Labellar Structure of the Maxillaria splendens Alliance (Orchidaceae: Maxillariinae) Indicates Floral Polyphenols as a Reward for Stingless Bees.

Several studies have reported stingless Meliponini bees gathering hairs from the labella of Maxillaria spp., including M. ochroleuca, a member of the M. splendens alliance. Such hairs usually contain food materials and are thought to have nutritional value. The papillose labella of representatives of the Maxillaria splendens alliance, however, bear scattered, simple 1-5-celled uniseriate trichomes (hairs) that lack food materials. By contrast, here, as well as polyphenolic compounds, typical labellar papillae usually contain small quantities of starch, protein, and minute droplets of lipid, the last probably involved in the production of fragrance. Towards the labellum apex occur elevated groups of papillae that lack food materials, but contain volatile compounds, probably fragrance precursors. In the past, the terms 'trichomes' or 'hairs' and 'papillae' have been used interchangeably, causing some confusion. Since the trichomes, however, unlike the papillae, are easily detachable and can fragment, it is most likely they, not the papillae, that have previously been observed being collected by bees, but their poor food content indicates that they do not function as food-hairs. Even so, our field observations of M. ochroleuca reveal that stingless bees scrape polyphenol-rich labellar tissue and possibly use this material to produce a resinous, complex, heterogeneous substance commonly referred to as 'bee glue', used for nest construction and repair.

Evidence of floral rewards in Brasiliorchis supports the convergent evolution of food-hairs in Maxillariinae.

PREMISE: Angiosperms distributed over a large geographical area may display considerable phenotypic variation that can be recognized at morphological and micromorphological levels. Here, we investigate the pollination biology and the presence of floral rewards in Brazilian populations of the widely distributed orchid, Brasiliorchis picta. Based on the new data presented here this study investigates the evolution of floral rewards in Maxillariinae, and tests for the occurrence of convergent evolution of food-hairs in this subtribe. METHODS: Micromorphological and histochemical analyses of the labellar tissues were conducted, together with chemical analysis of fragrance and experiments involving the use of chemical baits. The evolution of floral rewards in Maxillariinae were addressed. RESULTS: Microscopy revealed that B. picta offers food-hairs as a reward. Fragrance is produced by abaxially located labellar epidermal papillae. The main compound present in our samples (2-phenylethanol) also occurs in the aggregation pheromone produced by the mandible glands of pollinators, Meliponini bees. Our analyses indicate a high diversity of flower rewards and pollinators displayed by members of Maxillariinae, and support that edible trichomes evolved independently five times in the subtribe. CONCLUSIONS: The high diversity of floral rewards and pollinators displayed by members of Maxillariinae suggests that different pollinator pressures are involved in the evolution of this neotropical subtribe. In addition, the offering of food-hairs, which are generally infrequently encountered in Orchidaceae, arose by convergent evolution in Maxillariinae.

Crepidium sect. Crepidium (Orchidaceae, Malaxidinae)-Chemical and Morphological Study of Flower Structures in the Context of Pollination Processes.

Crepidium is a large genus of mainly pantropical orchids. The lips of its flowers are upwardly directed and do not serve as landing platforms for pollinators. This role is assumed by the dorsal sepal and/or gynostemium. Information about the pollination and floral morphology of this genus is scarce. To date, no papers have been published on these topics. Field observations have revealed that the flowers are visited by small flies, midges, fruit flies, other small dipterans, ants, spiders, and mites. Preliminary observations revealed at least two forms of small liquid droplets secreted on the lip surface of Crepidium species: simple secretions from epidermal cells, and cell sap released upon the rupturing of raphide-producing cells. Further research revealed that this was the first time liquid secretion was recorded in this genus. Floral secretions were subjected to sequential organic solvent extraction and gas chromatography-mass spectrometry (GC-MS). Floral parts were investigated by means of scanning (SEM) and transmission electron microscopy (TEM), and histochemical tests. The presence of liquid droplets on the lip of Crepidium, the presence of a food reward, and the sequence of raphide development are reported here for the first time.

Nectar secretion in a dry habitat: structure of the nectary in two endangered Mexican species of Barkeria (Orchidaceae).

Barkeria scandens and B. whartoniana are endangered, endemic taxa from Mexico. They are epiphytes adapted to dry habitats. Since these plants are xerophytic, their flowers were investigated for structural adaptations to nectar secretion. The flowers of both species are structurally similar, and contrary to most claims for the genus, have functional floral nectaries comprising a nectary chamber and a narrow tubular cuniculus. Nectar is present in both these structures, and contains sugars and lipid-like compounds. The nectary tissue is composed of a single-layered epidermis overlying 1-2 layers of subepidermal secretory parenchyma. The outer tangential wall of the epidermal cells is thick and multi-layered, whereas the cuticle, which often shows blistering, is lamellate and possesses micro-channels. Lipid-like material occurs both between the microfibrils of the cell wall and in the micro-channels. Robust secretory tissue, thick cell walls, and lipid-like nectar components limit nectar evaporation. Moreover, the rigidity of the nectary potentially makes it possible for red-flowered B. scandens to switch from entomophily to ornithophily.

