Evolution of anatomical characters in Acianthera section Pleurobotryae (Orchidaceae: Pleurothallidinae).

Acianthera section Pleurobotryae is one of ten sections of the genus Acianthera and include four species endemic to the Atlantic Forest. The objective of this study was to describe comparatively the anatomy of vegetative organs and floral micromorphology of all species of Acianthera section Pleurobotryae in order to identify diagnostic characters between them and synapomorphies for the section in relation of other sections of the genus. We analyzed roots, ramicauls, leaves and flowers of 15 species, covering eight of the nine sections of Acianthera, using light microscopy and scanning electron microscopy. Acianthera section Pleurobotryae is a monophyletic group and the cladistic analyses of anatomical and flower micromorphology data, combined with molecular data, support internal relationship hypotheses among the representatives of this section. The synapomorphies identified for A. sect. Pleurobotryae are based on leaf anatomy: unifacial leaves, round or elliptical in cross-section, round leaves with vascular bundles organized in concentric circles, and mesophyll with 28 to 30 cell layers. Within the section, the clade (A. crepiniana + A. mantiquyrana) presented more differences in vegetative organ morphology and higher support values in combined analyses when compared to the second clade, (A. atropurpurea + A. hatschbachii). For each of these clades an exclusive set of homoplasies of vegetative and floral organs were also identified. The results support the argument that vegetative organs are more evolutionarily stable in comparison to reproductive organs and thus helpful for inference of internal phylogenetic relationships in Acianthera, while flowers are highly variable, perhaps due to the diversity of pollinator attraction mechanisms. The analyses indicate that the elliptical leaves observed in A. crepiniana have originated from round leaves observed in the other species of this section, suggesting an adaptation to increase the area of exposure of the leaf and better the efficiency of capture of sunlight in shaded environments such as the Atlantic Forest. The presence of papillose regions in both vegetative and floral organs indicated that micromorphological characters are also useful for the delimitation of species and sections within the genus.

Comparative survey of secretory structures and floral anatomy of Cohniella cepula and Cohniella jonesiana (Orchidaceae: Oncidiinae). New evidences of nectaries and osmophores in the genus.

The morpho-anatomical structure of nectaries, osmophores, and elaiophores, and the anatomical and micromorphological features of floral pieces of Cohniella cepula Hoffmans. and Cohniella jonesiana Rchb.f. were comparatively analyzed. In both species, bracteal and sepal nectaries are structured, i.e., they present a secretory epidermis, secretory parenchyma, and vascular bundles. Nectar secretion is released through stomata. The anatomical and micromorphological traits are similar in both nectaries, which can be detected only if the nectar drops are secreted. Considering the location of these nectaries, the secreted nectar would not be a reward to pollinators. Osmophores are located at the base of both callus and laterals lobes, and consist of a layer of secretory epidermis composed of quadrangular cells and papillae. Elaiophores are found on the callus of the labellum and are of the epithelial type. The anatomical features of floral pieces are similar in both species. The anatomical analysis of sepals and petals showed a few differences, which could be of potential taxonomic value. Our results contribute valuable and novel information for the knowledge of these species and the genus, which will be useful in future taxonomic evaluations.

Proteomic and morphometric study of the in vitro interaction between Oncidium sphacelatum Lindl. (Orchidaceae) and Thanatephorus sp. RG26 (Ceratobasidiaceae).

Orchidaceae establish symbiotic relationships with fungi in the Rhizoctonia group, resulting in interactions beneficial to both organisms or in cell destruction in one of them (pathogenicity). Previous studies have focused mostly on terrestrial species with a few, preliminary studies, on epiphytes. To further our understanding of the molecular mechanisms involved in these symbioses, we evaluated the interaction between Oncidium sphacelatum Lindl. and the mycorrhizal fungus Thanatephorus sp. strain RG26 (isolated from a different orchid species) in vitro using morphometric and proteomic analyses. Evidence from the morphometric and microscopic analysis showed that the fungus promoted linear growth and differentiation of orchid protocorms during 98 days interaction. On day 63, protocorm development was evident, so we analyzed the physiological response of both organisms at that moment. Proteome results suggest that orchid development stimulated by the fungus apparently involves cell cycle proteins, purine recycling, ribosome biogenesis, energy metabolism, and secretion that were up-regulated in the orchid; whereas in the fungus, a high expression of proteins implicated in stress response, protein-protein interaction, and saccharides and protein biosynthesis were found in the symbiotic interaction. This is the first work reporting proteins differentially expressed in the epiphytic orchid-fungus interaction and will contribute to the search for molecular markers that will facilitate the study of this symbiosis in both wild orchids and those in danger of extinction.

