and detailed ultrastructure and functioning of the ﬂoral glands, other

J). Proteins, polysaccharides and terpenes were histochemically than the calyx glands, for a species of Malpighiaceae.

detected in globular cells, while phenolic substances were present

in smaller epithelial cells (Table 2). PAS and ruthenium red staining

showed that the walls of globular cells are irregularly thickened Floral glands and interaction with pollinators

and contain pectin-rich regions; the areas corresponding to the

“bubbles” are thin-walled and weakly stained with these reagents As typical ﬂowers adapted to pollination through oil-collecting

(Table 2). Approximately 3–4 layers of endothecial-like cells and a bees, D. pubipetala ﬂowers are yellow, scented and oil producing

central core of parenchymal cells supplied with xylem and phloem (Faegri and Pijl, 1979; Endress, 1994). The D. pubipetala pollina-

are observed in the connective tissue subjacent to the epithelium tors Centridini (Centris) and Tapinotaspidini (Monoeca) are the most

(Fig. 4I). important tribes of oil-collecting bees in the Americas (Alves-dos-

The “bubbles” were identiﬁed as protoplast protrusions with Santos et al., 2007). Monoeca bees scrape the calyx glands with

thin cell walls covered with a thin cuticle (Fig. 5A–C). These pro- anterior and median basi tarsi, which in this genus comprise sev-

truded regions are interspersed with areas of thicker walls (Fig. 5A). eral irregular rows of closely pressed, simple, tapering setae (Neff

The cytoplasm of globular cells is more abundant and concentrated and Simpson, 1981), causing a striated rupture of the cuticle in a

in protruded areas (Fig. 5A–C), and these cells contain free ribo- longitudinal direction, as observed in D. pubipetala calyx glands.

somes; polyribosomes, endoplasmic reticulum; mitochondria, with The interaction between oil-offering ﬂowers and bees comprises

well-developed cristae; plastids; hyperactive Golgi bodies; and numerous reciprocal, morphological, behavioural and chemical

large, oval storage vesicles (Fig. 5A–H). The plastids are large, oval, adaptations (Renner and Schaefer, 2010).

C.C.F. Possobom et al. / Flora 211 (2015) 26–39 33

Fig. 4. Glandular androecium of Diplopterys pubipetala. (A) Dehiscent anther with aggregated, white pollen grains and glandular connectives (*). (B) Stamen treated with NADI

reagent. Note the strong reaction at the apical portion of the glandular connective. (C–H) Scanning electron micrographs. (C) Glandular connective with globular secretory

cells. (D) Globular cells at the pre-secretory stage. (E) Detailed view of the previous ﬁgure showing a reticulated surface. (F) Depleted globular cells at the secretory stage.

(G) Detailed view of the previous ﬁgure showing vesiculated surface. (H) Turgid globular secretory cells with vesiculated surface cells and released secretions (*). (I–J) Light

micrographs. (I) Cross section of the stamen showing connective with a unistratiﬁed epithelium comprising larger globular secretory cells and smaller epithelial cells with

dense contents. (J) Turgid and depleted globular secretory cells with heterogeneous contents and released secretions (*). Scale bars: A, B and C = 500 ␮m; D, F, H and J = 50 ␮m;

E and G = 10 ␮m; I = 100 ␮m.

34 C.C.F. Possobom et al. / Flora 211 (2015) 26–39

Fig. 5. Connective secretory epithelial cells of Diplopterys pubipetala. (A–H) Transmission electron micrographs. (A) Apical pole of a globular cell showing a thick-walled

ﬂat region interspersed with thin-walled protuberant regions (arrows). Note the large vacuole (va) and the dense cytoplasm with abundant mitochondria (mi) and plastids

(pl). (B–C) Detailed view of the protuberant area showing protoplasts covered with a thin cell wall and cuticle. Note the periplasmic space (*) and adjacent vesicles fusing

with the plasma membrane. (D) Large plastids with tubules and dense droplets (pl), abundant mitochondria (mi) and ribosomes, smooth (ser) and rough (rer) endoplasmic

reticulum, and dispersed oil droplets (ol). (E) Storage vesicles (sv), plastids (pl), mitochondria (mi) and dispersed oil droplets (ol), marked with imidazole. (F) Storage vesicles

(sv), plastid (pl) and proliferated smooth endoplasmic reticulum (ser). (G–H) Periplasmic space near anticlinal cell walls (arrows). Note the proliferated smooth (ser) and

rough (rer) endoplasmic reticulum and Golgi body (Gb). Scale bars: A = 10 ␮m; B = 5 ␮m; C = 3 ␮m; D = 2 ␮m, E = 1,5 ␮m; F–H = 1 ␮m.

