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Floral elaiophores, although widespread amongst orchids, have not previously been described for Maxillariinae sensu lato. Here, two claims that epithelial, floral elaiophores occur in the genus Rudolfiella Hoehne (Bifrenaria clade) are investigated. Presumed elaiophores were compared with those of Oncidiinae Benth. and the floral, resin-secreting tissues of Rhetinantha M.A. Blanco and Heterotaxis Lindl., both genera formerly assigned to Maxillaria Ruiz & Pav. (Maxillariinae sensu stricto).
Putative, floral elaiophore tissue of Rudolfiella picta (Schltr.) Hoehne and floral elaiophores of Oncidium ornithorhynchum H.B.K. were examined by means of light microscopy, histochemistry, scanning electron microscopy and transmission electron microscopy.
Floral, epithelial elaiophores are present in Rudolfiella picta, indicating, for the first time, that oil secretion occurs amongst members of the Bifrenaria clade (Maxillariinae sensu lato). However, whereas the elaiophore of R. picta is borne upon the labellar callus, the elaiophores of O. ornithorhynchum occur on the lateral lobes of the labellum. In both species, the elaiophore comprises a single layer of palisade secretory cells and parenchymatous, subsecretory tissue. Cell wall cavities are absent from both and there is no evidence of cuticular distension in response to oil accumulation between the outer tangential wall and the overlying cuticle in R. picta. Distension of the cuticle, however, occurs in O. ornithorhynchum. Secretory cells of R. picta contain characteristic, spherical or oval plastids with abundant plastoglobuli and these more closely resemble plastids found in labellar, secretory cells of representatives of Rhetinantha (formerly Maxillaria acuminata Lindl. alliance) than elaiophore plastids of Oncidiinae. In Rhetinantha, such plastids are involved in the synthesis of resin-like material or wax. Despite these differences, the elaiophore anatomy of both R. picta (Bifrenaria clade) and O. ornithorhynchum (Oncidiinae) fundamentally resembles that of several representatives of Oncidiinae. These, in their possession of palisade secretory cells, in turn, resemble the floral elaiophores of certain members of Malpighiaceae, indicating that convergence has occurred here in response to similar pollination pressures.
Certain orchid species produce floral food-rewards such as nectar, oil, food-hairs (including pseudopollen) and resin-like secretions and these are important in attracting potential pollinators (Davies and Stpiczyńska, 2008, and references therein). Although nectar is the most common floral food-reward amongst Orchidaceae (van der Pijl and Dodson, 1969), floral oil is nonetheless produced by a significant number of species. These include members of Cranichidinae Lindl. (Dressler, 1993), Satyrinae Schltr. (Garside, 1922), Coryciinae Benth. (Buchmann, 1987; Steiner, 1989, 1993; Pauw, 2006), Bulbophyllinae Schltr. (Pohl, 1935; van der Cingel, 2001), Ornithocephalinae Schltr. (Buchmann, 1987; van der Cingel, 2001; Pacek and Stpiczyńska, 2007), Oncidiinae Benth. (van der Pijl and Dodson, 1969; Dressler, 1990; Singer and Cocucci, 1999; van der Cingel, 2001; Silvera, 2002; Singer et al., 2006; Pacek and Stpiczyńska, 2007; Stpiczyńska et al., 2007; Stpiczyńska and Davies, 2008) and Catasetinae (sensu Chase et al., 2003; Mickeliunas et al., 2006). Oil production in orchids is polyphyletic and is thought to have evolved at least five times in Oncidiinae alone (Silvera, 2002).
