Comparative Anatomy of the Floral Elaiophore in Representatives Of

Annals of Botany 112: 839–854, 2013 doi:10.1093/aob/mct149, available online at www.aob.oxfordjournals.org

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

Małgorzata Stpiczyn´ska1, Kevin L. Davies2,*, Agata Pacek-Bieniek1 and Magdalena Kamin´ska1 1University of Life Sciences, Akademicka 15, 20-950 Lublin, Poland and 2School of Earth and Ocean Sciences, Cardiff University, Main Building, Park Place, Cardiff CF10 3AT, UK * For correspondence. E-mail [email protected] Downloaded from https://academic.oup.com/aob/article/112/5/839/139696 by guest on 29 September 2021 Received: 19 March 2013 Returned for revision: 8 April 2013 Accepted: 14 May 2013 Published electronically: 24 July 2013

† 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 (Ko¨niger & 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 en- closing 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 com- plement, 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, manyof these elaiophore characters are shared with representatives of otherclades of Oncidiinae, including the Ornithocephalus clade. Consequently, elaiophores are considered homoplasious and of limited value in investigating the phylogeny of this subtribe.

Key words: Anatomy, elaiophore, lipid secretion, micromorphology, oil glands, Oncidiinae, ultrastructure.

INTRODUCTION studies of elaiophore structure did not commence until relatively The flowers of Oncidiinae (sensu Chase et al., 2003; Neubig et al., recently (Singer and Cocucci, 1999; Pacek and Stpiczyn´ska, 2012) display the greatest diversity of pollination systems known 2007; Stpiczyn´ska et al., 2007; Stpiczyn´ska and Davies, 2008; for Orchidaceae (Neubig et al.,2012). Most are rewardless and Aliscioni et al., 2009; Davies and Stpiczyn´ska, 2009; Pansarin attract insect pollinators by deceit (Chase, 2009; Neubig et al., and Pansarin, 2011; Pacek et al.,2012; Gomiz et al.,2013), 2012). However, of those species that offer food rewards to polli- and were mainly restricted to the epithelial elaiophores of nators, a significant number produce floral oil (Chase et al.,2003; genera then assigned to the Oncidium clade, such as Gomesa Chase, 2005; Neubig et al.,2012) in special epidermal glands or R. Br., Oncidium Sw. and Ornithophora Barb. Rodr., as well glandular hairs called epithelial and trichomal elaiophores, re- as Trichocentrum Poepp. & Endl. (Singer and Cocucci, 1999; spectively (Vogel, 1974). Furthermore, these flowers are thought Pacek and Stpiczyn´ska, 2007; Stpiczyn´ska et al., 2007; to mimic those of oil-secreting Malpighiaceae or sympatric Stpiczyn´ska and Davies, 2008; Aliscioni et al.,2009). These species of Calceolaria L. (Calceolariaceae; Neubig et al., 2012, elaiophores were found to be morphologically very diverse and and references therein). A complex mix of Batesian and did not always closely reflect the phylogeny accepted at that Mu¨llerian mimicry probably best explains the floral morphology time. However, their oil was characterized by the presence of dia- of Oncidiinae (Roy and Widmer, 1999). Floral elaiophores cylglycerols in which the acetyl group was invariably located at occur in members of several clades of Oncidiinae, where they position 1 of the glycerol moiety, and the fatty acid at position 2, are thought to have arisen at least seven times (Renner and with the long-chain fatty acid having either hydroxyl or acetoxy Schaefer, 2010). Although there are records of their involvement groups at position 3 and 7 (Reis et al.,2000, 2003; Silvera, in the attraction and rewarding of pollinators (van der Pijl and 2002; Singer et al.,2006). The major component of the floral oil Dodson, 1969; Dressler, 1990; van der Cingel, 2001; Davies and of Gomesa radicans (Rchb.f.) M.W. Chase & N.H. Williams Stpiczyn´ska, 2008; Davies, 2009, and referencestherein), detailed [as Ornithophora radicans (Rchb.f.) Garay & Pabst] is # The Author 2013. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: [email protected] 840 Stpiczyn´ska et al. — Floral elaiophore structure in the Gomesa and Oncidium clades

(2S,3’R,7’R)-1-acetyl-2-(3’,7’-diacetoxy-eicosanoyl)-glycerol N.H. Williams and G. ranifera (Lindl.) M.W. Chase & N.H. (Reis et al., 2003), and acylglycerols are thought also to occur in Williams (formerly Baptistonia echinata Barb. Rodr. and the ﬂoral oils of Gomesa loefgrenii (Cogn.) M.W. Chase & N.H. Oncidium raniferum Lindl., respectively), and two species of Williams (as Oncidium loefgrenii Cogn.) and other species Oncidium, namely O. amazonicum (Schltr.) M.W. Chase & of Gomesa (R. B. Singer, pers. comm., 2006), including N.H. Williams and O. oxyceras (Ko¨niger & J.G. Weinm.) Gomesa praetexta (Rchb.f.) M.W. Chase & N.H. Williams (as M.W. Chase & N.H. Williams (formerly Sigmatostalix amazo- Oncidium enderianum auct.) and related taxa. These orchids nica Schltr. and S. oxyceras Ko¨niger & J.G Weinm. bis, respect- are pollinated by oil-gathering Centris, Paratetrapedia and ively) in order to investigate whether the recent phylogenetic Tetrapedia bees, and the oil is fed to larvae or used as waterproof- revision is supported by elaiophore anatomy. ing for larval cells (Buchmann, 1987; Dressler, 1990; Singer and Cocucci, 1999; van der Cingel, 2001; Torretta et al., 2011; MATERIALS AND METHODS