C.C.F. Possobom et al. / Flora 211 (2015) 26–39 35

Table 3

Summary of main characteristics of ﬂoral glandular tissues of Diplopterys pubipetala.

Features Calyx Corolla Androecium

Glandular tissue location Abaxial surface of the sepals Termination of ﬁmbriate Apical and median portion of the connective of the ten

edge of the ﬁve petals, stamens, viewed macroscopically through neutral red

viewed macroscopically staining

through neutral red staining

Number 7–10 per flower (2 per sepal) Numerous (one per fimbriae) 10 per flower (one per stamen)

Shape and colour of glandular structures Spherical to ovoid/green Digitiform/yellow Globular/pale yellow

Anatomy Unistratiﬁed secretory Unistratiﬁed secretory Large globular secretory epithelial cells,

epithelial tissue, epithelial tissue and a central endothecial-like cells and a central vascularised

sub-epithelial and core of parenchymal cells parenchyma.

vascularised sub-glandular that is vascularised in

parenchyma posterior petal

Secretion type Lipophilic, conﬁrmed with Lipophilic, conﬁrmed with Mixed

osmium-imidazole osmium-imidazole (hydrophilic and lipophilic)

technique technique

Secretion accumulation Large subcuticular space and Small gaps between Periclinal space

canals between anticlinal periclinal cell wall and

walls cuticle

Secretion release Cuticle rupture Through intact cuticle Through intact and thin cuticle in protoplast

protrusion regions

Cytoplasm of secretory cells Abundance of Abundance of Abundance of ellipsoid mitochondria; rough and

polyribosomes; globular polyribosomes; ellipsoid smooth endoplasmic reticulum; large oval plastids

mitochondria; rough and mitochondria; rough and with a granular stroma, tubules and dense droplets;

smooth endoplasmic smooth endoplasmic large storage vesicles ﬁlled with a homogenous

reticulum; spherical plastids reticulum; large and granular material and hypertrophied; Golgi bodies,

devoid of inner membranes multi-shaped plastids devoid

and with small lipophilic of inner membranes and with

droplets; dispersed oil small lipophilic droplets;

droplets; few Golgi bodies; dispersed oil droplets and

plasmodesmata Golgi bodies; plasmodesmata

Proposed Function Elaiophore (oil as a reward) Osmophore (chemical Pollen mimicry (visual attraction)

attraction) Osmophore (chemical attraction)

Mucilage secretion (adherence of pollen grains to

pollinator body)

These bees gathered the oil produced by elaiophores and use the concentrated emission of odours in this region is associated

it for larval provisioning (Vogel, 1974; Vinson et al., 1997; Reis with the precise positioning of the visitor to the reward. For insects,

et al., 2007) and cell lining (Neff and Simpson, 1981; Simpson antennae are the primary olfactory organs, and ‘stereo’ olfaction

and Neff, 1981; Buchmann, 1987; Vogel, 1990a). The oil produced mediates the perception of spatial differences in scent concentra-

by the sepal glands of D. pubipetala acts as a primary attractor tion and the use of intraﬂoral scent guides (Lunau, 1991; Raguso,

for Centridini and Tapinotaspidini. These bees collect oil for larva 2001), which could be important for precise anchorage in ﬂow-

nutrition (Vogel, 1974; Vinson et al., 1997; Reis et al., 2007), nest ers with no landing area, as is the case of our study species. The

construction (Neff and Simpson, 1981; Simpson and Neff, 1981; precise landing behaviour of bees on D. pubipetala ﬂowers might

Buchmann, 1987; Vogel, 1990a) and possibly as an adult food reﬂect integrated perception, including both, visual and chemical

resource (Buchmann, 1987), and represent the most diverse group orientation.

in terms of adaptation for oil collecting (see Alves-dos-Santos et al., Beyond the Gates (1982) hypothesis concerning the function of

