Acianthera flower anatomy

ORIGINAL ARTICLE

Morphological and histological characterizationof the osmophores and nectaries of four speciesof Acianthera (Orchidaceae: Pleurothallidinae)

Marcos Cabral de Melo • Eduardo Leite Borba •

Elder Antonio Sousa Paiva

Received: 18 February 2009 / Accepted: 13 April 2010 / Published online: 15 May 2010

� Springer-Verlag 2010

Abstract Nectar and floral odor are frequently associated

with the presence and maintenance of specialized pollina-

tion systems in Orchidaceae. We studied flowers of

four Acianthera species, a genus of myophilous orchids

belonging to the largest fly-pollinated orchid group

Pleurothallidinae, in order to characterize the secretory

structures related to their pollination mechanism. Flowers

at anthesis were sampled to detect volatile compounds and

nectar; samples were fixed for light microscopy and scan-

ning and transmission electron microscopy. The labellum

presents epidermal cells and the first mesophyll layer

involved with secretory processes. Cellular characteristics

of these regions associated with the occurrence of sugars

allowed us to recognize them as nectaries. Some portions

of the sepals also shown to be involved with secretory

processes and the presence of nitrogenated volatile com-

pounds characterize them as osmophores. The production

of nectar in these species makes the occurrence of sapr-

omyophily questionable, even though these flowers present

characteristics of this floral syndrome. The presence of

osmophores on the sepals reinforces that this localization is

common among the Pleurothallidinae, whilst they occur in

a different region (labelum) in the other major fly-polli-

nated orchid group (Bulbophyllum).

Keywords Acianthera � Cell ultrastructure � Myophily �Nectaries � Orchidaceae � Osmophores

Introduction

The subtribe Pleurothallidinae, with approximately 4,100

species, and the unrelated genus Bulbophyllum Thouars,

with approximately 2,100 species are two of the largest

fly-pollinated orchid groups. They exhibit some of the most

interesting examples of floral convergence due to adapta-

tion to the same group of pollinators (Dressler 1993).

Although the flowers of these myophilous orchids share a

number of morphological characters, floral biology studies

have demonstrated that there is still a large diversity of

pollination mechanisms associated with fly-pollination,

involving both biotic and wind-assisted biotic mechanisms

(van der Pijl and Dodson 1966; Borba and Semir 1998).

Among them, different adaptations to attract these insects

stand out; some of them are associated with the insects’

feeding and/or reproductive instincts (Borba and Semir

1998, 2001; Singer and Cocucci 1999). Evolution of these

differences in many myophilous orchids seems to be rela-

ted to the great specificity of the plant-pollinator relation-

ship to a degree that was not initially expected.

Odor and nectar are important elements to maintain the

plant-pollinator relationship. Floral odor is responsible for

long distance attraction while nectar constitutes a principal

floral reward (Proctor et al. 1996). Nectar is the principal

reward to pollinators among the Orchidaceae (Dressler

1993) and is commonly present in orchids pollinated by

anthophilous species of Diptera. The presence of nectar

may result in high pollen transfer, as previously demon-

strated in epidendroid orchids (Peter and Johnson 2009).

Although nectar is not commonly found among sapromy-

ophilic plants, as their pollinators are attracted by ovipo-

sition instinct (Faegri and van der Pijl 1979; Proctor

et al. 1996), it seems to be an important element in the

mechanism of deceit-pollination in some wind-assisted

M. C. de Melo � E. L. Borba � E. A. S. Paiva (&)

Departamento de Botanica, Instituto de Ciencias Biologicas,

Universidade Federal de Minas Gerais,

Av. Antonio Carlos, 6627, Pampulha, Belo Horizonte,

Minas Gerais 31270-901, Brazil

e-mail: [email protected]

123

Plant Syst Evol (2010) 286:141–151

DOI 10.1007/s00606-010-0294-1

fly-pollinated Bulbophyllum species (Borba and Semir

1998) or in partially deceitful species of Acianthera (Borba

and Semir 2001).