Floral oils of Oncidiinae sensu lato (including the former Ornithocephalinae; Williams et al., 2001; Chase et al., 2003; Chase, 2005) consist largely of diacylglycerols (Reis et al., 2000, 2003, 2006) and these are produced and secreted by specialized, epidermal glands or hairs called epithelial and trichomal elaiophores, respectively (Vogel, 1974). Several bee genera, such as Rediviva, Tetrapedia, Paratetrapedia and Centris (Buchmann, 1987; Steiner, 1989, 1993; Singer and Cocucci, 1999; van der Cingel, 2001; Pauw, 2006; Singer et al., 2006), have been reported to gather floral oil from orchids. A number of Oncidiinae, including several species currently assigned to Tolumnia Raf., are known to mimic the flowers of New World Malpighiaceae (Silvera, 2002). In Panama, for example, flowers of Trichocentrum stipitatum (Lindl.) M.W. Chase & N.H. Williams mimic those of the malpighiaceous species Byrsonima crassifolia (L.) Kunth (Silvera, 2002). Malpighiaceae flowers possess sepaline glands or floral, epithelial elaiophores and the oily secretion which they produce consists of 8-acetoxy-substituted free fatty acids with a carbon-chain length of C14–C20. This secretion accumulates beneath the protective elaiophore cuticle. Female Centris spp. scrape the flower surface with their tarsi and, in Dinemandra ericoides A. Juss., this results in the rupturing of the cuticle, thus releasing the oil (Cocucci et al., 1996) which the bee gathers by means of the first and second pair of legs before transferring it to scopae on the hind legs. The bee then carries the oil to brood cells, where it is fed to larvae (Vogel, 1974, 1990; van der Cingel, 2001). Similarly, Singer and Cocucci (1999) observed Tetrapedia diversipes both gathering oil and pollinating the flowers of the orchid Oncidium paranaense Kraenzl. Again, as for Malpighiaceae, oil was gathered by the forelegs and subsequently transferred to scopae on the hind legs whilst the bee grasped the tabula infrastigmatica at the base of the column with its mandibles (Dressler, 1990). Such is the degree of mimicry between certain Oncidiinae and Malpighiaceae that, even at histological level, the floral elaiophores of both taxa closely resemble each other in that the secretory epidermis is composed of narrow palisade cells (Stpiczyńska et al., 2007; Davies and Stpiczyńska, 2008; Stpiczyńska and Davies 2008). Such anatomical organization has been described for species of section Rostrata Rolfe (e.g. O. trulliferum Lindl.), section Paucituberculata Lindl. (e.g. O. loefgrenii Cogn.), as well as Ornithophora radicans (Rchb.f.) Garay & Pabst and Trichocentrum cavendishianum (Bateman) M.W. Chase & N.H. Williams (Stpiczyńska et al., 2007; Davies and Stpiczyńska, 2008; Stpiczyńska and Davies, 2008). However, in other representatives of section Rostrata, such as O. cheirophorum Rchb.f., and section Paucituberculata, such as O. paranaense, as well as in Gomesa recurva R.Br., the secretory cells are cuboidal to oval and the elaiophore exhibits a lesser degree of anatomical differentiation and specialization (Singer and Cocucci, 1999; Pacek and Stpiczyńska, 2007; Stpiczyńska et al., 2007; Davies and Stpiczyńska, 2008).
