Comparative Account of Nectary Structure in Hexisea imbricata (Lindl.) Rchb.f. (Orchidaceae)

Abstract

• Background and Aims Despite the number of orchid species that are thought to be pollinated by hummingbirds, our knowledge of the nectaries of these orchids is based solely on a single species, Maxillaria coccinea (Jacq.) L.O. Williams ex Hodge. Nevertheless, it is predicted that such nectaries are likely to be very diverse and the purpose of this paper is to compare the nectary and the process of nectar secretion in Hexisea imbricata (Lindl.) Rchb.f. with that of Maxillaria coccinea so as to begin to characterize the nectaries of presumed ornithophilous Neotropical orchids.

• Methods Light microscopy, transmission electronmicroscopy and histochemistry were used to examine the histology and chemical composition of nectary tissue and the process of nectar secretion in H. imbricata.

• Key Results and Conclusions The nectary of H. imbricata has a vascular supply, is bound by a single-layered epidermis with few stomata and comprises two or three layers of subepidermal secretory cells beneath which lie several layers of palisade-like parenchymatous cells, some of which contain raphides or mucilage. The secretory cells are collenchymatous and their walls have numerous pits with associated plasmodesmata. They contain the full complement of organelles characteristic of secretory cells as well as intravacuolar protein bodies but some of the secretory epidermal cells, following secretion, collapse and their anticlinal walls seem to fold. Nectar secretion is thought to be granulocrine and, following starch depletion, lipid droplets collect within the plastids. The nectar accumulates beneath the cuticle which subsequently forms swellings. Finally, nectar collects in the saccate nectary spur formed by the fusion of the margins of the labellum and the base of the column-foot. Thus, although the nectary of H. imbricata and M. coccinea have many features in common, they nevertheless display a number of important differences.

Hummingbird, nectary, nectar secretion, ornithophily, pollination, transmission electron microscopy

INTRODUCTION

The appearance of ornithophily represents a major but relatively recent stride in the evolution of orchids and it has been supposed that bird-pollinated species generally grow at high elevations where insect pollinators seldom occur or, owing to the low temperatures, tend to be less active (van der Pijl and Dodson, 1969). At lower altitudes, however, it seems that birds may visit orchid flowers not only for the copious nectar that they produce but also for the insects that they contain.

The flowers of Neotropical, ornithophilous orchids are largely visited by hummingbirds (Trochilidae). Hummingbirds have long been observed visiting orchid flowers [e.g. Comparettia falcata Poepp. & Endl. (Ackerman, 1992; Meléndez-Ackerman et al., 2000), Elleanthus hallii (Rchb.f.) Rchb.f., E. rosea Schltr., Epidendrum ardens Kraenzl. and E. scabrum Ruiz & Pav. (all cited in van der Pijl and Dodson, 1969)] but detailed accounts of the particular species involved are rare. Nevertheless, records are available for a number of species and these indicate that a given species of hummingbird will often visit a variety of orchids. Thus, the rufous-tailed hummingbird (Amazilia tzacatl) has been observed visiting flowers of Comparettia falcata, Elleanthus hymenophorus Rchb.f., Epidendrum cnemidophorum Lindl., E. pfavii Rolfe and Isochilus linearis (Jacq.) R. Br. var. carnosiflorus (Lindl.) Correll, whereas the fiery-throated hummingbird (Panterpe insignis) visits the flowers of Sobralia amabilis (Rchb.f.) L.O. Williams and an unidentified species of Maxillaria Ruiz & Pav. Conversely, the flowers of Comparettia falcata are visited by both the rufous-tailed hummingbird and the Puerto Rican emerald (Chlorostilbon maugaeus), whereas the booted racquet-tail (Ocreatus underwoodii) and an unidentified species of Amazilia are known to visit the flowers of Elleanthus arpophyllostachys (Rchb.f.) Rchb.f. and Epidendrum secundum Jacq., respectively (van der Pijl and Dodson, 1969; Rodríguez-Robles et al., 1992; Ackerman et al., 1994).