Nectar-Secreting and Nectarless Epidendrum: Structure of the Inner Floral Spur.

Epidendrum, the largest genus of Neotropical orchids, contains both nectar-secreting and nectarless species. Here, we compare the fine structure of the inner floral spur, termed the cuniculus, in nectariferous (E. difforme, E. nocturnum,E. porpax, E. rigidum, E. vesicatum) and seemingly nectarless (E. capricornu, E. ciliare, E. criniferum, E. pseudepidendrum, E. radicans, E. xanthoianthinum) species. This is the first time for such a detailed investigation of cuniculus structure to be undertaken for Epidendrum. Our aim was to characterize features indicative of secretory activity and to ascertain whether flowers presumed to be nectarless produce alternative pollinator food-rewards. The cuniculus is formed by fusion of the basal part of the labellum and column and extends alongside the ovary and transmitting tract. Our study indicates that all investigated species produce nectar or nectar-like secretion to varying degrees, and no alternative pollinator food-rewards were observed. Even though macroscopic investigation of presumed rewardless species failed to reveal the presence of secretion within the cuniculus, close observations of the cells lining the cuniculus by LM, SEM, and TEM revealed the presence of cuticular blisters and surface material. Moreover, the similarity of both the thick tangential cell walls (with the exception of E. vesicatum) and organelle complement of cuniculus epidermal cells in both copiously nectariferous species and those producing only small quantities of surface secretion confirmed the presence of secretory activity in species generally regarded to be rewardless. The secretory character was particularly obvious in the cells of the cuniculus of E. nocturnum, but also in E. ciliare, E. radicans and E. xanthoianthinum, since electron-dense cytoplasm and mitochondria, ER and secretory vesicles were abundant. Furthermore, cell wall protuberances occurred in E. nocturnum, which was indicative of intense transmembrane transport. This investigation highlights the need to examine more closely whether Epidendrum spp. considered to lack food-rewards based solely on macroscopic examination really are rewardless and deceptive.

Nectar and oleiferous trichomes as floral attractants in Bulbophyllum saltatorium Lindl. (Orchidaceae).

Although many Orchidaceae have deceit flowers that produce no reward, the most common reward, when present, is nectar. Bulbophyllum, however, is unusual in that the labellar secretions of most species investigated to date lack sugars, and, therefore, cannot be considered true nectar. The African species Bulbophyllum saltatorium is an exception in that it produces not only nectar but also possesses specialized, capitate oleiferous trichomes. The nectary of B. saltatorium is borne on the labellum and is represented by a deep, narrow, median longitudinal groove, having a small aperture, and flanked by trichomes. Isodiametric epidermal cells lining this groove secrete nectar which collects both in the groove and on the surface of the labellum. As well as a nectary, the labellum of B. saltatorium also bears three types of unicellular trichomes: the longest trichomes are borne distally and abaxially; the marginal ones form a rim around the entire labellum, and finally, massive, capitate trichomes occur proximally and adaxially. These are oleiferous, containing large quantities of oil which might function as precursors of volatile components of fragrance or provide a food-reward. To the best of our knowledge, this is the first time for such oleiferous trichomes to be described for Bulbophyllum. Therefore, apart from their color and markings, flowers of this species are able to attract pollinators in at least two, possibly three ways: food-reward in the form of nectar; fragrance; and possibly food-rewards in the form of food-hairs.

Floral ultrastructure of two Brazilian aquatic-epiphytic bladderworts: Utricularia cornigera Studnicka and U. nelumbifolia Gardner (Lentibulariaceae).