2007). According to Buchmann (1987), energy derived by consump- connective secretion as a glue to adhere pollen to the ventral surface

tion, digestion, and metabolism of fats, such as ﬂoral oil, is higher of the body of pollinating insects, no other function has been pro-

per gram than for equivalent amounts of carbohydrates, such as posed for these secretory structures in Malpighiaceae. Considering

nectar. other plant families, information concerning secretory connectives

Scent emission has been reported in other plant families that or staminodes is more abundant and shows considerable diversity

offer ﬂoral oil as a reward, including Iridaceae (Dobson, 2006), in the composition and function of the secretions (Ambruster, 1984;

Myrsinaceae (Dötterl and Schäfﬂer, 2007; Schäfﬂer et al., 2012) Fahn, 1988; Rudall et al., 1999; Smets et al., 2000; Ronse-Decraene

and Orchidaceae (Dobson, 2006; Pansarin et al., 2009). Remark- and Smets, 2001; Moyano et al., 2003; Buzgo et al., 2007; Guimarães

ably, however, in Malpighiaceae as one of the the most important et al., 2008; De-Paula et al., 2011). Connective tissues associated

ﬂoral oil-producing family, little is known about the structures with mixed secretions containing carbohydrates and proteins have

responsible for the emission of chemical cues involved in this highly been reported in Leguminosae (Mimosoideae), and these secre-

specialized pollination system. While morphological characteris- tions have been associated with the maintenance of pollen grain

tics, such as colour, size and shape, make the posterior petal an dampness and serve as a food source for many insects (Chaudhry

important visual guide for visitors, the presence of osmophores and Vijayaraghavan, 1992). In other species, the connective tissues

in D. pubipetala petals might chemically guide pollinators. In D. could act as resin glands (Ambruster, 1984), nectaries (Fahn, 1988;

pubipetala, the petal glands are more numerous and larger at the Smets et al., 2000; Freitas and Sazima, 2003; Buzgo et al., 2007)

limb base near the claw, the narrowest region of the petals, which or osmophores (Sazima et al., 1993). The secretions produced from

provide the bees access to the elaiophores, located in the dorsal por- connective cells could also be associated with a role similar to the

tion of the ﬂower (Anderson, 1979 Zhang et al., 2010). It is likely pollen kit, as previously suggested (Moyano et al., 2003) for Leonu-

that in D. pubipetala, and perhaps in other Malpighiaceae species, rus sibiricus (Lamiaceae). Based on the results obtained herein, we

36 C.C.F. Possobom et al. / Flora 211 (2015) 26–39

propose a complex function for the glandular connectives of D. microbial and herbivore attacks (Mazid et al., 2011 and references

pubipetala. The mucilaginous secretions might be associated with therein).

the enhanced efﬁciency of pollen transfer to other ﬂowers, consis- Both granulocrine and eccrine secretion mechanisms occur

tent with the hypothesis of Gates (1982), but might also maintain concurrently in the elaiphores of D. pubipetala. Vesicles and multi-

the dampness of the pollen grains, favouring adherence to the vesicular bodies near the plasma membrane and a well-developed

stigma and subsequent germination. In addition, the morpholog- periplasmic space indicate that the secretion reaches the apoplas-

ical features of these plants might also be associated with visual tic compartment through exocytosis, indicating a granulocrine

attraction, while the presence of volatile substances might also be mechanism (Fahn, 2000). Oil droplets dispersed throughout the

associated with the chemical attraction of pollinators. peripheral cytoplasm, near the plasma membrane of epithelial

cells and in the extracellular spaces (tubular channels and sub-

Anatomical and functional characterisation of the ﬂoral cuticular space), suggest that the secretions reach the apoplastic

glands compartment through an eccrine mechanism. Through an eccrine

mechanism, materials cross the plasma membrane via active trans-

Calyx elaiophores port (Fahn, 2000; Evert, 2006), passing over the porous wall (via

Vogel (1974) coined the term elaiophores to describe the struc- loosely arranged cellulose microﬁbrils) and accumulating beneath

tures that produce non-volatile oil as a ﬂoral reward. Diplopterys the cuticle.

pubipetala presents epithelial elaiophores, areas of oil-producing The accumulation of secretions under a wide subcuticular space

secretory epithelial cells that accumulate below a thin protec- is commonly observed in Malpighiaceae elaiophores (Vogel, 1974;

tive cuticle (Vogel, 1974; Simpson and Neff, 1981; Buchmann, Subramanian et al., 1990; Cocucci et al., 1996; Castro et al., 2001).