The genus Acianthera (Pleurothallidinae) comprises

approximately 200 species distributed throughout tropical

South America, especially southeastern Brazil (Pridgeon

et al. 2005). As with other members of the subtribe,

Acianthera in humid forests are predominantly epiphytes,

or otherwise, lithophytes on rocky soils exposed to direct

sunlight. Their flowers show features typical of myophilous

flowers such as diurnal anthesis and, frequently, unpleasant

odor. Borba and Semir (2001) studied the pollination

biology of a group of Brazilian Acianthera species and

found that species pollinated by Phoridae flies have nectar

on the labellum, while those pollinated by Chloropidae

flies are nectarless and use deceit-pollination. In all these

species scent emission occurs in the sepals (E. L. Borba,

UFMG, Minas Gerais, Brazil and J. R. Trigo, UNICAMP,

Sao Paulo, Brazil, unpublished data).

Acianthera hamosa (Barb Rodr.) Pridgeon & M. W.

Chase, A. limae (Porto & Brade) Pridgeon & M. W. Chase,

A. modestissima (Rchb. f. & Warm.) Pridgeon & M. W.

Chase, and A. prolifera (Herb. ex Lindl.) Pridgeon &

M. W. Chase constitute a group of species that have very

similar overall flower morphology. However, they have

subtle morphological differences that may be related to the

attraction of distinct groups of pollinators (different fami-

lies of Diptera) and, thus, contribute to their reproductive

isolation (Melo 2008). These species have similar flower

morphology to those studied by Borba and Semir (2001).

Some areas of the sepals and labellum are shiny, and the

base of the labellum is frequently moist, which may indi-

cate that these regions can be involved in the release of

secretions, like those from osmophores or nectaries (Vogel

1990; Pacini et al. 2003). This may suggest that the papillose

regions of the labellum of those species pollinated by

Phoridae flies (A. hamosa, A. limae, and A. modestissima)

and Chloropidae flies (A. prolifera) are nectaries, even

though such structures were believed to be absent in the latter

(Melo 2008).

All four species seem to have osmophores located at the

adaxial face of sepals. The micromorphology of osmo-

phores in fly-pollinated orchids has been examined only in

a few species of Bulbophyllum and Pleurothallidinae before

(Pridgeon and Stern 1985; Vogel 1990; Teixeira et al.

2004). These studies have shown that osmophores are

generally found in the labellum, in species of Bulbophyl-

lum, or the sepals, in the Pleurothallidinae (Masdevallia,

Pleurothallis, Restrepia, and Scaphosepalum) (Vogel

1990; Teixeira et al. 2004). If osmophores occur in dif-

ferent parts of the flowers in other species of the Pleuro-

thallidinae, such as in Acianthera, is unknown. But if it

does, this could reinforce the idea of divergence in

anatomical and functional aspects of the flowers of

Bulbophyllum and Pleurothallidinae species, despite the

apparent morphological convergence of their flowers (van

der Pijl and Dodson 1966).

In this study we investigated the micromorphological

features of the labellum and sepals of four species of

Acianthera pollinated by Chloropidae (A. prolifera) and

Phoridae flies (A. hamosa, A. limae, A. modestissima) using

light microscopy and transmission and scanning electron

microscopy. We aim to (1) determine the occurrence of

nectary glands and osmophores in flowers of Acianthera;

(2) correlate these structures with the pollination mecha-

nism observed in the group; and (3) correlate the

morphology and location of these structures with that

observed in the two largest myophilous groups in the

family, Pleurothallidinae and Bulbophyllum.

Materials and methods

Plant material

Flowers were collected from plants cultivated at the

greenhouse of the Universidade Federal de Minas Gerais.

These plants were collected from wild populations previ-

ously included in reproductive biology studies by our group

(Melo 2008). In the wild these plants are usually found

growing on rock outcrops (A. modestissima and A. prolif-

era) or on the forest floor of gallery forests (A. hamosa and

A. limae) in areas of campos rupestres vegetation in Minas

Gerais State, Brazil. Voucher specimens were deposited in

the herbarium BHCB (A. hamosa, M.C. Melo 08; A. limae,

M.C. Melo 07; A. modestissima, M.C. Melo 06; A. prolif-

era, M.C. Melo 04).