Hitherto, few anatomical and ultrastructural investigations of orchid elaiophores have been undertaken. Furthermore, they were generally confined to members of Oncidiinae sensu lato (Singer and Cocucci, 1999; Pacek and Stpiczyńska, 2007; Stpiczyńska et al., 2007; Davies and Stpiczyńska, 2008; Stpiczyńska and Davies 2008) and, with the exception of Ornithocephalus kruegeri Rchb.f. (Pacek and Stpiczyńska, 2007), which has trichomal elaiophores, were restricted to species that have epithelial elaiophores. However, poorly defined regions of the labella of some orchid flowers, such as certain species of Maxillaria Ruiz & Pav., including Rhetinantha M.A. Blanco and Heterotaxis Lindl., (Porsch, 1905; van der Pijl and Dodson, 1969; Davies et al., 2003a, b; Davies and Turner, 2004; Flach et al., 2004; Matusiewicz et al., 2004, Singer and Koehler, 2004), Polystachya Hook. (Davies et al., 2002), Xylobium Lindl. (Davies and Stpiczyńska, 2006), Cymbidium Sw. (Macpherson and Rupp, 1934; Davies et al., 2006) and Eria vulpina Lindl. (syn. Trichotosia vulpina (Rchb.f.) Kraenzl; von Kirchner, 1925), produce a viscid, lipoidal or resin-like substance. Histochemistry has shown that such labellar secretions derived from Rhetinantha and Heterotaxis spp. may have nutritive value since they contain lipids and aromatic amino acids (Davies et al., 2003a, b), whereas more sophisticated chemical analyses (Flach et al., 2004) have demonstrated that the main component is triterpenoids (cycloartenol derivatives). Other closely related Rhetinantha spp., however, produce wax instead of resin-like material (Porsch, 1905; Senghas, 1993; Davies et al., 2003b, Flach et al., 2004) and, since this probably cannot be digested, it has been proposed that it is collected by bees and used for nest building and repair. Resin-like secretions also occur in Teuscheria wageneri (Rchb.f.) Garay (Bifrenaria clade), but whether or not they contain food substances has yet to be demonstrated (Davies and Stpiczyńska, 2006). Labellar secretions of Cymbidium lowianum (Rchb.f.) Rchb.f. and C. dayanum Rchb.f. also contain lipids (Davies et al., 2006) and Macpherson and Rupp (1934) observed the stingless bee Trigona hockingsii gathering and gnawing viscid material produced along the median axis of the lip of Cymbidium iridifolium A. Cunn. ex Lindl. (syn. C. madidum Lindl.), whereas Braga (1977) reported a vespid wasp, Stelopolybia cf. pallipes, gathering a similar substance from the lip of Maxillaria pendens Pabst. [Ornithidium pendens (Pabst) Senghas]. Although, anatomically, these tissues and the chemical composition of the secretions that they produce greatly differ from those of Oncidiinae, they nevertheless appear to fulfil a similar role.
Rudolfiella Hoehne (Maxillariinae sensu lato) is monophyletic and is considered by many authors to be closely related to, yet distinct from, Bifrenaria Lindl. As yet, however, its position within the Bifrenaria complex has not been satisfactorily resolved (Koehler et al., 2002). Although Braga (1977) claimed that the hymenopteran pollinator of R. aurantiaca (Lindl.) Hoehne feeds upon labellar hairs, Davies and Stpiczyńska (2006) did not find any labellar food-hairs in that species. This coincided with the publication of a brief statement (Singer et al., 2006) proposing that floral elaiophores, although common in Oncidiinae, might also occur in Rudolfiella aurantiaca. However, Singer and co-workers did not elaborate, nor did they describe the location of the putative elaiophore. At about the same time, Davies (K. L. Davies, unpubl. res. in 2006 – cited in Davies and Stpiczyńska, 2008; Stpiczyńska and Davies, 2008) also reported independently on the possibility that floral elaiophores might occur in a second species, Rudolfiella picta (Schltr.) Hoehne. If proven correct, these would be the first records of elaiophores for representatives of the Bifrenaria clade (Dressler, 1993; Whitten et al., 2000, 2007; Koehler et al., 2002, Chase et al., 2003; Chase, 2005).
The purpose of the present paper is to establish whether floral elaiophores occur in Rudolfiella and, if so, to compare their structure with those of Oncidiinae and with those tissues involved with the secretion of resin-like substances in Rhetinantha and Heterotaxis (Maxillariinae sensu stricto.)
Fresh, intact flowers of Rudolfiella picta (Schltr.) Hoehne and Oncidium ornithorhynchum H.B.K. were examined using an Olympus SZX12 stereo-microscope and the position of putative elaiophores determined. That these are indeed elaiophores was confirmed by staining whole, fresh flowers and hand-cut sections through fresh, putative elaiophore tissue for oil using a saturated ethanolic solution of Sudan III. Sections were also tested for starch using IKI.