That pollination has occurred, however, can only be verified when the transfer of pollinia is observed. Unfortunately, such records are very rare indeed. For example, Singer and Sazima (2000) observed hummingbirds both visiting and pollinating the flowers of Stenorrhynchos lanceolatus (Aubl.) L.C. Rich. Similarly, Singer (2003) observed the pollination of Elleanthus brasiliensis Rchb.f. by the hermit (Phaethornis petrei).

Generally, in the absence of direct evidence, pollination by hummingbirds can only be inferred from floral morphology. Based on such evidence, other possible candidates for hummingbird pollination include species of Ada Lindl., Alamania punicea La Llave & Lex., Cattleya aurantiaca (Bateman ex Lindl.) P.N. Don, Cochlioda rosea (Lindl.) Benth., C. vulcanica (Rchb.f.) Benth., Cyrtochilum mystacinum Lindl., C. retusum (Lindl.) Kraenzl., species of Elleanthus C. Presl. and Epidendrum L., Laelia milleri Blumensch., Masdevallia rosea Lindl., Maxillaria fulgens (Rchb.f.) L.O. Williams, species of Meiracyllium Rchb.f. (unlikely since its members produce a strong eugenol-like fragrance suggesting that the pollinators are male euglossine bees), Nageliella L.O. Williams, Sophronitis Lindl., Spiranthes speciosa (Jacq.) A. Rich. [syn. Stenorrhynchos speciosum (Jacq.) L.C. Rich.], Spiranthes standleyi (Ames) L.O. Williams [syn. Coccineorchis standleyi (Ames) Garay], species of Symphoglossum Schltr. and Rodriguezia secunda H.B.K. as well as Hexisea bidentata Lindl. and H. imbricata (Lindl.) Rchb.f., the subject of this present paper (van der Pijl and Dodson, 1969; van der Cingel, 2001). The flowers of these species display a unique combination of morphological characters, which they share with unequivocally ornithophilous taxa. The flowers are usually weakly zygomorphic, exhibit diurnal anthesis and tend to be tubular, scarlet, orange, pink, purple or yellow in colour, often in strongly contrasting combinations. They may be held horizontally but often hang freely. Labella may be absent or reflexed and the floral tissues are frequently tough and able to withstand damage resulting from contact with a hard beak. Frequently, the labellum is strongly folded or has a heavy callus, which partially closes the floral tube at the level of the anther and stigma. Many species possess cryptic pollinia. These structures are blue, grey (e.g. Sophronitis cernua Lindl.), brown, cream or greyish-white (e.g. Stenorrhynchos lanceolatus and Sophronitis coccinea (Lindl.) Rchb.f.) in colour, rather than the typical yellow of entomophilous species, and it has been suggested that this causes birds not to clean their beaks before effecting pollination. Indeed, cryptic pollinia have been observed in the hummingbird-pollinated species Hexisea aurea (Rchb.f.) Dressler [syn. Scaphyglottis aurea (Rchb.f.) Foldats] and slightly cryptic pollinia are known to occur in Hexisea imbricata (Dressler, 1971). Nectar guides and fragrances are absent but nectar is typically copiously produced, often in relatively broad floral tubes of moderate length (van der Pijl and Dodson, 1969).

For a century or so, there has been much confusion and debate over the identity of H. imbricata and its close relative H. bidentata. Indeed, for some time, these taxa were considered to be conspecific. Furthermore, it has been speculated, largely on the basis of floral morphology, that both orchids are pollinated by hummingbirds (van der Pijl and Dodson, 1969; Dressler, 1990). H. bidentata grows as an epiphyte upon trees in both forests and coffee plantations at elevations of up to 1500 m and occurs widely from Mexico, through Central America to Panama and northern South America (Ames and Correll, 1985). It has terminal, fascicled inflorescences of several scarlet flowers covered by imbricate, scarious sheaths, and the labellum margins are fused with the column forming a short, saccate nectary spur. The labellum has a very fleshy transverse thickening just in front of the column (Bechtel et al., 1983; Ames and Correll, 1985). However, although H. imbricata closely resembles H. bidentata and both species are widely distributed, H. imbricata can be distinguished in that its pseudobulbs are somewhat flattened and shiny with broad grooves (cylindrical pseudobulbs with a dull surface and narrow, deep grooves in H. bidentata), wider and stiffer leaves, floral segments that are wider and more fleshy and a more sharply bent labellum. Moreover, H. bidentata has a peculiar flap of tissue extending over each side of the stigma but this is absent from H. imbricata. Also, a yellow callus occurs on the labellum of H. imbricata, whereas the corresponding structure in H. bidentata is red-purple or maroon (Dressler, 1974).