Light microscopy

Flowers in full anthesis of all four species were fixed in

Karnovsky solution (Karnovsky 1965) for 48 h, dehydrated

in an ethanol series, and embedded in 2-hydroxyethyl-

methacrylate resin (Leica). Transverse and longitudinal

sections of 5 lm were made and stained with 0.05%

toluidine blue at pH 4.3 (O’Brien et al. 1964). Cross-

sections of fresh sepals and labellum were done by hand

and used in histochemical tests using Lugol’s solution to

detect starch (Johansen 1940) and Sudan Red B for lipids

(Pearse 1980), with their respective controls. These tests

were performed in flowers that have been in anthesis for up

to 48 h. To detect starch dynamics, the Lugol test was

repeated on flowers close to senescence (approximately

10 days in A. hamosa and A. modestissima and 20–25 days

in A. limae and A. prolifera). Glycerin was applied

to paradermal sections to observe stomatal movements

142 M. C. de Melo et al.

123

(Jernstedt and Clark 1979). Descriptions of structural

characteristics were made, and attention was especially

given to cells and tissue in the sepals and labellum likely to

be involved in secretory processes.

Scanning electron microscopy

Tissue samples of the lateral sepals and labellum of

A. limae, A. modestissima, and A. prolifera were prepared

for observation under scanning electron microscopy. Due

to low availability of flowers, A. hamosa was not included

in this analysis. Tissue was taken from the flowers up to

48 h after the beginning of anthesis, fixed in 2.5% glutar-

aldehyde (0.1 M phosphate buffer, pH 7.2), dehydrated in

an ethanol series, dried to critical point, and subsequently

sputter-coated with ca. 10 nm of gold as described by

Robards (1978). The samples were examined in a scanning

electron microscope, model Quanta 200 (Fei Company,

Hillsboro, OR, USA), and all images were processed

digitally.

Transmission electron microscopy

Overall, the floral anatomy of all the Acianthera species

included in this study is very similar. Therefore, only

flowers of A. prolifera were used for the TEM study. This

similarity was confirmed by observations of the sepals and

labellum under light and scanning electron microscopy and

the chemical nature of the substances they secrete. Tissue

samples from the lateral sepals and labellum were obtained

from flowers up to 48 h after beginning of the anthesis,

fixed in Karnovsky solution (Karnovsky 1965) for 24 h,

post-fixed in 1% osmium tetroxide (in 0.1 M phosphate

buffer, pH 7.2), and processed using standard methodolo-

gies (Roland 1978) for observation under transmission

electron microscope. Ultra-thin sections were stained

with uranyl acetate and lead citrate and examined under

a Philips CM 100 transmission microscope at 60 kV.

Descriptions were focused on cells and tissues that appeared

to be involved in secretory processes.

Preliminary analyses of nectar and volatile compounds

The presence of sugars was detected by thin layer chro-

matography (TLC) following Dafni (1992). Residual floral

secretions on the adaxial surface of the labellum and on the

lateral sepals of about ten flowers of each of the four

species were soaked in situ with distilled water (2 ll

droplet) for 10 min. These samples were then removed and

applied to 10 9 10 cm silica gel TLC plates prepared with

0.02% sodium acetate. It is important to emphasize that

samples from the labellum and sepals were analyzed sep-

arately. Standard marker solutions of fructose, glucose, and

sucrose, as well as distilled water (control) were also pre-

pared and applied to the same plates. These plates were run

with a mobile phase of chloroform:methanol (6:4), dried at

room temperature, sprayed with an orcinol-sulfuric acid

solution, and then heated to 120�C for 5 min. Sugars

stained as dark-purple bands against a yellow background

(Stahl 1988).

The Whiff test (Amsel et al. 1983) was used to detect

volatile amines, generally responsible for the unpleasant

scent emitted by flowers. The dorsal sepal, lateral sepals

and petals, and the labellum of ten flowers from each of the

four species were immersed in a solution of 10% KOH, in a

closed vessel for 1 min. As control, we used a flask with

just the KOH solution and another flask with the same

solution but with a piece of leaf from one of the species.

Floral parts that gave off fishy odors were considered as

sites where volatile compounds are probably emitted.

Results

The organization and anatomy of the floral parts are very

similar in all four species. The epidermis of the entire

perianth is uniseriate (Fig. 1). The parenchyma mesophyll

is homogenous and with collateral vascular bundles

(Fig. 1a, c), and many idioblasts containing raphides.

Given the great morphological similarity among the flow-

ers of the Acianthera studied here, we present a description

applicable to all four species, highlighting, where appro-

priate, the peculiarities of certain species.