Neubig et al., 2012, and references therein). Downloaded from https://academic.oup.com/aob/article/112/5/839/139696 by guest on 29 September 2021 The recent increased use of molecular approaches has permitted Plants used in this study were grown at the Botanic Garden of the resolution of Oncidiinae into a number of strongly supported the Maria Curie-Skłodowska University, Lublin, Poland. They clades, with the concomitant realignment of genera (Williams include Gomesa echinata (Barb. Rodr.) M.W. Chase & N.H. et al., 2001; Chase et al., 2009; Neubig et al., 2012). Thus, based Williams (accession no. 103/07), G. ranifera (Lindl.) M.W. mainly on the generic concepts of Chase (2009) and the molecular Chase & N.H. Williams (accession no. 28/06), Oncidium amazoni- work of Neubig and co-workers (2012), Oncidiinae currently cum (Schltr.) M.W. Chase & N.H. Williams (accession no. 65/10) includes 61 genera containing approx. 1600 species. Consequently, and O. oxyceras (Ko¨niger & J.G. Weinm.) M.W. Chase & N.H. of those Oncidium species for which elaiophore data were avail- Williams (accession nos 101/07 and 153/09). Spirit-preserved ma- able, most (with some exceptions, such as O. cheirophorum terial was deposited at the herbarium of Warsaw Botanic Garden, Rchb.f. and Oncidium sotoanum R. Jimeńez & Ha´gsater [syn. Poland under the accession numbers WABG002573 (G. echinata), O. ornithorhynchum Kunth], which remain in Oncidium), as WABG002574 (G. ranifera), WABG002575 (O. amazonica)and well as Ornithophora radicans, were transferred to the newly WABG002576 (O. oxyceras). Abbreviations for authors of plant re-circumscribed genus Gomesa to join G. recurva R. Br. names follow Brummitt and Powell (1992) throughout. Other taxa for which elaiophore structure has been examined, The position of elaiophores in intact, fresh flowers of all species but which are currently assigned to other clades, include investigated was determinedbymeansof a Nikon SMZ100 stereo- Trichocentrum cavendishianum (Bateman) M.W. Chase & microscope. Elaiophore tissue was subsequently examined using N.H. Williams, T. pumilum (Lindl.) M.W. Chase & N.H. light microscopy (LM), scanning electron microscopy (SEM) Williams, and the genus Lockhartia Hook., the last two taxa pos- and transmission electron microscopy (TEM) for three develop- sessing trichomal elaiophores (Singer and Cocucci, 1999; Pacek mental stages of the flower: closed bud, first day of anthesis and and Stpiczyn´ska, 2007; Stpiczyn´ska et al., 2007; Stpiczyn´ska final stage of anthesis. Floral oil secretion was monitored for the and Davies, 2008; Aliscioni et al., 2009; Davies and duration of anthesis, from the closed bud stage (approx. 3–4 Stpiczyn´ska, 2009; Pansarin and Pansarin, 2011; Neubig et al., weeks prior to anthesis – the earliest stage at which it was possible 2012; M. A. Blanco, K. L. Davies, M. Stpiczyn´ska, B. S. to dissect the floral bud and when oil was already visible) until the Carlsward, G. M. Ionta and G. Gerlach, unpubl. res.). end of anthesis and flower abscission. Gomesa, as currently circumscribed (Neubig et al., 2012), is Following macroscopic observations, pieces of elaiophore relatively broad and comprises some 125 species, and at least tissue (approx. 1 mm3) were excised and fixed in 2.5 % (v/v) glu- 23 other generic concepts. Although occurring from Argentina taraldehyde/4 % (v/v) formaldehyde in phosphate buffer (pH 7.4; to Amazonian Peru, it has its centre of distribution in Brazil, es- 0.1 M)for2hat48C, washed three times in phosphate buffer and pecially the Mata Atlaˆntica, where it largely replaces Oncidium. post-fixed in 1.5 % (w/v) osmium tetroxide solution for 1.5hat Despite its diverse floral morphology, members of this genus are 0 8C. The fixed material was then dehydrated using a graded easily recognized by their fused lateral sepals. ethanol series, and infiltrated and embedded in LR White resin In contrast, with the sole exception of O. baueri Lindl., (LR White acrylic resin, medium grade, Sigma). Following poly- Oncidium, as currently circumscribed (Neubig et al., 2012), is merization at 60 8C, sections were cut at 70 nm for TEM using a absent from Brazil and is distributed from Mexico and Florida, Reichert Ultracut-S ultramicrotome and a glass knife, stained through the Caribbean, Central America south to Bolivia and with uranyl acetate and lead citrate (Reynolds, 1963) and exam- Peru. It contains approx. 520 species and representatives of the ined using an FEI Tecnai Spirit G2 transmission electron micro- previously recognized genera Odontoglossum Kunth, Sigmatostalix scope, at an accelerating voltage of 90 kV. Rchb.f., Cochlioda Lindl., Symphyglossum Schltr., Mexicoa Garay Semi-thin sections (0.9–1.0 mm thick) were prepared for LM and Miltonioides Brieger & Lu¨ckel, as well as Chamaeleorchis and fluorescence microscopy. For general histology, they were Senghas & Lu¨ckel, Collare-stuartense Senghas & Bockemu¨hl and stained with aqueous 1 % (w/v) methylene blue/1 % (w/v) Heteranthocidium Szlach., Mytnik & Romowicz. Unlike Gomesa, azure II (1:1) for 5–7 min. the lateral sepals are free. Histochemical tests were used to detect the presence of lipids The considerable similarity between the flowers of certain and starch in elaiophore tissue by treating the latter with a satu- species of Gomesa and Oncidium is due to homoplasy, since rated ethanolic solution of Sudan III and aqueous IKI solution, floral traits in Oncidiinae are highly plastic and reflect evolution- respectively, and examining by means of a Nikon E-200 or ary shifts in pollinators (Neubig et al., 2012). Nikon Eclipse 90i light microscope. Semi-thin sections were Herewe compare elaiophore structure forafurther two species also treated with auramine O (Gahan, 1984) to detect the pres- of Gomesa, namely G. echinata (Barb. Rodr.) M.W. Chase & ence of lipid, and examined using a fluorescence microscope Stpiczyn´ska et al. — Floral elaiophore structure in the Gomesa and Oncidium clades 841