The discovery of other Hexisea spp. (Dressler, 1974) and the recent investigation of the phylogenetic relationships of Scaphyglottis Poepp. & Endl. and related genera based on nrDNA ITS sequence data would indicate that Hexisea Lindl. is an artificial genus and that it should now, together with Reichenbachanthus Barb. Rodr., Hexadesmia Brongn. and Platyglottis L.O. Williams be absorbed into Scaphyglottis (Dressler, 1990, 2002; Dressler et al., 2004). Indeed, Hexisea differs from Scaphyglottis only in that its labellum, which may be sigmoid, is geniculate below the column and in that the base of the lip is not jointed. Moreover, the margins of the lip of Hexisea are united to the column to form a deep nectary which does not extend further than the base of the sepals. Representatives of Scaphyglottis, as newly circumscribed on the basis of DNA evidence (Dressler et al., 2004), are united by the presence of nectar, a column-foot and a distinctive superposed growth habit. However, this very recent taxonomic revision has not, as yet, been widely adopted. Unlike both Hexisea and members of the genus Isochilus R. Br. (Dressler, 1990) which are thought to be hummingbird-pollinated solely on the basis of floral morphology, stingless bees of the genus Trigona (Meliponini) have actually been seen pollinating flowers of species currently assigned to Scaphyglottis (Dressler, 1990). Even so, the discovery of a natural intergeneric hybrid (or interspecific hybrid if adopting the recent revision of Dressler et al., 2004) between Hexisea and Scaphyglottis in Panama provides indisputable evidence that these two genera are very closely related (Dressler, 1990).

Given the number of Neotropical orchid species thought to be visited by hummingbirds, it is all the more remarkable that our current knowledge of the nectary of such orchids is based solely on the presumed ornithophilous orchid Maxillaria coccinea (Jacq.) L.O. Williams ex Hodge (Stpiczyńska et al., 2004). Flowers of this species fulfil many of the morphological criteria characteristic of ornithophilous species. A ‘faucet and sink’ arrangement occurs here and nectar is secreted from a small protuberance upon the ventral surface of the column. The nectar then collects in a semi-saccate reservoir formed by the fusion of the labellum and the base of the column-foot. The nectary comprises a single-layered epidermis and three or four layers of small, subepidermal cells. The epidermal cells lack ectodesmata but a reticulate cuticle is present. Nectar is thought to pass both along the apoplast and symplast and eventually through the reticulate cuticle. The secretory cells are collenchymatous, nucleated and have numerous pits with plasmodesmata, mitochondria, rough endoplasmic reticulum (ER) and plastids with numerous plastoglobuli but few lamellae. Intravacuolar protein bodies are common.

Comparison of the nectaries of the presumed hummingbird-pollinated orchids H. imbricata and M. coccinea would perhaps help us better understand the structural features that characterize the nectary of such Neotropical orchids.

MATERIALS AND METHODS

The position of the nectary in four intact, fresh flowers of H. imbricata was determined using an Olympus SZX12 stereo-microscope. Hand-cut sections through the nectary were tested for starch and lipids using IKI and a saturated alcoholic solution of Sudan III, respectively. Pieces of nectary tissue were fixed in 2·5 % glutaraldehyde/5 % sucrose in phosphate buffer (pH 6·8; 0·075 m) for 4 h at 20 °C, washed in phosphate buffer and post-fixed in 1 % osmium tetroxide at 0 °C for 2 h. The fixed material was then dehydrated using a graded ethanol series, infiltrated and embedded in Spurr resin. For general histology, semi-thin sections (about 1 µm thick) were stained using 1 % (w/v) toluidine blue in 1 % (w/v) aqueous sodium tetraborate solution (O'Brien et al., 1965; Vaughn, 1987). Sections were stained with Coomassie Brilliant Blue R-250 and ruthenium red for protein and mucilage, respectively (Jensen, 1962; Fisher, 1968). Micrometry and photomicrography of the nectaries were accomplished using a Nikon Eclipse 600 microscope with Screen Measurement version 4·21 software. TEM sections were cut at about 60 nm using a glass knife and a Reichert Om U-3 ultramicrotome. Sections were stained with uranyl acetate and lead citrate and examined using a TESLA BS-340 transmission electron microscope at an accelerating voltage of 60 kV.