Labellum structure

The labellum has two calli along its median portion; these

are discrete and consist of epidermal projections that are

structurally similar to those observed on the entire adaxial

face (Fig. 2a, g). The epidermis of the labellum is unise-

riate, with juxtaposed cells, and no stomata are present

(Figs. 1b, 2). Papillae are found on the entire adaxial face

(Fig. 2), from the distal region of the labellum to its base

where it joins the column. These papillae are intercalated

with unspecialized epidermal cells. The papillae have

sculptured surfaces with irregular areas delimited by raised

borders, especially in A. limae (Fig. 2c, f, h). Both the

papillae and the other epidermal cells of the adaxial face

have a dense cytoplasm and conspicuous nuclei. Both cell

types are covered by a continuous cuticle that is firmly

joined to the cell walls. On the abaxial face, on the con-

trary, the epidermal cells are flattened with large vacuoles.

The labellum mesophyll is parenchymatous, and the

superficial layer of the adaxial face is formed by cells with

a denser cytoplasm than the internal cells; starch grains are

also frequently observed here. There is considerably less

Osmophores and nectaries in Acianthera (Orchidaceae) 143

123

starch in the mesophyll cells of flowers during the secretory

phase and practically none in the mesophyll of presenes-

cent flowers. Vascular bundles (one dorsal and two laterals)

are present near the abaxial face, but no ramifications were

observed to the adaxial face (Fig. 1a).

Sepal structure

The epidermis of the sepals is uniseriate with juxtaposed

cells and covered by cuticle. The epidermis on the adaxial

face contains regions that are characterized by the presence

of cells with dense cytoplasm and conspicuous nuclei

(Fig. 1a, c, d). The epidermal cells on both sides of the

sepal are projected slightly upwards, giving the sepals a

subtle papillose appearance (Fig. 1c). The mesophyll, and

especially the area next to the adaxial face, is formed by a

two-to-three-cell layer with large numbers of starch grains

(Fig. 1e).

The apex of the dorsal sepal has a region formed by cells

with dense cytoplasm, while in the lateral sepals this region

extends along both sides of the fusion line from the basal

third of the sepals up to the apex. This area is restricted

only to the adaxial face of the sepals, where the stomata

occur exclusively. The stomata have wide pores and

inflated outwardly projecting guard cells (Fig. 3). The

cuticle is smooth, not ornamented, and without pores or

signs of ruptures of any kind (Fig. 3c, f). In some stomata,

especially in A. modestissima and A. limae, the cuticle

covers the stomatic pore, obliterating it (Fig. 3a). The

stomata were permanently open and no movements were

observed.

The underlying parenchymatous mesophyll on the

adaxial face of the sepals, towards the epidermal cells

described above, includes cells with dense cytoplasm,

conspicuous nuclei, and numerous plastids with starch

grains (Fig. 1c–e). The quantity of starch grains decreases

with the age of the flowers. Although starch grains can

be observed throughout the mesophyll and epidermis, they

are mostly concentrated in the cells containing dense

cytoplasm.

Labellum ultrastructure

The cells of the epidermis and the first layer of the meso-

phyll, on the adaxial face of the labellum, have morpho-

logical features that suggest their involvement in secretory

processes, while in other regions, the cells are vacuolated

suggesting low levels of metabolic activity. The epidermal

Fig. 1 Sections of flowers

(48 h after beginning of the

anthesis) of Aciantheraprolifera (Orchidaceae) under a

light microscope. a Transversal

section on the median portion of

an entire flower; note that only

the labellum and lateral sepals

have cells on the adaxial face

with dense cytoplasm associated

with secretory activity (arrows).

b Detail of the adaxial face of

the labellum showing the

nectary composed of papillose

epidermal cells with dense

cytoplasm. c Transversal section

of the lateral sepal in the

osmophore region; note that the

epidermis and underlying layer

on the adaxial (secretory) face

are composed of cells with

dense cytoplasm. d–eLongitudinal section of the

osmophore region of the dorsal

sepal showing the tissue

underlying the epidermal layer;

in e note the large number of

starch grains (dark points)

evidenced with Lugol’s solution

(vb vascular bundle)


123

cells of the adaxial face have thin walls and a dense and

organelle-rich cytoplasm (Fig. 4). Ordinary epidermal cells

and papillae are similar in their cytoplasmic composition.

The nuclei of these cells are large, slightly lobed, and

nucleoli are evident (Fig. 4c). Vacuoles are numerous, with

one or more central vacuoles and numerous small ones in

the periphery (Fig. 4a, b). Also the fusion of vacuoles and

the presence of intravacuolar membranes are observed

here.

Numerous mitochondria, ranging from globose to elon-

gated and with well-developed cristae, were observed in the

epidermal cells on the adaxial face of the labellum (Fig. 4b).