1987). Apart from Malpighiaceae, epithelial elaiophores could also Secretions from the subcuticular space permeate the intact cuticle

occur in a more restricted number of species, including Iridaceae (Cocucci and Vogel, 2001) or are released through cuticle rupture

(Goldblatt and Manning, 2006), Krameriaceae and Orchidaceae (Vogel, 1974; Simpson, 1982; Cocucci, 1991; Cocucci and Vogel,

(Vogel, 1974; Simpson and Neff, 1981; Buchmann, 1987). 2001). In the present study, we showed that the cuticle of D.

The overall distribution and anatomical organisation of calyx pubipetala elaiophores does not present pores or channels, and the

elaiophores in D. pubipetala is similar to that of other neotropical liberation of secretion occurs only after visiting bees rupture the

Malpighiaceae species, such as Heteropterys chrysophylla, Malpighia cuticle.

glabra and Stigmaphyllon littorale (Vogel, 1974), Dinemandra eri- The present study showed a strong anatomical and ultrastruc-

coides (Cocucci et al., 1996) and Galphimia brasiliensis (Castro et al., tural similarity between the calyx elaiophores and extraﬂoral

2001). In paleotropical species of Malpighiaceae, such as Hiptage nectaries of Diplopterys pubipetala (Possobom et al., 2010). Both

benghalensis and Acridocarpus smeathmannii, the calyx glands are structures exhibit secretory epithelial cells that accumulate secre-

also anatomically similar to the neotropical calyx elaiophores, tions below the subcuticular space and present cellular machinery

associated with extranuptial nectar but not with oil secretion to synthesis of mixed secretions, with the predominance of sug-

(Vogel, 1974; 1990a). In this context, Hiptage sericea, a paleotropical ars in extraﬂoral nectaries (Possobom et al., 2010) and lipids in

species, represents an exception, as the calyx glands of this species the elaiophores. These ﬁndings support the Vogel (1974, 1990a)

also produce oil (Subramanian et al., 1990). In addition, morpho- hypothesis that the calyx glands are homologous with extraﬂoral

logical similarity between elaiophores and extraﬂoral nectaries was nectaries.

observed in other Malpighiaceae (Vogel, 1974; Castro et al., 2001)

The histochemical analyses detected lipids as the primary com-

ponent of the secretion in the subcuticular space of the D. pubipetala Corolla osmophores

elaiophores, although the presence of polysaccharides and proteins The morphology of the petal glands of D. pubipetala is similar

might indicate that these glands are also involved in hydrophilic to that described for Heteropterys chrysophylla (Vogel, 1974) and

secretion. Indeed, small amounts of sugars (Baker, 1978; Vinson Dinemandra ericoides (Cocucci et al., 1996). The petal glands of D.

et al.,1997; Castro et al., 2001) and amino acids (Baker, 1978) were pubipetala are characterised by an extensive plastid compartment

also previously detected in the lipophilic secretions in the calyx ﬁlled with oil droplets and the proliferation of smooth endoplas-

glands of other Malpighiaceae. mic reticulum typical of lipophilic glands, particularly those that

The subcellular traits of the epithelial cells of D. pubipetala calyx secrete monoterpenes (Turner et al., 1999; Machado et al., 2006;

glands, such as the abundance of cytoplasmic oil droplets, smooth Melo et al., 2010). Furthermore, the presence of polyribosomes,

endoplasmic reticulum, and polymorphic plastids devoid of inner Golgi bodies, numerous vesicles and rough endoplasmic reticulum

membranes and ﬁlled with osmiophilic inclusions, are typical fea- might be associated with the cytoplasmic synthesis required for cell

tures of elaiophores (Castro et al., 2001; Stpiczynska et al., 2007; metabolism and monoterpene biosynthesis (Turner and Croteau,

Stpiczynska and Davies, 2008; Aliscioni et al., 2009; Davies and 2004; Melo et al., 2010). These organelles might be associated with