RESULTS

The terminal, fascicled inflorescence of H. imbricata bears several scarlet flowers covered with imbricate, scarious sheaths (Fig. 1). The labellum has a distinct yellow callus, and this closes the entrance of the floral tube. The saccate nectary is formed by the fusion of the margins of the labellum and the column (Fig. 2).

Fig. 1.

Inflorescence of Hexisea imbricata with fascicles of scarlet flowers and imbricate sheaths. Scale bar = 4 mm.

Fig. 2.

Saccate nectary at base of floral tube formed by partial fusion of labellum and column. Note also the yellow callus that closes the entrance into the floral tube. Scale bar = 1 mm.

The nectary tissue is some 41·39 µm (34·0–44·46 µm) deep and consists of a single-layered epidermis and two or three layers of subepidermal, secretory cells (Figs 3 and 4). The secretory epidermis, generally has few stomata. Several layers of parenchyma occur beneath the secretory cells and these parenchymatous cells tend to have a palisade-like arrangement (Fig. 4). The cells of the secretory epidermis (4·58–7·24 µm; mean = 6·02 µm diameter) and secretory parenchyma (8·53–21·96 µm; mean = 12·53 µm diameter) tend to be small, whereas those of the subsecretory parenchyma are larger (19·02–43·81 µm; mean = 26·26 µm diameter). Some of the latter are idioblastic and enclose raphides, whereas others are larger and form cavities containing mucilage. A large vascular bundle comprising xylem and phloem elements supplies the nectary (Fig. 3). The walls of the secretory cells consist of cellulose, whereas staining with ruthenium red revealed the presence of acidic polysaccharides in the middle lamella and the outer tangential walls of epidermal cells. Some of the nectary cells contain an intravacuolar protein body which stains with Coomassie Brilliant Blue R-250 and is finely granular and irregular in shape (Fig. 4).

Fig. 3.

Transverse section through nectary at base of floral tube. Large parenchyma cells with mucilage and vascular bundle are visible beneath a subepidermal layer of secretory cells. Vb = vascular bundle. Scale bar = 15 μm.

Fig. 4.

Transverse section of the nectary. Some of the cells of the secretory epidermis have collapsed (arrowheads) and protein bodies (arrows) are present in several of the nectary cells. Vb = vascular bundle; Ne = nectary. Scale bar = 25 μm.

The secretory cells are collenchymatous in that they possess thick cellulose walls (0·4–2·11 µm; mean = 1·53 µm) but subsecretory cells have thinner walls (0·3–0·4 µm; mean = 0·35 µm). Numerous pits with associated plasmodesmata connect the protoplasts of contiguous secretory cells (Fig. 5). The cytoplasm contains abundant mitochondria, dilated, smooth ER profiles and dictyosomes (Golgi apparatus) as well as numerous secretory vesicles (Figs 6 and 7), and these organelles often become associated with cell walls. Moreover, the cytoplasm contains numerous lipid droplets. The plastids are irregular in shape and contain starch grains but few lamellae, whereas others are smaller and contain numerous, small plastoglobuli (Figs 5 and 8). Secretory cells either contain many small vacuoles or a single larger vacuole with flocculent contents and numerous vesicles (Fig. 9).

Fig. 5.

Subepidermal cells of nectary with cell wall pits and plasmodesmata. Mitochondria and plastids containing numerous plastoglobuli are visible in the cytoplasm. Cw = cell wall; N = nucleus; m = mitochondrion; P = plastid; V = vacuole. Scale bar = 2 μm.

Fig. 6.