Rough endoplasmic reticulum was associated with the

smaller vacuoles, often surrounding them (Fig. 4b). The

plastids were globe-shaped, had poorly developed mem-

brane systems and dense stroma, and contained starch grains

(Fig. 4c). Free ribosomes and dictyosomes were also

observed here, although they were not frequent (Fig. 4b).

The epidermal cells have ample periplasmatic spaces and

present vesicles and flocculated material (Fig. 4b), which

suggests secretory activity taking place here.

The parenchymatous cells of the mesophyll have a

single large vacuole (Fig. 4d). Mitochondria and plastids

are also seen in these cells, the latter containing starch

grains, especially in young flowers.

Sepal ultrastructure

The cytoplasm of the epidermal cells, on the adaxial face of

the sepals, is organelle-rich (Fig. 5) and has lobed nuclei

and evident nucleoli. The cell membrane is sinuous,

forming a conspicuous periplasmatic space, with no signs

Fig. 2 Nectary region on the

adaxial face of the labellum of

Acianthera flowers 48 h after

the beginning of anthesis, under

SEM. a–c A. limae, d–f A.modestissima, g, h A. prolifera.

a, d, g, General aspects of the

surface of the labellum. Note

the papillae intercalated with

typical epidermal cells, and

presence of calli formed by

epidermal projections in the

median portion of the labellum

(arrows). In b, e note the

papillae intercalated with the

typical epidermal cells. In c, f, hdetail of the labellum papillae,

showing the sculptured cuticle

surface


123

of secretions accumulating here (Fig. 5a). The nucleus is

usually found in the lower third of the cells, while the

central portion of the cell contains numerous vacuoles.

Among the organelles in these epidermal cells, plastids and

mitochondria with well-developed cristae are the most

abundant (Fig. 5a, b). Many of the plastids are polymor-

phic, although globose forms predominate; the stroma is

dense and there are many osmiophilic inclusions (Fig. 5a,

b). A large part of the plastid stroma is occupied by starch

that appears to be partially hydrolyzed; it has an amorphous

aspect (Fig. 5b). Free ribosomes and rough endoplasmic

reticulum can be seen in the cytoplasm of the epidermal

cells (Fig. 5d). Dictyosomes are rare in both the epidermal

and the secretory cells of the mesophyll.

Mesophyll cells of the adaxial faces of the sepals show

ultrastructural characteristics similar to those seen in the

epidermal layer (Fig. 5c, d). These cells are connected to

each other and with the epidermis by plasmodesmata.

Some plastids contained no starch reserves at this stage,

and this was more evident in the cells of the internal layers

of the mesophyll. Frequent fusion of plastids or fusion of

plastids and vacuoles was also observed at this phase.

Analyses of nectar and volatile compounds

The TLC analyses indicated the presence of sugars on the

surface of the labellum of the four species examined but

were negative for the sepals. Samples of the labellum

yielded bands with Rfs corresponding to sucrose (0.46) and

to glucose and/or fructose (0.55). Two bands were observed

for A. hamosa and A. modestissima, one intense band for

sucrose and one weak band indicating a monosaccharide.

Analysis of the samples of A. limae and A. prolifera, on the

other hand, showed two bands with similar intensity.

The Whiff test was positive for all four species and

strong fishy odor was perceived from the dorsal and lateral

sepals. However, odor intensity was stronger in the lateral

sepals. No odor was perceived from the labellum or any

vegetative organ soaked in KOH for any of the species.

Discussion

The structural and chemical analyses of the four species

provide strong evidence to conclude that some regions of

Fig. 3 SEM images of the

osmophore region on the

adaxial face of the sepals of

Acianthera flowers 48 h after

the beginning of anthesis.

a–c A. limae, d A. modestissima,

e, f A. prolifera. In a, b, d, e,

general aspect of the epidermis

of the osmophores formed by

papillose cells with smooth

cuticles. In a the detail shows

a stoma obliterated with cuticle.

e, f Detail of a stomatal pore;

notice the smooth and intact

cuticle


123

the labellum and the calyx are secretory. These are easily

identified by their high cytoplasmic density and starch

presence. Also, the ultrastructural analysis of A. prolifera

showed an abundance of mitochondria, which are usually

found in areas of elevated energetic demands due to

secretory processes. The abundance of mitochondria in the

tissue-forming part of osmophores and nectary glands in

the Orchidaceae has been previously reported (Pridgeon

and Stern 1983; Stpiczynska et al. 2005b).