[fluorescein isothiocyanate (FITC) filter, excitation light 465– marked purple and bears three, prominent, raised, longitudinal 495 nm, barrier filter 515–555 nm]. Furthermore, the periodic ridges. The central ridge is bifid, and each of the two lateral, up- acid–Schiff (PAS) reaction was employed to detect the presence wardly curved ridges has a free, rounded tip (Figs 1A, B and 2A). of insoluble polysaccharides (Jensen, 1962). Micrometry and The elaiophore is of the epithelial type and is composed of a photomicrography of elaiophore tissue were accomplished using single layer of palisade-like cells of mean dimensions 52.5 × both Nikon Eclipse 90i (NIS-Elements AR software) and FEI 19.4 mm, and one or two layers of smaller, isodiametric, sub- Tecnai Spirit G2 (TEM Imaging & Analysis computer program). epidermal cells (Fig. 1C–E). The ground parenchyma contains For SEM, fixed pieces of labellum were dehydrated and sub- several collateral vascular bundles and numerous idioblasts jected to critical-point drying using liquid CO2. They were then with raphides (Fig. 1C). sputter-coated with gold and examined using a Tescan Vega II LS Secretory activity of the palisade-like cells commences at the scanning electron microscope, at an accelerating voltage of 10 kV. bud stage, some 3 weeks prior to anthesis. At this stage, small dro-

plets of secretion are visible on the callus surface (Fig. 2A, B). Downloaded from https://academic.oup.com/aob/article/112/5/839/139696 by guest on 29 September 2021 RESULTS Following anthesis,the volume ofsecretion increases until iteven- tually coats almost the entire surface of the mid-lobe of the label- Gomesa echinata lum (Fig. 1B). However, most secreted material occurs at the tips Here, the elaiophore is located on the labellar callus and occupies of the lateral callus ridges (Fig. 2B), but is also present on lateral almosthalf the lengthof the labellum.Thewhitishcallusisheavily parts of the labellum, close to the callus, where epidermal cells

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F IG.1. (A–E)Gomesa echinata, floral habit and LM. (A) Flower with elaiophore located proximally on the labellum. Scale bar ¼ 4 mm. (B) Elaiophore with two prominent callus projections (arrows). The whole surface of the gland is coated with secretion. Scale bar ¼ 1 mm. (C) Section of the elaiophore showing secretory epithelium. Idioblasts with raphides occur in the ground parenchyma beneath the secretory cells. Scale bar ¼ 50 mm. (D) Detail of palisade-like epithelial cells with centrally placed nuclei surrounded by plastids and small vacuoles. (E) Lipids stained with Sudan III accumulatewithin cavities in the outercell walls of epidermal cells (arrows). (D, E) Scale bar ¼ 20 mm. The following list of abbreviations applies to all the figures: C, cuticle; Cl, lamellate layer of cuticle; Cr, reticulate layer of cuticle; Cw, cell wall; D, dictyosome; E, secretory epithelium; El, elaiophore; Ep, elaioplast; L, lipid droplet; m, mitochondrion; mf, myelin-like figure; N, nucleus; P, plastid; R, raphides; RER, rough endoplasmic reticulum; SER, smooth endoplasmic reticulum; st, starch; V, vacuole; Vb, vascular bundle. 842 Stpiczyn´ska et al. — Floral elaiophore structure in the Gomesa and Oncidium clades