Utricularia cornigera and Utricularia nelumbifolia are giant, aquatic-epiphytic species of carnivorous bladderwort from southeastern Brazil that grow in the central 'urns' of bromeliads. Both species have large, colourful flowers. The main aim of our study is to ascertain whether the prominent floral palate of U. cornigera and U. nelumbifolia functions as an unguentarius-i.e. an organ that bears osmophores. Floral tissues of both species were investigated using light microscopy, scanning electron microscopy, transmission electron microscopy and histochemistry. Floral palates of U. cornigera and U. nelumbifolia provide clear visual signals for pollinating insects. In both species, the palate possesses diverse micro-morphology, comprising unicellular, conical to villiform papillae and multicellular, uniseriate, glandular trichomes that frequently display terminal branching. The most characteristic ultrastructural feature of these papillae was the presence of relatively large, polymorphic plastids (chromoplasts) containing many plastoglobuli. Similar plastids are known to occur in the fragrance-producing (osmophores) and oil-producing (elaiophores) tissues of several orchid species. Thus, these palate papillae may play a key role in providing the olfactory stimulus for the attraction of insect pollinators. Nectariferous trichomes were observed in the floral spurs of both species, and in U. nelumbifolia, free nectar was also recorded. The location, micro-morphology, anatomy and ultrastructure of the floral palate of the two species investigated may thus indicate that the palate functions as an unguentarius. Furthermore, the flowers of these taxa, like those of U. reniformis, have features consistent with bee pollination.

Floral micromorphology of the Australian carnivorous bladderwort Utricularia dunlopii, a putative pseudocopulatory species.

Flowers of sexually deceptive taxa generally possess a set of morphological and physiological characters that mimic their insect pollinators. These characters often include a specific insect-like floral configuration, together with scent glands (osmophores) that produce fragrances which chemically resemble insect sex pheromones. Furthermore, these flowers tend not to produce pollinator food rewards. According to some authors, flowers of the Australian bladderwort Utricularia dunlopii (and species of the Utricularia capilliflora complex) resemble insects, and pollination perhaps occurs by pseudocopulation. The aims of this paper are to compare the structure and distribution of floral glandular trichomes in the Australian carnivorous plant U. dunlopii with those of closely related species assigned to the same section and to discuss their putative function. Floral tissues of U. dunlopii P. Taylor, Utricularia paulinae Lowrie, Utricularia dichotoma Labill. and Utricularia uniflora R.Br. (section Pleiochasia) were investigated using light microscopy, scanning electron microscopy, transmission electron microscopy and histochemistry. In U. dunlopii, two long, erect, filiform appendages arising from the upper lip of the corolla, together with three arising from the lower lip, bear numerous glandular trichomes that may function as osmophores. In other species, such as U. uniflora and U. paulinae, glandular papillae on the corolla palate may also function as osmophores. The floral anatomical and morphological organisation of U. dunlopii differs from that of the other investigated species, indicating that its insect pollinators are also likely to differ. Morphological and ultrastructural observations, while generally contributing to our understanding of the flower of U. dunlopii, do not refute the possibility that pollination here may occur by pseudocopulation. Further field-based investigations, however, are now necessary to test this hypothesis.

Labellar anatomy and secretion in Bulbophyllum Thouars (Orchidaceae: Bulbophyllinae) sect. Racemosae Benth. & Hook. f.

BACKGROUND AND AIMS: Floral secretions are common in Bulbophyllum Thouars, and the labella of a number of Asian species are said to produce secretions rich in lipids that act as food rewards for insect pollinators. Although some of these reports are based on simple histochemical tests, a much greater number are anecdotal and, hitherto, neither the ultrastructure of the labellum nor the secretory process has been investigated in detail. Furthermore, sophisticated histochemical approaches have generally not been applied. Here, both the labellar structure and the secretory process are investigated for four species of Asian Bulbophyllum sect. Racemosae Benth. & Hook. f., namely Bulbophyllum careyanum (Hook.) Spreng., B. morphologorum Kraenzl., B. orientale Seidenf. and B. wangkaense Seidenf., and compared with those of unequivocal lipid-secreting orchids. METHODS: Labellar, secretory tissue was investigated using light microscopy, scanning electron microscopy, transmission electron microscopy and histochemistry. KEY RESULTS: The adaxial median longitudinal groove of the labellum contained secretory tissue comprising palisade-like epidermal cells, similar to those of certain lipid-secreting Oncidiinae Benth. However, these cells and their secretions gave positive results mainly for protein and mucilage, and their organelle complement was consistent with that of cells involved in protein and mucilage synthesis. Sub-cuticular accumulation of secretion resulted in cuticular distension and blistering. The sub-epidermal layer of isodiametric parenchyma contained starch and, like the epidermal cells, ultrastructure consistent with mucilage synthesis. Lipids were mainly confined to the cuticle, and hardly any intracellular lipid droplets were observed. CONCLUSIONS: It is proposed that mucilage is produced by dictyosomes present in the palisade-like epidermal cells. Mucilage precursors may also be produced by these same organelles in sub-epidermal cells and are thought to pass along the symplast via plasmodesmata into the adjoining palisade-like secretory cells, which contain abundant arrays of rough endoplasmic reticulum. Here, they become chemically modified and form a protein-rich, mucilaginous secretion that, following vesicle-mediated transport across the cytoplasm, traverses the cell wall and accumulates in blisters formed from the distended cuticle. Rupture of these blisters releases the secretion onto the labellar surface. However, in certain species, there is some evidence that the secretion may traverse the cuticle via cuticular pores, and micro-channels may permit the passage of fragrance. Hydrolysis of sub-epidermal starch probably generates the carbohydrate and, together with mitochondria, much of the energy required for the secretory process. This anatomical organization resembles that found in certain lipid-secreting, Neotropical species of Bulbophyllum and Oncidiinae, but since the chemical composition of their secretions is different, and these taxa occur on a separate continent and have different insect pollinators, parallelism of floral anatomy is likely.