The presence of sugars on the outer surface of the

labellum of the four species of Acianthera allowed us to

delimit the nectary gland. This was corroborated by the

ultrastructural characteristics of the epidermal cells of the

labellum in A. prolifera studied under TEM. Meanwhile,

the presence of volatile nitrogenized compounds, high

cytoplasmic density of the epidermal cells, and the pres-

ence of starch in the subepidermal layers allowed us to

identify the location of osmophores in the papillose

regions of the adaxial face of the sepals, which are larger

in the lateral sepals than in the dorsal sepal. These his-

tological and chemical characteristics have been observed

before in other studies and seem to be typical of the

osmophores in the Orchidaceae and other plant families

(Pridgeon and Stern 1983, 1985; Vogel 1990; Garcia et al.

2007).

The overall morphology of the nectary glands examined

here is similar to that previously described in the Orchid-

aceae, and it is formed by a secretory epidermis associated

with layers of parenchymatic tissue (Stpiczynska and

Matusiewicz 2001; Teixeira et al. 2004; Stpiczynska et al.

2005b; Vieira et al. 2007). The presence of papillae, like

the ones observed in this study, or unicellular trichomes

that increase the secretion surface has also been described in

the nectary glands of other Orchidaceae species (Stpiczynska

1997; Stpiczynska and Matusiewicz 2001; Teixeira et al.

2004; Stpiczynska et al. 2005b).

Idioblasts containing raphides of calcium oxalate have

been observed in the sepals and petals of other species of

orchids (Stpiczynska et al. 2003, 2005a), and their presence

near plant secretory structures has often been noted in

diverse taxonomic groups (Schnell et al. 1963). According

to Paiva and Machado (2005), the presence of these crys-

tals may be related to the elimination of excess calcium

from the cytosol.

The nectaries of the four species of Acianthera exam-

ined here are not vascularized, but they are associated with

large starch reserves that can be used as energy sources for

secretory cells. Starch accumulation in the presecretory

phase is commonly seen in the nectaries of orchid species

(Figueiredo and Pais 1992; Stpiczynska 1997; Davies et al.

Fig. 4 Ultrastructural aspects

of the nectary region on the

adaxial face of the labellum of

Acianthera prolifera flowers

48 h after the beginning of

anthesis. a General view of a

papilose cell showing its dense

cytoplasm and large number of

organelles. b Epidermal cell

with ample periplasmatic space

containing flocculated material

(probably secretion residues)

and numerous mitochondria,

some free ribosomes, but few

dictyosomes. c Secretory cell

with evident nucleus and

nucleolus; globoid plastids with

poorly developed membrane

systems and starch grains.

d Detail of subepidermal

parenchymatic cells; notice

large central vacuole

(di dictyosomes, mimitochondria, pl plastid,

re endoplasmic reticulum,

se secretion, va vacuole)


123

2005; Stpiczynska et al. 2005a, b), as well as in other plants

(Nepi et al. 1996; Paiva and Machado 2008). A significant

reduction in the levels of starch, such as those observed

here, in the tissue of the nectary gland and during the

secretory phase, has also been reported in earlier studies

(Nepi et al. 1996; Stpiczynska et al. 2005a, b; Vieira et al.

2007; Paiva and Machado 2008).

The secretion mode of the nectaries of A. prolifera can

be characterized as granulocrine, in which vesicles with

secretion materials derived from the endoplasmic reticu-

lum, the dictyosomes, or both, fuse to the plasma mem-

brane. This process has been observed in the nectaries of

other Orchidaceae, such as Hexisea imbricata (Stpiczynska

et al. 2005a) and Platanthera bifolia (Stpiczynska 1997).

We observed that the cuticle of A. prolifera does not

constitute a barrier to nectar flow, a situation that has also

been observed in other orchid species (Stpiczynska et al.

2003, 2005a).

The presence of starch reserves in the tissues underlying

the glandular epidermis, as observed in the four species of

Acianthera, is a common characteristic of the osmophores,

including those in Orchidaceae (Vogel 1990). A number of

studies have indicated that starch is utilized in these

osmophores as a source of both energy and carbon for the

synthesis of volatile substances (Vogel 1990). The mor-

phological alterations observed in the plastids suggest the

transition of these organelles to become vacuoles has been

similar to that observed in the floral nectaries of other

plants (Jiang et al. 2002; Gaffal et al. 2007; Paiva and

Machado 2008).