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F IG.2. (A–F)Gomesa echinata, SEM. (A) Surface of an elaiophore with lipid droplets (arrows) present on the callus projections and on lateral surfaces. Scale bar ¼ 500 mm. (B) Detail of projection surface coated with secretion. Scale bar ¼ 100 mm. (C) Lateral part of the labellum showing conical papillae and lipid droplets (arrows). Scale bar ¼ 60 mm. (D) Distal part of an elaiophore coated with lipid droplets. Scale bar ¼ 50 mm. (E) Epidermal cells with ruptured cuticle (arrowheads). Scale bar ¼ 20 mm. (F) Elaiophore at the final stage of anthesis coated with scale-like flakes of dried secretion. Scale bar ¼ 50 mm. See Fig. 1 for abbreviations. with conical papillae predominate (Fig. 2C). Sub-cuticular accu- The position of these cavities often coincides with that of mulation of secreted material results in the stretching and ultimate radial (anticlinal) walls (Fig. 1E). Smaller cavities may also be rupture of the cuticle (Fig. 2C–E). Maximum secretory activity associated with the radial cell walls of epidermal cells, and het- occurs between Day 4 and 10 of anthesis, but then diminishes as erogeneous, secreted material often accumulates between and the flower approaches abscission. By the final stage of anthesis, upon the cuticular lamellae (Fig. 3A, B). a film, or, more commonly, scales of dried secretion, coats the The cytoplasm of secretory, palisade-like cells is granular, entire surface of the elaiophore (Fig. 2F). with prominent, centrally located nuclei (Fig. 1D), and contains Transmission electron microscopy revealed that the outer, tan- numerous profiles of rough (RER) and smooth (SER) endoplas- gential cell wall is thick (approx. 1 mm) and bears a bi-layered mic reticulum (Fig. 3C, G). In elaiophore tissue, the latter are cuticle comprising an outer, lamellate and an inner, reticulate closely associated with plastids (Fig. 3D, E, G). Small vacuoles, layer (Fig. 3A, B). Cavities are absent from this wall at the bud often with osmiophilic contents, are scattered throughout the stage, but are visible at anthesis and contain secreted material. cytoplasm, whereas larger vacuoles tend to be located at the Stpiczyn´ska et al. — Floral elaiophore structure in the Gomesa and Oncidium clades 843

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F IG.3. (A–G)Gomesa echinata at bud stage (D), and at the start (A–C) and ﬁnal stage of anthesis (E–G), TEM. (A) Thick, outer cell wall of an epidermal cell with cuticle comprising an outer, lamellate layer and an inner, reticulate layer. Heterogeneous secretion (asterisk) accumulates between and upon the cuticular lamellae (arrowheads). Scale bar ¼ 0.5 mm. (B) Detail of cell wall with surface secretion (asterisks). (C) Outer cell wall and cytoplasm of elaiophore cell showing numerous RER proﬁles. (D) Plastids with small starch grains and dark stroma, the vacuoles with osmiophilic contents. (E) Elaioplasts of irregular shape containing lipid droplets (asterisks). (F) Lipid droplets scattered throughout cytoplasm. (B–F) Scale bars ¼ 1 mm. (G) Epithelial cell with electron-dense cytoplasm, and sub-epithelial parenchyma cell with large vacuole and narrow layer of parietal cytoplasm. Plasmodesmata (arrow) connect these cells. Abundant SER occurs in the cytoplasm of epithelial cell. Scale bar ¼ 0.5 mm. See Fig. 1 for abbreviations.

basal end of secretory cells (Fig. 1C). Elaiophore vacuoles fre- (Fig. 3G), frequently with myelin-like figures. Cytoplasmic con- quently contain membranous, myelin-like figures, flocculent or tinuity between these cells and those of the secretoryepidermis is osmiophilic material and lipid bodies (Fig. 3D, E). Lipid maintained via primary pit-fields containing plasmodesmata. bodies also occur scattered throughout the cytoplasm (Fig. 3F), Starch grains are present in epidermal cells, within numerous together with mitochondria. Staining with Sudan III revealed plastids that have a perinuclear distribution, as well as in cells that during the bud stage, lipid droplets accumulate in the cyto- of the sub-epidermal layer (Figs 1D and 3D). These plastids plasm of secretory cells, in particular, adjacent to the outer, tan- also contain small plastoglobuli. At anthesis, there is a marked gential wall and radial walls of epidermal cells, and are still reduction in both the number and size of starch grains, and, by present at the final stage of anthesis (Fig. 1E). the end of anthesis, they have often disappeared completely. Sub-epidermal parenchyma cells have a narrow layer of less Simultaneously, plastids differentiate to form elaioplasts dense, parietal cytoplasm and a large, centrally located vacuole containing numerous lipid droplets (Fig. 3E). 844 Stpiczyn´ska et al. — Floral elaiophore structure in the Gomesa and Oncidium clades

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F IG.4.(A–G)Gomesa ranifera, ﬂoral habit, SEM and LM. (A) Elaiophore located upon the labellar callus. Scale bar ¼ 1 mm. (B) Detail of the surface of tiered callus showing oval projection. (C) Detached cuticle (asterisks) of epithelial cells. (D) Transverse section of elaiophore showing palisade-like epithelial cells, sub-epithelial parenchyma cells containing large starch grains, and idioblasts with raphides in ground parenchyma. (B–D) Scale bars: B ¼ 500 mm; C and D ¼ 50 mm. (E) Epithelial cells at the start of anthesis, with prominent nuclei and numerous plastids (arrows). A small cuticular blister (arrowhead) is visible on the outer cell wall. Sub-epithelial parenchyma cells contain large starch grains. Scale bar ¼ 20 mm. (F) Epithelial cells and underlying parenchyma cells at bud stage with numerous starch grains stained red by the PAS reaction. Scale bar ¼ 50 mm. (G) Lipids in palisade-like epithelial cells and underlying parenchyma stained intensely with Sudan III. Idioblasts containing raphides occur in the ground parenchyma. Scale bar ¼ 100 mm. See Fig. 1 for abbreviations.