Comparative anatomy of floral elaiophores in Vitekorchis Romowicz & Szlach., Cyrtochilum Kunth and a florally dimorphic species of Oncidium Sw. (Orchidaceae: Oncidiinae).

BACKGROUND AND AIMS: Recently, molecular approaches have been used to investigate the phylogeny of subtribe Oncidiinae, resulting in the re-alignment of several of its genera. Here, a description is given of the structure of the floral elaiophores (oil glands) of four species formerly assigned to Oncidium Sw. Those of Vitekorchis excavata (Lindl.) Romowicz & Szlach., Cyrtochilum meirax (Rchb.f.) Dalström and a species of Oncidium displaying floral dimorphism, namely O. heteranthum Poepp. & Endl. var. album, are compared with that of Gomesa longipes (Lindl.) M.W. Chase & N.H. Williams, whose epithelial elaiophores are typical of many Oncidiinae, in order to extend our understanding of elaiophore diversity within this subtribe. METHODS: Floral elaiophore structure was examined and compared at anthesis for all four species using light microscopy, scanning electron microscopy, transmission electron microscopy and histochemistry. KEY RESULTS: In all species investigated, with the exception of C. meirax, the floral elaiophore occurs on the labellar callus and is of the intermediate type, possessing both glabrous and trichomatous regions. By contrast, although all four species produce lipid secretions, C. meirax lacks an obvious elaiophore. In each case, the secretory tissue is represented by a single-layered epidermis of cuboidal cells (trichomatous and/or atrichomatous). Palisade cells are absent. The secretion may be wax- or oil-like and is usually produced by smooth endoplasmic reticulum (SER). However, in C. meirax, where rough endoplasmic reticulum (RER) predominates, oil accumulates as plastoglobuli within elaioplasts. These plastoglobuli are then discharged into the cytoplasm, forming oil bodies. In some species, oil usually accumulates within vesicles at the plasmalemma or in the periplasmic space before traversing the cell wall and accumulating beneath the cuticle, sometimes with distension of the latter. Gomesa longipes is unusual in its production of a heterogeneous secretion, whereas Vitekorchis excavata is equally remarkable for the protuberances found on the walls of its secretory cells. CONCLUSIONS: Anatomically, the secretory tissues of all four species, despite currently being assigned to four different genera, are remarkably similar and indicative of homoplasy. This supports previous investigations of the floral elaiophore in Oncidiinae, which showed that the same elaiophore characters may be shared by different clades, but not always by species of the same genus. Consequently, elaiophores are considered to be of limited value in investigating the phylogeny of this subtribe. Furthermore, floral dimorphism does not greatly modify elaiophore structure in the fertile flowers of Oncidium heteranthum var. album. Based on the presence or absence of well-defined elaiophores, the nature of the secretion and the cell ultrastructure, it is likely that floral oil may be produced in Oncidiinae in one of two ways: by the ER (mainly SER) or by plastids, most notably elaioplasts. Once the oil is discharged into the cytoplasm as oil bodies or oil droplets, there is little difference between the subsequent stages of oil secretion; the oil traversing the cytoplasm (often vesicle-mediated) and cell wall before accumulating beneath the cuticle.

Floral elaiophores in Lockhartia Hook. (Orchidaceae: Oncidiinae): their distribution, diversity and anatomy.