Elaiophore tissue of both species (approx. 2-mm cubes) was fixed in 2·5 % glutaraldehyde/4% formaldehyde in phosphate buffer (pH 7·4; 0·1 m) for 4 h at 4 °C and washed in phosphate buffer. It was then post-fixed in 1 % osmium tetroxide solution at 0 °C for 1·5 h, dehydrated using a graded ethanol series and infiltrated and embedded in L.R. White resin contained in gelatine capsules. Following polymerization at 60 °C, sections were cut at 60 nm for transmission electron-microscopy (TEM) using a Reichert Ultracut-S ultramicrotome and a glass knife, stained with uranyl acetate and lead citrate (Reynolds, 1963) and examined using a TESLA BS-340 or Zeiss Leo 912AB transmission electron microscope at an accelerating voltage of 60 kV.
Semi-thin sections (0·9–1·0 µm thick) were prepared for light microscopy (LM) and stained for general histology using 0·25 % toluidine blue O in 0·25 % (w/v) aqueous sodium tetraborate solution (TBO). Micrometry and photomicrography were undertaken using a Nikon Eclipse 600 microscope with Screen Measurement version 4·21 software.
Finally, fixed pieces of elaiophore tissue were dehydrated in acetone, subjected to critical-point drying using liquid CO2, sputter-coated with gold and examined by means of a TESLA BS-300 scanning electron microscope (SEM) at an accelerating voltage of 25 kV.
It would appear that Rudolfiella picta is a very variable species (Fig. 1). The present study centres on the first plant (Fig. 1A, B), whose flowers during early anthesis were as brightly coloured as those illustrated for the second specimen (Fig. 1C, D). Labellar elaiophores occur in R. picta and Oncidium ornithorhynchum (Figs 1 and and6).6). However, whereas in R. picta the oil-secreting tissue is located on the callus (Fig 1), in O. ornithorhynchum, a pair of well-defined, white, inflated elaiophores occur on the auricle-like, lateral lobes of the labellum (Figs 6 and and7A).7A). In R. picta, the callus is fleshy, linguiform (Figs 1 and and2A)2A) and verrucose (Fig. 2B–D) and oil occurs both on its surface and on adjacent regions of the labellum (Fig. 2B). Observations indicate that elaiophores of O. ornithorhynchum produce much more oil than those of R. picta. However, flowers of R. picta, when tested, were already several days older than those of O. ornithorhynchum and, consequently, the degree of secretion here is perhaps partly related to the age of the flower. Staining with ethanolic Sudan III demonstrated the presence of small oil droplets on the surface of the elaiophore of O. ornithorhynchum (Fig. 6D). Staining was less obvious for R. picta, largely because of the darker-coloured flowers. Although most of the floral parts of R. picta stained with this reagent, a region corresponding to the glossy part of the callus (Fig. 1A, B) stained rather more intensely, the oil occurring as small droplets within and upon the surface of secretory cells or as a thin, uniform film (Figs 2B and 3A–C). Oil droplets were also present in the subsecretory parenchyma, but absent from cells comprising the narrow, central part of the labellum, even though they were observed on its surface. Oil, once secreted, is able to flow onto other parts of the lamina of the mid-lobe, although none was observed towards the tip of the labellum, where conical papillae predominate (Fig. 3D). Rudolfiella picta lacks a tabula infrastigmatica (Fig. 1), whereas a prominent two-lobed tabula infrastigmatica is present in O. ornithorhynchum (Fig. 6).