Gomesa ranifera and secreted material accumulates in grooves between the tiers Again, in G. ranifera, the elaiophore is of the epithelial type, (Fig. 4A, B). Generally, the callus elaiophore has similar hist- and is located on the callus (Fig. 4A–C). The callus is tiered, ology to that of G. echinata, comprising palisade-like, secretory Stpiczyn´ska et al. — Floral elaiophore structure in the Gomesa and Oncidium clades 845

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F IG.5. (A–I)Gomesa ranifera at start of anthesis, TEM. (A) Heterogeneous secretion containing rounded or irregular profiles or cavities (asterisks) accumulating betweenthick,tangentialwall of epidermis and cuticle.Dictyosomesandsecretoryvesiclesare presentin theparietalcytoplasm. Scalebar ¼ 1 mm. (B)Heterogeneous secretion accumulating in intercellular spaces, including those alongside radial walls. Note the formation of numerous cavities (asterisks) in the intercellular spaces. Cell wall protuberances (arrows) are also present. Plastids with starch grains and lipid droplets occur in the cytoplasm. Scale bar ¼ 2 mm. (C) Small protuberances (arrows) of the tangential cell wall project into the cytoplasm. Abundant SER profiles and mitochondria occur in the parietal cytoplasm. Scale bar ¼ 0.5 mm. (D) Vacuole containing membranes and flocculent material, and surrounded by SER. Scale bar ¼ 1 mm. (E) Sub-epithelial parenchyma cells with plastids containing starch grains, and vacuoles containing lipid droplets. Protuberances of the cell wall are indicated by arrows. Plasmodesmata occur in primary pit-fields. Scale bar ¼ 2 mm. (F) Plasmodesmata and protuberance (arrow) of radial cell wall of secretory cell. Scale bar ¼ 0.5 mm. (G) Epithelial cell with mitochondria, plastids containing starch, and large, intravacuolar lipid droplets. Cell wall protuberances are indicated by arrows. (H) Plastids with starch grains in cytoplasm of sub-epidermal cell. The cytoplasm of epithelial and sub-epithelial cells is connected by numerous plasmodesmata in primary pit-fields. (G, H) Scale bars ¼ 1 mm. (I) Section of epithelial cell showing prominent nucleus and plastids containing starch grains, as well as lipid droplets. Scale bar ¼ 2 mm. See Fig. 1 for abbreviations. 846 Stpiczyn´ska et al. — Floral elaiophore structure in the Gomesa and Oncidium clades

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F IG.6. (A–F)Gomesa ranifera at final stage of anthesis, TEM. (A) Myelin-like figures containing small vesicles, as well as lipid droplets and mitochondria, occur in the cytoplasm of epithelial cells. Scale bar ¼ 1 mm. (B) Vacuole containing flocculent material and myelin-like figure. Scale bar ¼ 0.5 mm. (C) Cytoplasm containing dilated SER profiles, and vacuole containing dense, flocculent material and osmiophilic droplet. Scale bar ¼ 1 mm. (D) Cytoplasm of epidermal cell containing large lipid droplets and plastids with starch grains. Scale bar ¼ 0.5 mm. (E) Plastids containing small starch grains and large oil droplets. Lipid droplets are also present in both the cytoplasm and vacuole. (F) Cytoplasm with numerous SER profiles, mitochondria and elaioplasts. Lipid droplets also occur scattered throughout the cytoplasm. (E, F) Scale bars ¼ 1 mm. See Fig. 1 for abbreviations. epidermal cells, isodiametric sub-epidermal cells and ground reticulate layer of the cuticle (Fig. 5A), with the largest cavities oc- parenchyma with collateral vascular bundles and large idioblasts curring at the final stage of anthesis. Likewise, secreted material containing raphides (Fig. 4D–G). Furthermore, secretory activ- also accumulates in the intercellular spaces formed by dissolution ity commences at the bud stage, a week or so prior to anthesis, of the middle lamella, including those formed alongside radial with maximum activity occurring between Day 8 and 12, and de- walls and, again, cavities are present (Fig. 5B). The inner surface clining prior to floral abscission. of the outer, tangential wall and radial walls is irregular due to Epidermal cells have mean dimensions of 49.7 × 18.5 mm. The small, peg-like protuberances, around which the plasmalemma outer, tangential cell wall is thick (approx.1.5 mm) and bears a becomes invaginated, thus increasing its surface area (Fig. 5B, C, bi-layered cuticle comprising an outer, lamellate layer and an E–G). Numerous plasmodesmata in primary pit-fields provide inner, reticulate layer (Fig. 5A). The outer surface of the cuticle continuity between the cytoplasm of epidermal and sub-epidermal is smooth, but forms blisters as it becomes detached from the cells, and are particularly frequent in the periclinal (tangential) wall during the secretory stage (Fig. 4C, E). Cavities are absent walls that separate these tissues (Fig. 5E–H). from the outer, tangential wall of the epidermis during the bud Epidermal cells are nucleate (Figs 4D and 5I) and contain plas- stage, but appear at anthesis (Fig. 4F). This coincides with the ac- tids (Fig. 4E, F). The cytoplasm contains abundant SER profiles, cumulation of heterogeneous, secreted material, containing secretory vesicles (Fig. 5A–D) and mitochondria (Fig. 5C, G). rounded or irregular profiles or cavities between this wall and the Dictyosomes were seldom observed. At the secretory stage, Stpiczyn´ska et al. — Floral elaiophore structure in the Gomesa and Oncidium clades 847 intravacuolar lipid bodies (Fig. 5E, G–H), flocculent material droplets occur in the cytoplasm of both epidermal and sub- and myelin-like figures are present (Fig. 6A–C), but, by the epidermal cells (Fig. 6D, E). end of anthesis, the latter are larger and more highly organized (Fig. 6B), and the cytoplasm is less granular. Oncidium amazonicum Plastids contain both starch and oil droplets (Figs 5B, E–I and 6D, E). During the bud stage, numerous amyloplasts with several The floral elaiophore of O. amazonicum is located on the label- large starch grains are present in both epidermal and sub- lar callus. The elaiophore is yellow in colour with a purple rim epidermal cells (Fig. 5E–I). During the secretory stage of anthe- (Fig. 7A, B), and is of the epithelial type (Fig. 7C–F).Asinthe sis, the starch becomes depleted and the amyloplasts differentiate previous species, the elaiophore consists of palisade-like epider- into elaioplasts containing oil droplets (Fig. 6D–F). Similar mal cells of mean dimensions 39.5 × 17.2 mm, and isodiametric Downloaded from https://academic.oup.com/aob/article/112/5/839/139696 by guest on 29 September 2021