BACKGROUND AND AIMS: A significant proportion of orchid species assigned to subtribe Oncidiinae produce floral oil as a food reward that attracts specialized bee pollinators. This oil is produced either by glabrous glands (epithelial elaiophores) or by tufts of secretory hairs (trichomal elaiophores). Although the structure of epithelial elaiophores in the Oncidiinae has been well documented, trichomal elaiophores are less common and have not received as much attention. Only trichomal elaiophores occur in the genus Lockhartia, and their distribution and structure are surveyed here for the first time. METHODS: Flowers of 16 species of Lockhartia were studied. The location of floral elaiophores was determined histochemically and their anatomical organization and mode of oil secretion was investigated by means of light microscopy, scanning electron microscopy and transmission electron microscopy. KEY RESULTS AND CONCLUSIONS: All species of Lockhartia investigated have trichomal elaiophores on the adaxial surface of the labellum. Histochemical tests revealed the presence of lipoidal substances within the labellar trichomes. However, the degree of oil production and the distribution of trichomes differed between the three major groups of species found within the genus. All trichomes were unicellular and, in some species, of two distinct sizes, the larger being either capitate or apically branched. The trichomal cuticle was lamellate, and often appeared distended due to the subcuticular accumulation of oil. The labellar trichomes of the three species examined using transmission electron microscopy contained dense, intensely staining cytoplasm with apically located vacuoles. Oil-laden secretory vesicles fused with the plasmalemma and discharged their contents. Oil eventually accumulated between the cell wall and cuticle of the trichome and contained electron-transparent profiles or droplets. This condition is considered unique to Lockhartia among those species of elaiophore-bearing Oncidiinae studied to date.

Comparative anatomy of the floral elaiophore in representatives of the newly re-circumscribed Gomesa and Oncidium clades (Orchidaceae: Oncidiinae).

BACKGROUND AND AIMS: Recently, molecular approaches have been used to investigate the phylogeny of Oncidiinae. This has resulted in the transfer of taxa previously considered to be species of Oncidium Sw. into Gomesa R. Br. and the re-circumscription of both genera. In this study, the structure of the floral elaiophore (oil gland) is described and compared for Gomesa echinata (Barb. Rodr.) M.W. Chase & N.H. Williams, G. ranifera (Lindl.) M.W. Chase & N.H. Williams, Oncidium amazonicum (Schltr.) M.W. Chase & N.H. Williams and O. oxyceras (Königer & J.G. Weinm.) M.W. Chase & N.H. Williams in order to determine whether phylogenetic revision is supported by differences in its anatomy. METHODS: The floral elaiophore structure was examined and compared at three developmental stages (closed bud, first day of anthesis and final stage of anthesis) for all four species using light microscopy, fluorescence microscopy, scanning electron microscopy, transmission electron microscopy and histochemistry. KEY RESULTS: In all species investigated, the floral elaiophore occurs on the labellar callus and is of the epithelial type, comprising cuboidal to palisade-like, secretory epidermal cells and a layer of sub-epidermal cells, both tissues enclosing ground parenchyma supplied with collateral vascular bundles and containing idioblasts, often with raphides or phenolic contents. A bi-layered cuticle comprising an outer, lamellate and an inner, reticulate layer is present, and sub-cuticular accumulation of secreted material results in distension of the cuticle. Secretion-filled cavities are present at anthesis in the elaiophore cell walls and, in most species, the outer, tangential walls of the elaiophore have small, peg-like projections that protrude into the cytoplasm. In all taxa examined, the elaiophore organelle complement, especially the smooth endoplasmic reticulum (SER), is typical of lipid-secreting cells. CONCLUSIONS: In terms of location, morphology, anatomy and ultrastructure, the floral elaiophores of both Gomesa and Oncidium species examined are very similar, and distinction between these genera is not possible based on elaiophore features alone. Furthermore, many of these elaiophore characters are shared with representatives of other clades of Oncidiinae, including the Ornithocephalus clade. Consequently, elaiophores are considered homoplasious and of limited value in investigating the phylogeny of this subtribe.

Floral features, pollination biology and breeding system of Chloraea membranacea Lindl. (Orchidaceae: Chloraeinae).

BACKGROUND AND AIMS: The pollination biology of very few Chloraeinae orchids has been studied to date, and most of these studies have focused on breeding systems and fruiting success. Chloraea membranacea Lindl. is one of the few non-Andean species in this group, and the aim of the present contribution is to elucidate the pollination biology, functional floral morphology and breeding system in native populations of this species from Argentina (Buenos Aires) and Brazil (Rio Grande do Sul State). METHODS: Floral features were examined using light microscopy, and scanning and transmission electron microscopy. The breeding system was studied by means of controlled pollinations applied to plants, either bagged in the field or cultivated in a glasshouse. Pollination observations were made on natural populations, and pollinator behaviour was recorded by means of photography and video. KEY RESULTS: Both Argentinean and Brazilian plants were very consistent regarding all studied features. Flowers are nectarless but scented and anatomical analysis indicates that the dark, clavate projections on the adaxial labellar surface are osmophores (scent-producing glands). The plants are self-compatible but pollinator-dependent. The fruit-set obtained through cross-pollination and manual self-pollination was almost identical. The main pollinators are male and female Halictidae bees that withdraw the pollinarium when leaving the flower. Remarkably, the bees tend to visit more than one flower per inflorescence, thus promoting self-pollination (geitonogamy). Fruiting success in Brazilian plants reached 60·78 % in 2010 and 46 % in 2011. Some pollinarium-laden female bees were observed transferring pollen from the carried pollinarium to their hind legs. The use of pollen by pollinators is a rare record for Orchidaceae in general. CONCLUSIONS: Chloraea membrancea is pollinated by deceit. Together, self-compatibility, pollinarium texture, pollinator abundance and behaviour may account for the observed high fruiting success. It is suggested that a reappraisal and re-analysis of important flower features in Chloraeinae orchids is necessary.