The presence of rough endoplasmic reticulum, and the

low frequency of dictyosomes in the secretory cells of the

osmophores in the species of Acianthera examined here,

is compatible with the nitrogen-rich nature of their

secretions. Low frequency of dictyosomes was also

reported for Restrepia, which likewise has a disagreeable

odor associated with amines (Pridgeon and Stern 1983;

Vogel 1990).

The secretion of volatile chemical compounds from the

osmophores of A. prolifera seems to occur by ecrine pro-

cesses, with a notable absence of vesicles in the secretory

cells, as has been reported for other Pleurothallidinae such

as Restrepia and Scaphosepalum (Pridgeon and Stern 1983,

1985). This is unusual because a granulocrine secretion

Fig. 5 Ultrastructural aspects

of the osmophores on the

adaxial face of the sepals of

Acianthera prolifera flowers

48 h after the beginning of

anthesis. a General view of an

epidermal cell of the

osmophore; notice the high-

density cytoplasm and large

numbers of organelles. b Detail

of an epidermal cell of the

osmophore showing plastids

with dense stroma and

osmiophilic inclusions; notice

hydrolyzed starch grains that

have an amorphous outline in

the interior of the plastid.

c, d Detail of the cells of the

subepidermal tissue showing

plastids with translucent stroma

resulting from starch hydrolysis;

notice in d the high density of

mitochondria and the well-

developed endoplasmic

reticulum (mi mitochondria,

pl plastid, re endoplasmic

reticulum, va vacuole)


123

mode seems to be common in other groups of orchids

(Vogel 1990; Stpiczynska 1993, 2001).

Emission of volatile compounds by osmophores by

cuticular diffusion processes has been observed in

Orchidaceae before, such as in species of Scaphosepalum

(Pridgeon and Stern 1985) and Stanhopea (Stern et al.

1987); or by cuticular pores in species of Restrepia and

Restrepiella (Pridgeon and Stern 1983) and Gymnadenia

conopsea (Stpiczynska 2001). The emission of volatile

compounds in these species of Acianthera, however, seems

to be associated with the presence of stomata. Stomatal

pores were frequently observed on the surface of the nec-

taries that are involved in exogenous secretion, and Vogel

(1990) suggested they could work as possible routes for

volatile secretions. We found evidence here that the

secretion products of species of Acianthera are liberated by

the cells of the osmophores and accumulated in the peri-

plasmatic and intercellular spaces. These compounds are

probably volatilized by daylight temperatures and finally

reach the outside environment through the stomatal pores.

This hypothesis can be further supported by the liberation

of these odors only during the hottest hours of the day. This

has also been reported for other species of Acianthera

(Borba and Semir 2001) and Bulbophyllum (Borba and

Semir 1998).

The diversity of nectaries in Orchidaceae is intimately

related to their adaptations to diverse groups of pollinators

(Baker and Baker 1983; Pacini et al. 2003). Nectaries in

most orchids are formed by protuberances (calli) or

papillose regions located on the column, labellum, or

between the two (Dressler 1993). The presence of exposed

nectaries on the labellum, as in the species of Acianthera

studied here, seems to be common in myophilous orchids,

and it has been found in several species of Acianthera,

Anathallis, Bulbophyllum, Octomeria, and Stelis (van der

Pijl and Dodson 1966; Dressler 1993; Borba and Semir

1998, 2001; Singer and Cocucci 1999).

Nectar is usually offered as a reward to anthophilous

species of Diptera (Singer and Cocucci 1999; Albores-

Ortiz and Sosa 2006) or in mechanisms of partial deceit-

pollination by saprophytic flies (Borba and Semir 1998,

2001). The species in the present study have purple flowers

with disagreeable odors that are generally associated with

sapromyophily (Proctor et al. 1996), but the presence of

nectar makes the occurrence of strict deceit-pollination

doubtful (van der Pijl and Dodson 1966; Faegri and

van der Pijl 1979). Additionally, no oviposition events

were observed in the species pollinated by Chloropidae

(A. prolifera) or by Phoridae (A. hamosa, A. limae, and

A. modestissima) in this group (Melo 2008). Some authors,

however, argue that the absence of nectar is a feature of

sapromyophily s.s., since the insects would be attracted by

oviposition instinct rather than food instinct (van der Pijl

and Dodson 1966; Faegri and van der Pijl 1979). At least in

Orchidaceae, absence of nectar in sapromyophilous sys-

tems can be effective especially in trap-like mechanisms,

as observed in some Bulbophyllum species (Teixeira et al.