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F IG.7.(A–F)Oncidium amazonicum, ﬂoral habit and LM. (A) Lateral view of ﬂower with elaiophore located on proximal part of labellum, and with two small projections (arrows). Scale bar ¼ 2 mm. (B) Detail of elaiophore with secretion. Scale bar ¼ 1 mm. (C) Palisade-like cells of the epithelial layer with sub-epidermal parenchyma. Occasional, small starch grains, that stained red with PAS, are present. Scale bar ¼ 50 mm. (D) Secretory cells with denselystaining cytoplasm and prominent nuclei. Large cavities (asterisks) occur in the thick, tangential cell wall. Scale bar ¼ 20 mm. (E) Paradermal section of epithelial layer stained with auramine O. Note epidermal cells with thick, tangential cell walls whose cavities contain secretion (asterisk). (F) Epithelial cells with plastids (arrows), and thick, outer cell wall with large cavities (asterisks). (E, F) Scale bars ¼ 10 mm. See Fig. 1 for abbreviations. 848 Stpiczyn´ska et al. — Floral elaiophore structure in the Gomesa and Oncidium clades sub-epidermal cells. The proximal callus projection is not secretory. The epidermal cells are nucleate, often with prominent A nucleoli (Fig. 7D–F). Secretoryactivity commences at the bud stage, and small, glis- tening lipid droplets are clearly visible on the elaiophore surface through a stereomicroscope. However, they were not seen, even at the secretory stage, using SEM and may have been dissolved during tissue processing. Secreted material collects throughout anthesis in two symmetrically arranged, marginal depressions of the callus, so that, towards the end of anthesis, almost the entire callus is coated with oil (Fig. 7A, B). The secreted material

was much more viscid in O. amazonicum than in other taxa inves- Downloaded from https://academic.oup.com/aob/article/112/5/839/139696 by guest on 29 September 2021 tigated, but, as in the other species, it stained with both Sudan III and auramine O (Fig. 7E, F). The outer, tangential wall of epidermal cells is thick (approx. 1.5 mm) and possesses a relatively thick cuticle (Fig. 7C, D), which at the bud stage appears undulate in section. However, at anthesis, it forms prominent cavities and blisters, the latter even- tually collapsing in upon themselvestowards the end of this stage (Figs 7C–F and Fig 8A–C). As in the two former species, the B cuticle is bi-layered and composed of an outer, lamellate and an inner, reticulate layer (Fig. 9A–D). Secretion-filled cavities occur in the outer, tangential walls, often at points coinciding with radial walls (Fig. 7C–F). Heterogeneous, secreted material containing rounded or irregular profiles or cavities accumulates between the outer, tangential cell wall and the reticulate layer of the cuticle (Fig. 9A–D). These profiles are particularly large during the final stage of anthesis, and, at this stage, the structure of the reticulate layer is much looser (Fig. 9D). Secreted material containing similar profiles or cavities also accumulates in intercellular spaces (Fig. 9E), including those formed alongside radial walls (Fig. 10A–D, G). A distinctive feature of the outer, tangential cell walls of the elaiophore towards the end of anthesis is the presence of small, peg-like projections that protrude into the cytoplasm (Fig. 9F). The cytoplasm of epidermal cells, as revealed by TEM, is electron-dense and granular (Fig. 10A–G), with a well-developed system (vacuome) of small vacuoles and vesicles (Fig. 10A–D, F–G). Some vacuoles of various sizes display a perinuclear C distribution, whereas others occur basally (Fig. 10A, B, D), and many contain flocculent material, osmiophilic lipid droplets (Fig. 10A–D, F) and myelin-like figures (Fig. 10G). The cytoplasm also contains short profiles of predominantly RER and occasional lipid droplets. Elaioplasts are absent from epidermal cells. Instead, during the bud stage and at the commencement of anthesis, plastids of irregular shape, and containing only small starch grains, are present in these cells (Fig. 10A–C). A greater number of starch-containing plastids, however, is present in the sub-epidermal parenchyma (Fig. 7C). Chromoplasts with numerous smallplastoglobuli are present at the end of anthesis (Fig. 10E).