Floral colleters in Pleurothallidinae (Epidendroideae: Orchidaceae).

PREMISE OF THE STUDY: The term colleter is applied to trichomes or emergences positioned close to developing vegetative and floral meristems that secrete a sticky, mucilaginous, and/or lipophilic exudate. Several ecological functions are attributed to these glands, but none are exclusive to colleters. Patterns of morphology and distribution of colleters may be valuable for systematics and phylogeny, especially concerning problematic and large groups such as the subtribe Pleurothallidinae, and are also essential to understand the evolution of these glands in Orchidaceae as a whole. METHODS: We used scanning electron and light microscopy to examine the structure and occurrence of trichomes on bracts and sepals and in the invaginations of the external ovary wall (IEOW) in flowers in several developmental stages from species in seven genera. KEY RESULTS: The exudate was composed of polysaccharides, lipophilic, and phenolic compounds. Colleters were secretory only during the development of floral organs, except for the glands in the IEOW that were also active in flowers at anthesis. After the secretory phase, fungal hyphae were found penetrating senescent trichomes. CONCLUSIONS: Trichome-like colleters seem to be a widespread character in Epidendroideae, and digitiform colleters are possibly the common type in this subfamily. Mucilage from IEOW colleters may aid in the establishment of symbiotic fungi necessary for seed germination. The presence of colleters in the IEOW may be a case of homeoheterotopy, in which extrafloral nectaries that produce simple sugar-based secretions (as in other orchid species) have changed to glands that produce secretions with complex polysaccharides, as in Pleurothallidinae.

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.

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.

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.

Elaiophores in Gomesa bifolia (Sims) M.W. Chase & N.H. Williams (Oncidiinae: Cymbidieae: Orchidaceae): structure and oil secretion.

BACKGROUND AND AIMS: Oils are an unusual floral reward in Orchidaceae, being produced by specialized glands called elaiophores. Such glands have been described in subtribe Oncidiinae for a few species. The aims of the present study were to identify the presence of elaiophores in Gomesa bifolia, to study their structure and to understand how the oil is secreted. Additionally, elaiophores of G. bifolia were compared with those of related taxa within the Oncidiinae. METHODS: Elaiophores were identified using Sudan III. Their structure was examined by using light, scanning electron and transmission electron microscopy. KEY RESULTS: Secretion of oils was from the tips of callus protrusions. The secretory cells each had a large, centrally located nucleus, highly dense cytoplasm, abundant plastids containing lipid globules associated with starch grains, numerous mitochondria, an extensive system of rough and smooth endoplasmatic reticulum, and electron-dense dictyosomes. The outer tangential walls were thick, with a loose cellulose matrix and a few, sparsely distributed inconspicuous cavities. Electron-dense structures were observed in the cell wall and formed a lipid layer that covered the cuticle of the epidermal cells. The cuticle as viewed under the scanning electron microscope was irregularly rugose. CONCLUSIONS: The elaiophores of G. bifolia are of the epithelial type. The general structure of the secretory cells resembles that described for other species of Oncidiinae, but some unique features were encountered for this species. The oil appears to pass through the outer tangential wall and the cuticle, covering the latter without forming cuticular blisters.

A simple floral fragrance and unusual osmophore structure in Cyclopogon elatus (Orchidaceae).