In section, the epithelial elaiophore of R. picta, like that of Oncidium trulliferum, O. loefgrenii, Ornithophora radicans and Trichocentrum cavendishianum, comprises a single layer of secretory palisade cells beneath which occur several layers of parenchymatous subsecretory cells (Fig. 4A–D). In R. picta, subsecretory tissue is supplied by collateral vascular strands (Fig. 4F) and contains idioblasts with raphides. Epithelial palisade cells have mean dimensions of 40·5 × 10·2 µm, a thin cuticle that does not become detached from the outer tangential wall, a centrally placed nucleus and dense cytoplasmic contents (Fig. 4A–D). The outer tangential wall is relatively thick (mean = 2·27 µm), whereas the inner tangential and radial walls (mean = 0·68 µm) are thinner (Fig. 4A–D). Radial walls have primary pit-fields and are traversed by numerous plasmodesmata connecting adjacent cells (Fig. 5A, C). Staining with TBO revealed that these walls consist mainly of cellulose. Transmission electron microscopy (TEM) revealed the absence of cell wall cavities and ectodesmata and that, even where verrucose protrusions occur, the cuticle remains intact (Fig. 5B). Large vacuoles are present (Figs 4A–D and 5A) and, in material post-fixed with osmium tetroxide and subsequently stained with TBO, grey, spherical or oval organelles (Fig. 4A–D). The latter tend to have a peri-nuclear distribution and are frequently closely associated with the vacuole (Fig. 4A–D). Identical structures also occur in the subsecretory parenchyma (Fig. 4E), as well as in xylem parenchyma (Fig. 4F) of the vascular strand supplying the callus. Similarly, TEM revealed the presence of numerous plastids containing abundant plastoglobuli. These organelles have a peri-nuclear distribution (Fig. 5A, C, D) and frequently occur close to the plasmalemma and tonoplast (Fig. 5C, D). Secretory vesicles and arrays of smooth endoplasmic reticulum are also present in the cytoplasm (Fig. 5A, C, D). Treatment with Sudan III clearly showed the presence of darkly stained oil droplets both within secretory palisade cells and upon their outer tangential walls (Fig. 3A–C). Intracellular oil bodies are spherical to ellipsoid (Fig. 5A, C, E) and gather beneath the outer, tangential cell wall and in the vicinity of the nucleus (Fig. 5A, C). Similar oil droplets and plastids with plastoglobuli are also present in subsecretory parenchyma.
Oncidium ornithorhynchum differs from other species of Oncidiinae previously studied for elaiophore structure (Singer and Cocucci, 1999; Stpiczyńska et al., 2007; Davies and Stpiczyńska, 2008; Stpiczyńska and Davies 2008) in that, with the exception of a yellow callus, its flowers are pink (Fig. 6), not yellow and brown as in many other species of section Rostrata. The sweet, candy-like fragrance it produces is also unusual. At the beginning of anthesis, the relatively long, pink, bifid, mid-lobe of the labellum is partly coiled and the lateral lobes are at approx. 90 ° to the median, longitudinal axis of the flower (Fig. 6B). As anthesis progresses, the mid-lobe uncoils and expands and the lateral lobes with elaiophores become reflexed so that they lie more or less parallel to each other (Fig. 6C). Elaiophore activity commences at late bud stage and oil, which stains intensely with Sudan III (Fig. 6D), is present throughout the whole lifespan of the flower. During this process, folds appear in the cuticle overlying the adaxial epidermis of the elaiophore (Fig. 7D) and the topography of the latter tissue, which in bud is clearly visible (Fig. 7C), becomes increasingly obscured by secreted oil (Figs 7B, E, F).
The elaiophore of O. ornithorhynchum comprises a single-layered, secretory epithelium of palisade cells derived from the adaxial epidermis as well as several layers of spongy parenchyma (Fig. 8A–C) containing numerous amyloplasts whose starch stains blue-black with IKI. Intercellular spaces within the spongy mesophyll adjacent to the secretory epithelium are particularly well developed (Fig. 8B–F). During the pre-secretory stage, flocculent material accumulates in intercellular spaces directly beneath the secretory epithelium (Fig. 8C, D), whereas during the secretory stage, this substance disappears from the intercellular spaces but is visible between the outer tangential wall and the distended cuticle (Fig. 8E, F). The subsecretory parenchyma is supplied by several collateral, vascular strands that run close to the abaxial epidermis (Fig. 8A, B). The abaxial surface, although not secretory, nevertheless consists of palisade cells (Fig. 8A–C) and these contain small lipid droplets. Although the secretory, adaxial epidermis and non-secretory, abaxial epidermis of the elaiophore lack stomata, these are present on the callus surface.