Oncidium oxyceras In O. oxyceras, lipids are secreted by a small, butterﬂy-shaped elaiophore located in a cup-like depression of the callus

(Fig. 11A, B). Morphologically and anatomically, it is similar F IG.8.(A–C)Oncidium amazonicum at start of anthesis SEM. (A) Surface of to that of O. amazonicum and, once more, comprises a single elaiophore with blistered or collapsed cuticle (asterisks). Scale bar ¼ 200 mm. layer of palisade-like epidermal cells of mean dimensions (B) Folded cuticle with large blister. Scale bar ¼ 50 mm. (C) The cuticle 39.0 × 16.9 mm, and a single layer of isodiametric, sub- remains intact even in collapsed blisters. Scale bar ¼ 100 mm. See Fig. 1 for abbreviations. epidermal cells. The ground parenchyma is supplied with Stpiczyn´ska et al. — Floral elaiophore structure in the Gomesa and Oncidium clades 849

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C D

E F

F IG.9. (A–F)Oncidium amazonicum at start (A–C) and final stage of anthesis (D–F), TEM. (A) Outer, tangential cell wall of epidermal cell with bi-layered cuticle comprising an outer, lamellate layer and an inner, reticulate layer. Heterogeneous secretion containing rounded or irregular profiles or cavities (asterisks) accumulates between thewall and reticulate layerof the cuticle. Plastids with few starch grains occur in the parietal cytoplasm. Intravacuolar membranous profiles are present. Scale bar ¼ 1 mm. (B) Detail of tangential cell wall and bi-layered cuticle showing heterogeneous secretion with rounded profiles orcavities (asterisks). Scale bar ¼ 0.5 mm. (C) Secretion containing large, rounded profiles or cavities (asterisks) accumulating between the tangential cell wall and cuticle. (D) Profiles or cavities (asterisks) of irregular shape are formed between the cell wall and the loosely organized reticulate layer of the cuticle. (E) Secreted, heterogeneous material containing profiles or cavities (asterisks) also occurs in the intercellular spaces associated with the radial cell walls of secretory cells. (C–E) Scale bars ¼ 1 mm. (F) Heterogeneous secretion with large, rounded profiles or cavities (asterisks) adjacent to cell wall. Note also the wall protuberances (arrows) and invaginated plasmalemma. Scale bar ¼ 0.5 mm. See Fig. 1 for abbreviations.

collateral vascular bundles (Fig. 11C) and contains large idio- tangential cell wall contains large cavities at anthesis, and their blasts with phenolic contents. The epidermal cells have promin- position often coincides with that of radial walls (Fig. 11D, E). ent nuclei, and nucleoli are often visible (Fig. 11C, E, F). Smaller, scattered cavities are also present in the outer, tangential Secretioncommencesatthebudstage,4weekspriortoopening, cell wall (Fig. 12B, C), as well as radial walls (Fig. 12D, E). Both and, on average, continues throughout the entire length of anthesis these walls have small, peg-like projections that protrude into the (10 d). The labellar, cup-like depressions of the oldest flowers cytoplasm (Fig. 12E). Primary pit-fields with plasmodesmata are often contain viscid, lipid-rich, secreted material (Fig. 11B). frequent, particularly in the radial cell walls of epidermal cells. Again, the cuticle is bi-layered, comprising an outer, lamellate The cytoplasm of the secretory,epidermal cells contains abun- and an inner, reticulate layer (Fig. 12A, B). During the bud stage, dant SER (Fig. 12B–I). There are numerous vacuoles and small the cuticle, although undulate or folded, is still attached to the vesicles, and the larger vacuoles may occur either at the basal or outer, tangential cell wall (Figs 11C and 12A), but, at anthesis, the apical end of the cell (Fig. 11C–F). Small vacuoles contain- numerous cuticular blisters develop (Fig. 11D, E). The outer, ing flocculent material or myelin-like figures were observed for 850 Stpiczyn´ska et al. — Floral elaiophore structure in the Gomesa and Oncidium clades

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D E

F G

F IG. 10. (A–G) Oncidium amazonicum at start (A–D) andfinalstage of anthesis(F–G)TEM. (A,B) Epithelial cellswithdense cytoplasmandlargenuclei.Thelatter are surroundedby plastids of irregularshape, and smallvacuoles. Narrow intercellularspaces (asterisks) developalongsidethe thin,radial cell walls. (A, B) Scale bar ¼ 5 mm. (C) Parietal cytoplasm with numerous small vacuoles, plastids and mitochondria. Scale bar ¼ 1 mm. (D) Nucleus surrounded by plastids and small vacuoles. Intercellular spaces (asterisks) are formed alongside radial walls by the dissolution of the middle lamella. Scale bar ¼ 2 mm. (E) Dense parietal cytoplasm with mitochondria and plastid containing small starch grains and numerous, smaller plastoglobuli. Scale bar ¼ 0.5 mm. (F) Vacuole of secretory cell containing large osmiophilic bodies. (G) Large, myelin-like intravacuolar figure. Heterogeneous, secreted material (asterisk) with rounded profiles or cavities occurs within the intercellular spaces of radial cell walls. (F, G) Scale bars ¼ 1 mm. See Fig. 1 for abbreviations. all stages investigated (Fig. 12C–I). The latter were more DISCUSSION common in older flowers. Numerous mitochondria (Fig. 12D, The morphology and anatomical organization of the elaiophore I) and plastids of irregular shape (Fig. 12C, F–I) are also of Gomesa and Oncidium are remarkably similar (Pacek and present. Starch is absent from these cells during the bud stage, Stpiczyn´ska, 2007; Stpiczyn´ska et al., 2007; Stpiczyn´ska and but small individual starch grains are present in plastids at anthe- Davies, 2008; Aliscioni et al., 2009; Davies and Stpiczyn´ska, sis, mostly in cells of the sub-epidermal parenchyma (Fig. 11F). 2009; Gomiz et al., 2013). The elaiophore is usuallyof the epithe- Also at anthesis, oval elaioplasts containing large lipid lial type and occurs on the callus or on each of the lateral lobes of droplets appear in the epidermal secretory cells (Fig. 12H, I). the labellum, adjacent to the callus. However, in Gomesa flexuosa By contrast, lipid droplets were present in the cytoplasm of all (Sims) M.W. Chase & N.H. Williams (Gomiz et al., 2013), the stages investigated. Stpiczyn´ska et al. — Floral elaiophore structure in the Gomesa and Oncidium clades 851