2004). However, in systems where it is necessary to keep

the insect in the flower for a longer period of time, the

presence of nectar may be crucial for pollination to occur

successfully, even if the animal has been attracted by

oviposition instinct (Borba and Semir 1998). Thus my-

ophily s.s. and sapromyophily s.s. may represent only the

extremes of a continuum in which the pollination mecha-

nisms of these species cannot be precisely placed.

The four orchids studied here show pollination mecha-

nisms similar to those reported by Borba and Semir (2001)

in another group of Acianthera. In the study by Borba and

Semir (2001), the two Phoridae-pollinated species offered

nectar while the three species pollinated by female flies of

Chloropidae were deceitful. Female flies were only

observed laying their eggs in the flowers of two of them.

Micromorphological analyses of the flowers of these latter

species, similar to the study we have reported here, could

help to clarify how these two groups of species, with

apparently considerable macromorphological convergence,

exploit a similar pollinator group but possess different

pollination mechanisms.

Osmophores may be located in different parts of the

perianth in orchids (Pridgeon and Stern 1983, 1985; Stern

et al. 1987). In the Pleurothallidinae, including the species

studied here, the osmophores seem to be only in the sepals

or on appendages that originate from them, while in

Bulbophyllum osmophores are commonly found on the

labellum. These two groups have flowers with very similar

external morphologies and share many adaptations to my-

ophily (van der Pijl and Dodson 1966; Dressler 1993), but

there are notable differences in floral micromorphology

associated with each group, as mentioned above. At least

initially, these differences may not play an important role

in attracting specific groups of pollinators or determining

different pollination mechanisms, as Bulbophyllum and

Pleurothallidinae present a wide variation in both param-

eters (Borba and Semir 1998; Singer and Cocucci 1999;

Borba and Semir 2001; Blanco and Barboza 2005; Albores-

Ortiz and Sosa 2006). Rather, they may represent only

phylogenetic constraints in both groups. However both

studies on floral biology and morphology are still very

limited for this to be determined in such large groups.

Flowers pollinated by saprophilous flies generally emit

odors similar to proteinaceous material in decomposition,

mainly nitrogen-containing compounds such as amines,

ammonia, and indols (Proctor et al. 1996). These sub-

stances are associated with the odors of various typically

sapromyophilous species belonging to Pleurothallidinae

and Bulbophyllum (Vogel 1990). However, other studies in


123

species of these groups have not always confirmed their

presence (Kaiser 1993). The latter could be due to the

sampling method, which used polymers associated with the

technique of head-space and used solvents for the polymer

elution (Kaiser 1993). This technique may be inappropriate

when dealing with nitrogenous substances of low molec-

ular weight, such as ethylamine and dimethylamine, which

are apparently common in Acianthera and other fly-polli-

nated species (E. L. Borba, UFMG, Minas Gerais, Brazil,

and J. R. Trigo, UNICAMP, Sao Paulo, Brazil, unpublished

data), where the technique of solid injection seems more

appropriate (Silva et al. 1999). Also, due to the use of very

small amounts of plant tissue in the solid injection tech-

nique, identifying the precise location of the osmophores in

the flower is necessary. Thus, the Whiff test may be an

important indicator as to which technique is best suited to

be employed in these myophilous groups, with the solid

injection being indicated when nitrogenous substances are

present, such as the Neotropical Pleurothallidinae, in con-

trast to the Neotropical Bulbophyllum species, where these

substances are apparently rarer.

Acknowledgments We thank the technical staff of the Centro de

Microscopia Eletronica, Instituto de Biociencias, UNESP Botucatu,

for their help in preparing the samples, and an anonymous reviewer

for improvements to the manuscript. This work was supported by

grants from Conselho Nacional de Desenvolvimento Cientıfico e

Tecnologico (CNPq) and from Pro-Reitoria de Pesquisa/UFMG to E.

Borba. M. Melo received a fellowship from Fundacao de Amparo

a Pesquisa do Estado de Minas Gerais (FAPEMIG). E. Borba and

E. Paiva are supported by a productivity grant (PQ2) from CNPq.

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Acianthera flower anatomy

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