Subepidermal cell of nectary with numerous, dilated, smooth ER profiles and secretory vesicles. Plastids may contain plastoglobuli and often starch grains. ER = endoplasmic reticulum; m = mitochondrion; P = plastid; V = vacuole; SV = secretory vesicle. Scale bar = 1 μm.

Fig. 7.

ER profiles, dictyosomes (Golgi apparatus) and numerous secretory vesicles in close proximity to the cell wall. Cw = cell wall; ER = endoplasmic reticulum; G = Golgi apparatus (dictyosomes); m = mitochondrion; SV = secretory vesicle. Scale bar = 1 μm.

Fig. 8.

Plastids with large starch grains. Cw = cell wall; G = Golgi apparatus (dictyosomes); m = mitochondrion; P = plastid; st = starch. Scale bar = 1 μm.

Fig. 9.

Secretory cell with mitochondria, secretory vesicles and lipid droplets in close proximity to the cell wall. Numerous vesicles are visible within the large lytic vacuole. ER = endoplasmic reticulum; G = Golgi apparatus (dictyosomes); m = mitochondrion; V = vacuole. Scale bar = 1 μm.

The outer tangential wall of the epidermis has a thick (0·3–0·5 µm), smooth cuticle (Fig. 10). This cuticular layer is unbroken and lacks surface pores or cracks. However, at intervals, it is separated from the cell wall due to the accumulation of nectar (Fig. 11). Following nectar secretion, some of the secretory epidermal cells become distorted or collapse and their anticlinal walls seem to fold (Figs 10 and 12). In extreme cases, the little that remains of the cell contents appears as a dark mass just visible between very thick cell walls (Fig. 12).

Fig. 10.

Cell of secretory epidermis. Tangential walls are thick and the outer wall has a smooth cuticle whereas the anticlinal wall has a folded appearance. Cw = cell wall; C = cuticle. Scale bar = 0·5 μm.

Fig. 11.

Cuticle separating from the cell wall as nectar accumulates. Cw = cell wall; C = cuticle. Scale bar = 0·5 μm.

Fig. 12.

Remains of protoplasts of the secretory epidermis visible between collapsed tangential cell walls. Cw = cell wall. Scale bar = 0·5 μm.

DISCUSSION

Comparison of floral morphology and nectary ultrastructure

The flowers of H. imbricata fulfil many of the criteria that characterize ornithophilous flowers. The scarlet, tubular flowers have six more or less equal perianth segments and are thus weakly zygomorphic. The labellum has a prominent callus that closes the floral tube at the level of the stigma and anther and a saccate nectar spur is formed by the partial fusion of the labellum and column. Nectar is copiously produced but no obvious nectar guides or fragrance were detected (van der Pijl and Dodson, 1969). As in M. coccinea (Stpiczyńska et al., 2004), the nectary consists of a single-layered epidermis and multiple layers of small, subepidermal cells beneath which occur several layers of large parenchyma cells that frequently contain raphides. However, unlike the parenchyma cells of M. coccinea, which tend to be rather isodiametric, those of H. imbricata are palisade-like. Other parenchyma cells are larger, and some of these contain mucilage. The secretory cells of both species are collenchymatous and this feature may prevent the nectary from becoming damaged by the beak of a visiting bird. The cell walls consist of cellulose but acidic polysaccharides occur in the middle lamella and outer tangential walls of epidermal cells. Again, in both orchids, finely granular and irregularly shaped, intravacuolar protein bodies are present within the nectary cells and, in each case, the nectary is supplied by a vascular strand. The epidermal cuticle of H. imbricata is smooth, whereas that of M. coccinea is reticulate but both, at intervals, become detached from the epidermis as nectar accumulates between the outer tangential wall and the cuticle. Finally, the secretory cells of both species have a similar complement of organelles comprising nuclei, mitochondria, dictyosomes, ER, secretory vesicles and plastids containing numerous plastoglobuli but few lamellae. However, whereas the plastids of M. coccinea were devoid of starch, this substance was common within the plastids of H. imbricata.

The secretory cells of M. coccinea generally contain rough ER, but those of H. imbricata tend to contain smooth ER. The smooth ER of secretory tissues can participate in the synthesis and transport of lipophilic substances (Fahn, 2000) and indeed, the cytoplasm of H. imbricata contains numerous lipid droplets. In both species, pits with associated plasmodesmata are present in the walls of contiguous cells.