Floral elaiophore structure in four representatives of the Ornithocephalus clade (Orchidaceae: Oncidiinae).

BACKGROUND AND AIMS: A significant number of species assigned to the Neotropical orchid sub-tribe Oncidiinae reward insect pollinators with oil produced in floral glands termed elaiophores. The latter may be glabrous (epithelial elaiophores) or hirsute (trichomal elaiophores). Although the detailed anatomy and ultrastructure of epithelial elaiophores have been studied for a number of genera, such as Oncidium Sw., Gomesa R. Br. and Trichocentrum Poepp. & Endl., hitherto, trichomal elaiophores have been investigated only for a single species of Oncidiinae, Ornithocephalus ciliatus Lindl. Furthermore, this is the only representative of the Ornithocephalus clade to be investigated to date. Here, an examination is made of the elaiophore anatomy and ultrastructure of a further four species currently assigned to this clade (Ornithocephalus gladiatus Hook., Phymatidium falcifolium Lindl., Zygostates grandiflora (Lindl.) Mansf. and Zygostates lunata Lindl.) and the results compared with those obtained for other Oncidiinae. METHODS: Elaiophore structure was examined for all species at three stages of flower development: closed bud, first day of anthesis and final stage of anthesis, using light microscopy, fluorescence microscopy, scanning electron microscopy, transmission electron microscopy and histochemistry. KEY RESULTS: Elaiophores of O. gladiatus occur upon the lateral lobes of the labellum and display characters intermediate between those of typical epithelial and trichomal elaiophores, in that they are largely glabrous, consisting mainly of cuboidal epidermal cells, but bear short, unicellular hairs proximally. By contrast, the elaiophores of all the other species investigated occur on the callus and are of the trichomal type. In P. falcifolium, these unicellular hairs are capitate. In all species, oil secretion commenced at the closed floral bud stage. Ultrastructurally, the mainly trichomal elaiophores of the four representatives of the Ornithocephalus clade closely resembled the epithelial elaiophores of other Oncidiinae, in that their cells displayed an organelle complement typical of lipid-secreting cells. However, in some taxa, a number of noteworthy characters were present. For example, the elaiophore cuticle of O. gladiatus and P. falcifolium was bi-layered, the outer layer being lamellate, the inner reticulate. The cuticle of Z. grandiflora and Z. lunata was also lamellate, but here, a reticulate layer was absent. Accumulation of secreted oil resulted in the localized distension of the cuticle. Cuticular cracks and pores, however, were absent from all species. The walls of the secretory cells of Z. grandiflora were also atypical in that they had short protuberances or ingrowths, and contained cavities which are thought to be involved in the secretory process. CONCLUSIONS: Of the species investigated, most displayed similar anatomical organization, their trichomal elaiophores occurring on the labellar callus. They, thus, differ from many other members of the Oncidiinae, where epithelial elaiophores are found either on the callus, or on the lateral lobes of the labellum. However, ultrastructurally, all elaiophores, whether those of representatives of the Ornithocephalus clade, or those of other oil-secreting Oncidiinae, possessed a similar complement of organelles, regardless of whether the elaiophores were trichomal or epithelial. In view of the latter, and the similar chemical composition of oils derived from all Oncidiinae investigated to date, it is probable that position and type of elaiophore, and possibly the structure of the overlying cuticle, play an important role in pollinator selection in these oil-secreting orchids.

A comparative survey of floral characters in Capanemia Barb. Rodr. (Orchidaceae: Oncidiinae).