Stpiczynska, 2009; Pacek et al., 2012). However, the abundance of the glycosylation of secretion compounds, particularly some toxic

polyribosomes, RER and vesicles in the epithelial cells are evidence components of essential oils (Figueiredo and Pais, 1994). In addi-

of hydrophilic secretions (Evert, 2006). However, we must consider tion, these organelles could also be involved in the synthesis of the

that these organelles could also be associated with the synthe- pectinases and cellulases that participate in wall degradation pro-

sis and elimination of cell wall degradation enzymes (Rodrigues cesses necessary for wall-gap development (Ascensão et al., 1997;

and Machado, 2012). Indeed, cell wall disintegration, resulting in Machado et al., 2006).

gaps in the cuticular layer and channels between the anticlinal Based on positive staining with neutral red, NADI and Sudan IV

walls of epithelial cells, was observed during elaiophore matura- and the results of the ultrastructural analyses, the petal glands in D.

tion in D. pubipetala. In addition to the epithelium, the anatomical pubipetala are osmophores, i.e., ﬂoral scent glands (Vogel, 1990b;

and ultrastructural organisations of the glandular parenchyma of Evert, 2006). The lipophilic nature of these glands has been pre-

calyx glands indicate that this region also participates in secretion. viously reported for other Malpighiaceae species, but no function

This evidence includes the presence of vascular supply through the has been suggested. According to Vogel (1974), the petal glands of

xylem and phloem and the abundance of plasmodesmata connect- Heteropterys chrysophylla produce non-polar lipids, different from

ing all gland cells. Moreover, phenolic substances in the vacuoles of those observed in the secretions produced from calyx glands, and

sub-epithelial parenchymal cells protects the gland tissues against Lobreau-Callen (1989) also detected lipids in the secretions from

C.C.F. Possobom et al. / Flora 211 (2015) 26–39 37

the petal glands of Burdachia primatocarpa and Glandonia macro- Conclusions

carpa.

With respect to the release mechanism, the substances secreted Our data indicated that secretions from the calyx, corolla and

from protoplasts through granulocrine and eccrine mechanisms androecium glands in D. pubipetala perform essential functions in

accumulate inside gaps resulting from the disintegration of the the interaction with ﬂoral visitors, acting as primary and secondary

cuticle layer. The secretion of volatile oils from the gaps to the attractants. In addition, the secretions produced in the glandular

gland surface likely occurs via diffusion through the intact and thin connective tissues might increase the efﬁciency of zoophilous pol-

cuticle. lination, improving the efﬁciency in the transfer of pollen grains.

The degree of development, including the number, size and The results presented herein are novel, as the detailed morphology

vasculature of the petal glands in D. pubipetala differs between and functioning of ﬂoral glands, other than the calyx glands, are

posterior and lateral petals, suggesting the increased production unknown for almost all Malpighiaceae species.

of secretions in the former glands.

Acknowledgements

Androecium glandular tissue – multiple functions

The results of the present study suggest that all ten connective This work was part of the Master’s Dissertation of the ﬁrst author

tissues of D. pubipetala stamens are glandular, suggesting impor- (Programa de Pós- Graduac¸ ão em Ciências Biológicas (Botânica),

tant roles for these structures in pollination. The shiny surface of IBB/UNESP) and supported through the Fundac¸ ão de Amparo à

the connectives, formed by large yellow globular epithelial cells, is Pesquisa do Estado de São Paulo – FAPESP (Biota Thematic Process

similar to those described for the staminodes of Commelina dianthi- number 08/55434-7 and grant number MS 06/54268-0) and the

folia (Commelinaceae) (Hyrcan and Davis, 2005), mimicking large Conselho Nacional de Desenvolvimento e Pesquisa – CNPq (Edi-

quantities of pollen grains that might attract insects to these ﬂow- tal Universal 470649/2008-9 and grant number PQ 301464/2008-1

ers. awarded to S.R. Machado). The authors would like to thank the

The results of the histochemical analyses indicated that the technicians of the Electron Microscopy Center (CME) of UNESP,

epithelial cells of the connective tissue produce mixed secretions. Botucatu, for lab assistance, D.A.O. Naliato for assistance with the

The copious and sticky exudates on surface were determined as ﬁeldwork, and Drs. J.M.F. Camargo (in memoriam) and S. Mateus

mucilage (Johansen, 1940), whereas the positive neutral red and for the identiﬁcation of the bees.

NADI staining indicated the presence of lipophilic substances, likely

volatile terpenes.

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