Thus, despite the absence of field data, comparison of floral morphology, anatomy and ultrastructure of H. imbricata with that of the presumed hummingbird-pollinated M. coccinea, argues strongly in favour of ornithophily in the former species.

Comparison of nectar secretion

In H. imbricata, nectar accumulates in the saccate nectar spur formed by the fusion of the column and margins of the labellum. The nectary comprises a secretory epidermis and several layers of underlying parenchyma. Whereas in some members of the Orchidaceae the secretory epidermis bears unicellular trichomes which greatly enlarge the secretory surface area (Galetto et al., 1997; Stpiczyńska, 1997; Stpiczyńska and Matusiewicz, 2001), the secretory epidermis here is glabrous (Pais, 1987; Figueiredo and Pais, 1992; Galetto et al., 1997) and, despite the presence of sparse stomata on the nectary surface, it seems unlikely, as in Maxillaria coccinea (Stipczyńska et al., 2004), that nectar is exuded via these structures.

As previously stated, the nectary is supplied by a prominent vascular bundle comprising phloem and xylem elements. Only some 12·6 % of angiosperm species have collateral vascular bundles within the nectary (Frei, 1955). Consequently, most studies concur that, generally, both sugar and water components of the nectar are provided by the phloem sap and that this is transported from sieve tubes to nectary cells (Fahn, 2000; Pacini et al., 2003; Vassilyev, 2003; de la Barrera and Nobel, 2004). It would appear, however, from the presence of collateral bundles, that this is not the case in H. imbricata. As starch grains were not detected in the nectary cells of M. coccinea, perhaps sugars in that species are delivered directly in the phloem sap and modified in the secretory cells. Alternatively, in some species, nectar sugars are produced in the nectary cells themselves by photosynthesis, whereupon starch remains in the plastids for a short period only. In such cases, nectar secretion is prolonged (Pacini et al., 2003). In H. imbricata, however, sugars transported via phloem strands are stored in the nectary cells as starch.

Numerous idioblasts and cavities with mucilage occur in the subsecretory parenchyma of H. imbricata, adjacent to the vascular bundles. According to Chapotin et al. (2003) and Sawidis (1998), mucilage helps maintain the relatively high water potential of flowers. In the case of epiphytic orchids that grow under relatively high water stress, the presence of mucilage may, due to its hydrophilic character, increase the capacity of the flower to store water and this in turn improves its longevity.

Transport of pre-nectar in H. imbricata is possible via plasmodesmata which traverse the thick cellulose cell walls. Cell walls may also play a role in the transport of nectar which passes along the apoplast as in the nectaries of M. coccinea (Stpiczyńska et al., 2004). Indeed, some workers believe that the apoplast forms the main route taken by pre-nectar (Vassilyev, 2003). However, according to others, nectar is transported mainly along the symplast, especially in cases where cutinized layers are present in the cell wall as these may hinder its passage (Findlay and Mercer, 1971; Gunning and Hughes, 1976; Kronestedt et al., 1986; Sawidis et al., 1987; Terry and Robards, 1987; Robards and Stark, 1988; Zellnig et al., 1991; Fahn, 2000; Waigmann and Zambryski, 2000).