BACKGROUND AND AIMS: Capanemia Barb. Rodr. comprises seven species that mostly inhabit the Brazilian Atlantic Rain Forest domain. The genus currently consists of two sections: Capanemia Cogn. and Planifolia Pabst, distinguished on the basis of leaf shape. We compare the floral morphology and anatomy of all species to determine whether separation into sections is supported by floral characters. METHODS: Both fresh flowers and herbarium specimens were investigated, and column and pollinarium features, together with the presence or absence of floral rewards, recorded. Anatomical features were examined using both light microscopy and scanning electron microscopy. KEY RESULTS AND CONCLUSIONS: With the sole exception of Capanemia therezae, all species shared a distinctive set of floral characters. Flowers were mostly white or yellowish-white and fragrant, and column wings were positioned parallel to the labellum, concealing the stigmatic cavity. Pollinaria had proportionally long tegular stipes and clavate to reniform pollinia, whereas the labellum possessed a conspicuous indument of trichomes, but was devoid of nectar or any other secretion that might function as a food-reward. Capanemia therezae, however, was exceptional in having greenish, unscented flowers with short, rounded and divergent column wings and an exposed stigmatic cavity. Its pollinaria had proportionally short tegular stipes and round pollinia, whereas the labellum lacked trichomes. Droplets of nectar were evident on the adaxial surface of the labellum, adjacent to the callus. Floral features did not support the currently accepted sectional division of Capanemia. If ongoing phylogenetic studies demonstrate that both sections are indeed monophyletic, then these taxa should be distinguished solely on the basis of foliar features.

Comparative labellar micromorphology of Zygopetalinae (Orchidaceae).

BACKGROUND AND AIMS: Molecular evidence indicates that the Neotropical sub-tribe Zygopetalinae is sister to Maxillariinae. Most members of the latter sub-tribe have deceit pollination strategies, but some species produce rewards such as nectar, pseudopollen, resin and wax, and are pollinated by a range of pollinators that include stingless bees (Meliponini), wasps and hummingbirds. By contrast, relatively little is known about the pollination of Zygopetalinae species. However, some are pollinated by fragrance-gathering, male euglossine bees or employ nectar deceit strategies. The aim of this study is to describe the labellar micromorphology of Zygopetalinae and to compare it with that of Maxillariinae sensu lato (s.l.) as part of an ongoing project to record the range of labellar characters found within the tribe Maxillarieae, and to assess whether these characters represent synapomorphies or homoplasies resulting from similar pollination pressures. METHODS: The labella of 31 species of Zygopetalinae, including Cryptarrhena R. Br. and representatives of the Zygopetalum, Huntleya and Warrea clades, were examined using light microscopy and scanning electron microscopy, and the range of labellar characters was recorded. These characters were subsequently compared with those of Maxillariinae s.l. which formed the subject of our previous investigations. KEY RESULTS AND CONCLUSIONS: The labellar micromorphology of Zygopetalinae is less diverse than that of Maxillariinae and does not reflect the currently accepted phylogeny of the former sub-tribe based on molecular studies. Instead, the relative uniformity in labellar micromorphology of Zygopetalinae is probably due to homoplasies resulting from similar pollinator pressures. Labellar trichomes are relatively uncommon in Zygopetalinae, but occur in certain members of both the Zygopetalum and Huntleya clades. Trichomes are unbranched, uniseriate and multicellular with rounded apices, or unbranched and unicellular, with tapering, pointed and flexuose apices. Hitherto, unicellular trichomes of this kind have been observed only for euglossophilous orchid taxa, and the adoption of a relatively limited range of pollination strategies by Zygopetalinae may have resulted in reduced investment in micromorphological labellar characters.

Comparative anatomy of the nectary spur in selected species of Aeridinae (Orchidaceae).

BACKGROUND AND AIMS: To date, the structure of the nectary spur of Aeridinae has not been studied in detail, and data relating to the nectaries of ornithophilous orchids remain scarce. The present paper compares the structural organization of the floral nectary in a range of Aeridinae species, including both entomophilous and ornithophilous taxa. METHODS: Nectary spurs of Ascocentrum ampullaceum (Roxb.) Schltr. var. aurantiacum Pradhan, A. curvifolium (Lindl.) Schltr., A. garayi Christenson, Papilionanthe vandarum (Rchb.f.) Garay, Schoenorchis gemmata (Lindl.) J.J. Sm., Sedirea japonica (Rchb.f.) Garay & H.R. Sweet and Stereochilus dalatensis (Guillaumin) Garay were examined by means of light microscopy, scanning electron microscopy and transmission electron microscopy. KEY RESULTS AND CONCLUSIONS: The diverse anatomy of the nectary is described for a range of Aeridinae species. All species of Ascocentrum investigated displayed features characteristic of ornithophilous taxa. They have weakly zygomorphic, scentless, red or orange flowers, display diurnal anthesis, possess cryptic anther caps and produce nectar that is secluded in a relatively massive nectary spur. Unicellular, secretory hairs line the lumen at the middle part of the spur. Generally, however, with the exception of Papilionanthe vandarum, the nectary spurs of all entomophilous species studied here (Schoenorchis gemmata, Sedirea japonica, Stereochilus dalatensis) lack secretory trichomes. Moreover, collenchymatous secretory tissue, present only in the nectary spur of Asiatic Ascocentrum species, closely resembles that found in nectaries of certain Neotropical species that are hummingbird-pollinated and assigned to subtribes Maxillariinae Benth., Laeliinae Benth. and Oncidiinae Benth. This similarity in anatomical organization of the nectary, regardless of geographical distribution and phylogeny, indicates convergence.