Adaxial, palisade epithelial cells of this species have mean dimensions of 20·10 × 17·98 µm, centrally placed nuclei, dense cytoplasmic contents and numerous, small vacuoles (Fig. 8D–F). Oil droplets occur both within these cells and upon the outer, tangential wall, and again these stain intensely with Sudan III. The outer, tangential walls are relatively thick (mean = 3·48 µm), whereas the radial walls are much thinner (mean = 1·03 µm) and consist primarily of cellulose (Fig. 8D, F). The inner tangential walls are also relatively thick and often stain more intensely than the other walls with TBO (Fig. 8D–F). TEM revealed that the cellulose microfibrils of the cell wall have a reticulate arrangement but, unlike certain other members of Oncidiinae, LM and TEM showed that these walls lack cavities (Figs 8F and and9A).9A). The cytoplasm, as revealed by TEM, stains intensely and is very granular. It contains mitochondria, ribosomes, oil droplets, endoplasmic reticulum, numerous secretory vesicles, plastids of irregular profile and small vacuoles (Fig. 9A–F). Extensive, myelin-like, membranous figures, together with oil bodies and cytoplasmic inclusions, occur in the vacuoles (Fig. 10A–D). Plastids occasionally contain plastoglobuli or small starch grains (Fig. 10A, B). Small vacuoles and secretory vesicles congregate close to cell walls, especially the outer tangential wall, adjacent to the plasmalemma (Fig. 9C–E). Finally, they discharge their contents, which then accumulate between the outer, tangential wall of the secretory epidermis and the cuticle. The cuticle, which is thin, thus becomes detached and distended (Fig. 8E, F).
The subsecretory parenchyma of R. picta differs from that of O. ornithorhynchum since, whereas the former consists of tightly packed parenchyma with small intercellular spaces (Fig. 4A, C, D), the latter has an extensive intercellular space system (Fig. 8A–F).
The epithelial elaiophore of Rudolfiella picta and Oncidium ornithorhynchum (section Rostrata), in their possession of a single, secretory layer and several layers of subsecretory parenchyma cells, have similar anatomical organization to that of O. trulliferum (section Rostrata), O. loefgrenii (section Paucituberculata), Ornithophora radicans and Trichocentrum cavendishianum (Stpiczyńska et al., 2007; Davies and Stpiczyńska, 2008; Stpiczyńska and Davies, 2008). However, whereas the secretory layer of R. picta, like the four last species and O. ornithorhynchum, consists mainly of palisade cells, those of Oncidium cheirophorum are more cuboidal (Pacek and Stpiczyńska, 2007). Cuboidal, secretory elaiophore cells also occur in Oncidium paranaense (section Paucituberculata) and Gomesa recurva (Singer and Cocucci, 1999; Stpiczyńska and Davies, 2008). Oncidium loefgrenii also differs from the other species of Oncidium mentioned, in that the elaiophore is borne upon the callus, not the lateral lobes of the labellum (Stpiczyńska et al., 2007). Wall cavities, which are thought to facilitate the transport of hydrophobic compounds across the water-saturated cell wall, are present in O. cheirophorum, O. trulliferum, O. loefgrenii and Trichocentrum cavendishianum (Pacek and Stpiczyńska, 2007; Stpiczyńska et al., 2007; Davies and Stpiczyńska, 2008; Stpiczyńska and Davies, 2008). However, they are absent from O. ornithorhynchum, R. picta and G. recurva. Furthermore, distension of the elaiophore cuticle occurs in O. ornithorhynchum, O. cheirophorum, O. paranaense, Ornithophora radicans, Trichocentrum cavendishianum and Ornithocephalus kruegeri as oil accumulates between it and the outer tangential wall. Cuticular distension does not, however, occur in R. picta, O. trulliferum, O. loefgrenii and Gomesa recurva (Pacek and Stpiczyńska, 2007; Stpiczyńska et al., 2007; Davies and Stpiczyńska, 2008; Stpiczyńska and Davies 2008).