When nectar secretion is at its greatest, the plastids within the nectary cells of H. imbricata contain both starch and numerous plastoglobuli. Starch is very common in the nectary cells of orchids (Figueiredo and Pais, 1992; Pais and Figueiredo, 1994; Galetto et al., 1997; Stpiczyńska, 1997) and other plants (Nepi et al., 1996; Graffal et al., 1998; Razem and Davis, 1999; Vesprini et al., 1999) and starch stored within nectary cells can be utilized both as a source of nectar and as a source of energy for highly metabolic processes (Durkee, 1983). Moreover, according to the ‘sugar excretion’ hypothesis (de la Barrera and Nobel, 2004), high rates of water uptake by flowers may result in the accumulation of considerable amounts of carbohydrates delivered via the phloem. Conversion of these carbohydrates into starch can, in turn, help maintain the high water potential of flowers. Even so, it is worth noting that the nectary cells of several orchid species do not accumulate starch during any stage of nectar secretion (Stpiczyńska and Matusiewicz, 2001; Stpiczyńska et al., 2004). Some of the plastids in H. imbricata are elongated or have an irregular profile and this is often associated with a depletion in starch content. Following the disappearance of starch, abundant plastoglobuli develop within the plastids and lipid droplets also occur in the cytoplasm. Lipids are also frequently found in the nectary cells of other orchids (Figueiredo and Pais, 1992; Stpiczyńska, 1997; Stpiczyńska and Matusiewicz, 2001; Stpiczyńska et al., 2004). Indeed, Pais and Figueiredo (1994) reported the accumulation of osmiophilic substances (presumably lipids) in the cytoplasm adjacent to the cell walls of the nectary cells of Limodorum abortivum (L.) Sw. However, currently there are no data available for the lipid content of orchid nectar.

Final modification of the nectar occurs within the nectary cells and, in H. imbricata, well-developed arrays of endoplasmic reticulum and dictyosomes are involved in this process. The nectar is then secreted into the space lying between the plasmalemma and cell wall. Since, during the secretory stage, numerous secretory vesicles are visible in the outer cytoplasm and some of these vesicles appear to fuse with the plasmalemma, it is likely that the mode of secretion that operates in this species is granulocrine. Granulocrine secretion has also been observed for the nectary cells of Platanthera bifolia L. (Stpiczyńska, 1997), but the ultrastructure of the nectary cells of M. coccinea would indicate that sugars are probably carried across the plasmalemma by active transport in that species (Stpiczyńska et al., 2004).

In H. imbricata, the nectar, having crossed the outer tangential wall of the secretory epidermis, accumulates beneath the cuticle. At several points, the cuticle covering the nectary cells forms swellings, but these did not appear to rupture despite the pressure exerted by the accumulating nectar. This observation may indicate that the cuticle is permeable to nectar as has been recorded for a number of other orchid species (Stpiczyńska, 2003; Stpiczyńska et al., 2004). Conversely, during holocrine secretion, nectar is released by the rupture of the cell wall as well as the cuticle covering the secretory epidermis (Vesprini et al., 1999).

A remarkable feature of the nectary cells of H. imbricata is that protoplasts degenerate following nectar secretion and, once secretion is complete, the entire epidermal cell collapses. During the post-secretory stages, the remains of the protoplast become compressed between the tangential cell walls. It seems, however, that not all nectary cells in this species are simultaneously involved in secretion since histological sections reveal that, although some epidermal cells may have collapsed, others remain intact and are presumably functional. Although nectary cells can undergo a number of structural changes during the post-secretory stage (Rachmilevitz and Fahn, 1973; Durkee et al., 1981; Sawidis et al., 1989; Nepi et al., 1996; Razem and Davis, 1999), atrophy of protoplasts, as found in nectary cells of H. imbricata, has not been recorded previously.

To conclude, although the nectary of H. imbricata has a number of features in common with that of M. coccinea, there are nonetheless significant differences. To date, only two detailed studies of nectaries of presumed hummingbird-pollinated orchids have been undertaken. Nevertheless, it is predicted, given the great variety of presumed ornithophilous orchids that occur in the Neotropics, that the nectaries of many of these too will be very diverse. Ideally, the authors would have liked to relate nectary structure and the process of nectar secretion to a named pollinator. Unfortunately, the relevant field data are currently not available and until this is remedied the story has to remain incomplete.

K.L.D. is grateful to the Stanley Smith (UK) Trust for their generous grant and we also thank the Friends of the City of Swansea Botanical Complex, UK for partly funding this work. We also acknowledge the help of Mgr. Janusz Matusiewicz of CLA AR, Lublin, Poland for electron microscopy facilities.

Author notes

1Department of Botany, Agricultural University, Akademicka 15, 20-950 Lublin, Poland, 2School of Earth, Ocean and Planetary Sciences, Cardiff University, PO Box 914, Park Place, Cardiff CF10 3YE, UK and 3Swansea Botanical Complex, Singleton Park, Swansea SA2 9DU, UK