Comparative histology of floral elaiophores in the orchids Rudolfiella picta (Schltr.) Hoehne (Maxillariinae sensu lato) and Oncidium ornithorhynchum H.B.K. (Oncidiinae sensu lato).

BACKGROUND AND AIMS: 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). METHODS: 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. KEY RESULTS AND CONCLUSIONS: 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.

Labellar micromorphology of two euglossine-pollinated orchid genera; Scuticaria Lindl. and Dichaea Lindl.

BACKGROUND AND AIMS: Until recently, there was no consensus regarding the phylogenetic relationships of the Neotropical orchid genera Scuticaria Lindl. and Dichaea Lindl. However, recent evidence derived from both gross morphological and molecular studies supports the inclusion of Scuticaria and Dichaea in sub-tribes Maxillariinae and Zygopetalinae, respectively. The present paper describes the labellar micromorphology of both genera and seeks to establish whether labellar characters support the assignment of Scuticaria and Dichaea to these sub-tribes. METHODS: The labella of four species of Scuticaria and 14 species of Dichaea were examined using light microscopy and scanning electron microscopy, and their micromorphology was compared with that of representative species of Maxillariinae sensu lato and Zygopetalinae (Huntleya clade). KEY RESULTS AND CONCLUSIONS: In most specimens of Scuticaria examined, the papillose labella bear uniseriate, multicellular, unbranched trichomes. However, in S. steelii (Lindl.) Lindl., branched hairs may also be present and some trichomes may fragment and form pseudopollen. Multicellular, leaf-like scales were also present in one species of Scuticaria. Similar, unbranched hairs are present in certain species of Maxillaria Ruiz & Pav. (Maxillariinae sensu stricto) and Chaubardia Rchb.f. (Huntleya clade). As yet, moniliform, pseudopollen-forming hairs have not been observed for Zygopetalinae, but their presence in Scuticaria steelii, Maxillaria and Heterotaxis Lindl. supports the placing of Scuticaria in Maxillariinae. As other genera are sampled, the presence of branched hairs, hitherto unknown for Maxillariinae sensu lato, may prove to be a useful character in taxonomy and phylogenetic studies. Euglossophily occurs in Dichaea, as well as Chondrorhyncha Lindl. and Pescatorea Rchb.f. (Huntleya clade), and all three genera tend to lack distinctive labellar features. Instead, lip micromorphology is relatively simple and glabrous or papillose. However, two of the Dichaea species examined bear unicellular, labellar trichomes very similar to those found in Bifrenaria Lindl. (pollinated by both euglossine bees and Bombus spp.), and this feature may have arisen by convergence in response to similar pollination pressures.

Elaiophore structure and oil secretion in flowers of Oncidium trulliferum Lindl. and Ornithophora radicans (Rchb.f.) Garay & Pabst (Oncidiinae: Orchidaceae).

BACKGROUND AND AIMS: Many orchid flowers have glands called elaiophores and these reward pollinating insects with oil. In contrast to other reward-producing structures such as nectaries, the anatomy of the elaiophore and the process of oil secretion have not been extensively studied. In this paper, elaiophore structure is described for two members of Oncidiinae, Oncidium trulliferum Lindl. and Ornithophora radicans (Rchb.f.) Garay & Pabst. METHODS: Elaiophores of both species were examined using light microscopy, scanning electron microscopy and transmission electron microscopy. KEY RESULTS AND CONCLUSIONS: In flowers of Oncidium trulliferum and Ornithophora radicans, oil is secreted by morphologically distinct elaiophores associated with the labellar callus. However, in O. trulliferum, elaiophores also occur on the lateral lobes of the labellum. In both these species, the epithelial elaiophores are composed of a single layer of palisade-like epidermal cells and a distinct subepithelial layer. Secretory elaiophore cells may contain numerous, starchless plastids, mitochondria and smooth endoplasmic reticulum profiles. In O. trulliferum, the cytoplasm contains myelin-like figures but these are absent from O. radicans. In the former species, cavities occur in the cell wall and these presumably facilitate the passage of oil onto the elaiophore surface. In O. radicans, the accumulation of oil between the outer tangential wall and the cuticle causes the latter to become distended. Since it is probable that the full discharge of oil from the elaiophores of O. radicans occurs only when the cuticle is ruptured by a visiting insect, this may contribute towards pollinator specificity. The structure of the elaiophore in these species resembles both that found in previously investigated species of Oncidiinae and that of certain members of the Malpighiaceae.