Intravacuolar myelin-like figures occur in elaiophore secretory cells of O. ornithorhynchum, O. trulliferum and T. cavendishianum, as well as those of O. cheirophorum and Ornithocephalus kruegeri (M. Stpiczyńska, unpub. res. in 2007), but not those of R. picta, O. loefgrenii, G. recurva and Ornithophora radicans. Consequently, the presence of myelin-like figures does not reflect currently accepted phylogenetic relationships, but is probably related to tissue differentiation and senescence (Davies et al., 1992, and references therein).
Floral elaiophores of R. picta share a number of characters with those of Oncidiinae and representatives of the latter subtribe, in turn, exhibit considerable elaiophore diversity (Table 1). Elaiophores may vary in position (whether on callus or lateral lobes of the labellum), morphology (whether epithelial or trichomal), histology of the secretory layer (whether palisade or cuboidal cells), porosity of the cell wall (whether cavities are present or not) and mode of oil discharge (whether or not the cuticle distends and ruptures). Notwithstanding the fact that, hitherto, few species have been studied, none of these combinations of elaiophore characters reflect currently accepted infrageneric relationships of Oncidium. Instead, elaiophores probably evolved in response to various pollinator pressures and thereby contribute towards pollinator selection.
Recent taxonomic revisions (Whitten et al., 2000; Chase et al., 2003; Chase, 2005), based on molecular approaches and gross morphology, favour the incorporation of genera formerly assigned to Bifrenariinae Dressler into a broadly circumscribed Maxillariinae. Anatomically, however, elaiophores of Rudolfiella more closely resemble those of Oncidiinae than they do the labellar resin-secreting tissues of Rhetinantha and Heterotaxis (Davies et al., 2003a, b; Davies and Turner, 2004). Unlike the elaiophores of Rudolfiella and many Oncidiinae, the resin-secreting regions of the labella of these core Maxillariinae species are morphologically poorly defined (Davies et al., 2003a, b; Davies and Turner, 2004). For example, in certain members of Rhetinantha (formerly Maxillaria acuminata Lindl. alliance) and Mormolyca (sensu Blanco et al., 2007; formerly Maxillaria acutifolia Lindl. alliance or Maxillaria sect. Rufescens Christenson), viscid, resin-like or lipid-rich material is secreted by obpyriform papillae found along the adaxial, median, longitudinal axis of the labellum (Davies et al., 2003b; Davies and Turner, 2004), whereas, in Heterotaxis violaceopunctata (Rchb.f.) F. Barros, H. villosa (Barb. Rodr.) F. Barros and Maxillaria nasuta Rchb.f., uniseriate, multicellular, hairs occur on the adaxial, labellar surface and these are embedded in viscid, resin-like material (Davies et al., 2003a). By contrast, in Maxillaria reichenheimiana Endres & Rchb.f. and M. pseudoreichenheimiana Dodson, cell walls of bicellular, labellar trichomes selectively stain with Sudan III, thus demonstrating the presence of lipid (Davies and Turner, 2004).
In a species previously referred to as Maxillaria cf. notylioglossa Rchb.f., but since re-determined as Rhetinantha divaricata (Barb. Rodr.) M.A. Blanco, secretory cells contain mitochondria, extensive arrays of smooth endoplasmic reticulum, oil bodies and spherical or oval plastids with numerous plastoglobuli (Davies et al., 2003b). Remarkably, the very characteristic oval, elaiophore plastids of Rudolfiella picta are also heavily laden with plastoglobuli and more closely resemble those of Rhetinantha divaricata (Davies et al., 2003b) than elaiophore plastids of Oncidiinae.