We studied gland morphology, anatomy and the chemical composition of the floral fragrance in the sweat bee-pollinated orchid Cyclopogon elatus. This is apparently the first such analysis for any Cyclopogon species, and one of very few studies in which both odour and osmophore are characterised in a nectar-rewarding orchid. Structures responsible for floral scent production were localised with neutral red staining and histochemical assays for lipids and starch. Their morphology and anatomy were studied with scanning electron microscopy and light microscopy thin sections, respectively. Fragrance samples were collected using SPME fibres and analysed with GC-MS. Anatomical evidence suggests that two parallel oval-shaped patches of unicellular trichomes on the abaxial surface of the labellum are osmophores. These are rich in stored lipids, while the parenchyma surrounding the vascular bundles contains starch. Only freshly opened flowers produced odours, while buds and withered flowers lacked scent. The chemical composition of the odour was dominated (>99.8%) by a single compound, trans-4,8-dimethyl-nona-1,3,7-triene (DMNT). Gland anatomy and position on the outside of the perianth are unusual for scent glands in general. The presence of DMNT, a nearly ubiquitous compound in herbivore-induced vegetative emissions and one of the major floral volatiles of Yucca, is not surprising in view of hypotheses on the evolutionary origin of flower scents, suggesting that wound volatiles are utilised as kairomonal attractants by florivores whose activities result in pollination.

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.

Root character evolution and systematics in Cranichidinae, Prescottiinae and Spiranthinae (Orchidaceae, Cranichideae).

BACKGROUND AND AIMS: Previous studies have suggested that velamen characteristics are useful as taxonomic markers in Orchidaceae. Members of tribe Cranichideae have been assigned to two velamen types constructed based on combinations of characters such as the presence of secondary cell-wall thickenings and pores. However, such characters have not been analysed on an individual basis in explicit cladistic analyses. METHODS: The micromorphology of roots of 26 species of Cranichideae was examined through scanning electron microscopy and light microscopy, scoring the variation and distribution of four characters: number of velamen cell layers, velamen cell-wall thickenings, presence and type of tilosomes, and supraendodermal spaces. The last three characters were analysed cladistically in combination with DNA sequence data of plastid trnK/matK and nuclear ribosomal internal transcribed spacer (ITS) regions and optimized on the resulting phylogenetic tree. KEY RESULTS: Thickenings of velamen cell walls group Prescottiinae with Spiranthinae, whereas tilosomes, documented here for the first time in Cranichideae, provide an unambiguous synapomorphy for subtribe Spiranthinae. Supraendodermal spaces occur mostly in species dwelling in seasonally dry habitats and appear to have evolved three times. CONCLUSIONS: Three of the four structural characters assessed are phylogenetically informative, marking monophyletic groups recovered in the combined molecular-morphological analysis. This study highlights the need for conducting character-based structural studies to overcome analytical shortcomings of the typological approach.

Pollinarium morphology and floral rewards in Brazilian Maxillariinae (Orchidaceae).

BACKGROUND AND AIMS: There is strong support for the monophyly of the orchid subtribe Maxillariinae s.l., yet generic boundaries within it are unsatisfactory and need re-evaluation. In an effort to assemble sets of morphological characters to distinguish major clades within this subtribe, the pollinarium morphology and floral rewards of representative Brazilian species of this subtribe were studied. METHODS: The study was based on fresh material from 60 species and seven genera obtained from cultivated specimens. Variation of pollinarium structure and floral rewards was assessed using a stereomicroscope and by SEM analysis. KEY RESULTS: Four morphological types of pollinaria are described. Type 1 appears to be the most widespread and is characterized by a well-developed tegula. Type 2 lacks a stipe and the pollinia are attached directly to the viscidium. Type 3 also lacks a stipe, and the viscidium is rigid and dark. In Type 4, the stipe consists of the whole median rostelar portion and, so far, is known only from Maxillaria uncata. Nectar, trichomes, wax-like and resin-like secretions are described as flower rewards for different groups of species within the genus Maxillaria. Data on the biomechanics and pollination biology are also discussed and illustrated. In Maxillariinae flowers with arcuate viscidia, the pollinaria are deposited on the scuttellum of their Hymenopteran pollinators. In contrast, some flowers with rounded to rectangular, pad-like viscidia fix their pollinaria on the face of their pollinators. CONCLUSIONS: Pollinarium morphology and floral features related to pollination in Brazilian Maxillariinae are more diverse than previously suggested. It is hoped that the data presented herein, together with other data sources such as vegetative traits and molecular tools, will be helpful in redefining and diagnosing clades within the subtribe Maxillariinae.