The elaiophore of Rudolfiella thus shares a number of characters with that of Oncidiinae, as well as with the secretory, labellar tissue of Rhetinantha (Maxillariinae sensu lato). Furthermore, Rudolfiella, although only distantly related to Oncidiinae, also shares a number of other features with that subtribe. For example, flowers of Rudolfiella resemble, both in shape, colour and patterning, the gold and red-brown flowers of some Oncidiinae species and, like them, are borne on multi-flowered inflorescences (Bechtel et al., 1981). This indicates that Rudolfiella and Oncidiinae exhibit adaptive convergence in response to similar pollinator pressures.
Specialized, oil-gathering bees pollinate many Oncidiinae and these are also thought to pollinate Rudolfiella. For example, Braga (1977) claimed that Rudolfiella is pollinated by hymenoptera but, as yet, no pollinator has been identified with certainty for this genus, nor indeed for O. ornithorhynchum. Even so, Singer (R. B. Singer, pers. comm. in 2008) suggests that the small size of Rudolfiella flowers and the general similarity of the elaiophore to that of representatives of Oncidium section Waluewa Pabst indicate that, as in O. pubes Lindl., they might be pollinated by Tetrapedia bees (Tetrapediini). It is reported that species of Tetrapedia, whilst visiting Oncidium flowers, grasp the tabula infrastigmatica with their mandibles for support. However, Rudolfiella picta and most Oncidium section Waluewa flowers, including O. pubes, lack this structure. R. B. Singer (pers. comm. in 2008), however, has observed that the proximal part of the labellum and the column of orchids that possess a tabula infrastigmatica and are pollinated by oil-gathering bees, such as certain Oncidium spp., tend to lie more or less parallel to each other, even though the distal part of the lip may be more or less vertical. Conversely, in oil-producing orchids that lack a tabula infrastigmatica (such as Oncidium section Waluewa), the labellum is more or less horizontal and the column perpendicular to it and, in the absence of a tabula, this affords better purchase for visiting insects. Rudolfiella falls into this second category. In contrast to Rudolfiella and Oncidium, Meliponini (stingless bees) pollinate Maxillaria sensu lato (including Rhetinantha and Heterotaxis) and these flowers also lack a tabula infrastigmatica.
Thus, it would appear that Rudolfiella, although possessing elaiophore plastids that most closely resemble those of Maxillariinae, nonetheless exhibits convergence with Oncidium at several levels; morphological, anatomical and histological. Whether convergence is also expressed at the biochemical level remains to be determined since, whereas the composition of the floral oil of R. picta is currently unknown, that of Oncidiinae sensu lato has been extensively investigated (Reis et al., 2000, 2003, 2006). Indeed, preliminary results for O. ornithorhynchum (Silvera, 2002) showed that the main components are hexadecanal (11·81 %), octadecanoic acid, 3-hydroxy-methyl ester (15·73 %), pentacosane (12·24 %) and hexacosane (11·48 %). Unfortunately, in the absence of biochemical data for Rudolfiella, comparison of oil composition is currently not possible. What is clear, however, is that, despite differences in the phylogeny and gross floral structure of R. picta and O. ornithorhynchum, both species have certain elaiophore characters in common and these appear to have evolved along similar lines in response to pollinator pressures.
K.L.D. is grateful to the Stanley Smith Horticultural Trust (UK) for helping to fund this work. The authors also thank Rodrigo Singer (Departamento de Botânica, Instituto de Biociências, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil) for sharing with them his field data, Mark Whitten (Florida Museum of Natural History, University of Florida, USA) for supplementary photographs and Alan Gregg (Swansea Botanical Complex, Swansea, UK) for help in preparing the manuscript. We also thank Dr Michal Rudaś (CLA University of Life Sciences, Lublin, Poland) and Mgr Julita Nowakowska (Laboratory of Electron Microscopy, University of Warsaw, Poland) for use of TEM facilities.