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C D

E F

F IG. 11. (A–F). Oncidium oxyceras, ﬂoral habit and LM. (A) Flower with elaiophore located proximallyon the labellum. Scale bar ¼ 1 mm. (B) Detail of elaiophore with single projection, and containing secretion. Scale bar ¼ 0.5 mm. (C) Transverse section of the elaiophore showing intensely stained, palisade-like, secretory epithelial cells, a singlelayerof sub-epidermal cells, vascular bundle and ground parenchyma. (D) Large cell wall cavities (asterisks), and plastids (arrows) stained yellow- greenwith auramine O. (C,D) Scalebars ¼ 30 mm.(E) Secretoryepidermalcells withprominentnuclei andcell wall cavities(asterisks).(F)The PAS reactionrevealed the presence of few starch grains in parenchyma cells. (E, F) Scale bars ¼ 20 mm. See Fig. 1 for abbreviations.

elaiophore is of the intermediate type, in that, although mainly intact as secreted material accumulates beneath its surface. Only glabrous, it also has trichomatous regions. The typical epithelial in G. echinata did we observe ruptured cuticular blisters. elaiophore consists of cuboidal, secretory, epidermal cells and In all four species, secretion-filled cavities are present in the one to several layers of isodiametric, sub-epidermal cells, outer, tangential cell wall, and these usually coincide with the beneath which occurs ground parenchyma supplied with vascu- position of radial walls and appear at the onset of lipid secretion. lar bundles and often containing idioblasts with raphides. Similar cavities also occur in G. bifolia (Sims) M.W. Chase & In the investigated species, a bi-layered or stratified cuticle is N.H. Williams, G. loefgrenii, G. venusta (Drapiez) M.W. present, comprising an outer, lamellate and an inner, reticulate Chase & N.H. Williams and O. cheirophorum. However, they region. The cuticle becomes distended, but usually still remains were not observed for G. flexuosa, G. radicans, G. recurva, 852 Stpiczyn´ska et al. — Floral elaiophore structure in the Gomesa and Oncidium clades

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D E F

G H I

F IG. 12. (A–I)Oncidium oxyceras at start (A) and final stage of anthesis (B–I), TEM. (A) Tangential cell wall with bi-layered cuticle comprising an outer, lamellate layer and an inner, reticulate layer. Scale bar ¼ 1 mm. (B) Tangential cell wall with small, scattered cavities containing secretion (asterisks); the cytoplasm mainly containing SER. Scale bar ¼ 0.5 mm. (C) Parietal cytoplasm with SER, plastids, small vacuoles and secretory vesicles. Cavities in the cell wall are marked with asterisks. (D) Radial cell wall with small cavities (asterisks). The cytoplasm contains SER, small vacuoles and mitochondria. (E) Cavities (asterisks) in the radial cell walls of secretory cells contain secretion, and peg-like wall protuberances (arrow) protrude into the cytoplasm. The cytoplasm contains abundant SER profiles, plastids, mitochondria and lipid droplets. (C–E) Scale bars ¼ 1 mm. (F) Vacuoles with flocculent material, and elaioplasts containing several lipid droplets. (G) Dilated SER profiles and irregularly shaped plastid. (H) Cytoplasm with SER, and elaioplast containing small lipid droplets (arrows). (I) Elaioplast with lipid droplets (arrows), together with mitochondria and small vacuoles with flocculent contents. (F–I) Scale bars ¼ 0.5 mm. See Fig. 1 for abbreviations.

G. riograndensis (Cogn.) M.W. Chase & N.H. Williams, in the formation of intercellular spaces containing osmiophilic Gomesa varicosa (Lindl.) M.W. Chase & N.H. Williams and material, with some indication of cavity formation (see fig. 6C O. ornithorhynchum (Pacek and Stpiczyn´ska, 2007; Stpiczyn´ska in Stpiczyn´ska and Davies, 2008). et al.,2007; Stpiczyn´ska and Davies, 2008; Aliscioni et al., The epidermal walls of G. ranifera, O. amazonicum and 2009; Davies and Stpiczyn´ska, 2009; Gomiz et al., 2013). O. oxyceras are unusual in that they have peg-like protuberances Often, concomitant with the development of these cavities, and, to date, these have not been reported for any other species of dissolution of the middle lamella and separation of cells results Oncidium or Gomesa, as currently circumscribed. Nevertheless, in the formation of intercellular spaces, including spaces similar structures have been recorded for Zygostates grandiflora formed alongside radial walls, in which accumulates secreted (Lindl.) Mansf. (Pacek et al., 2012). Their function is not clear material containing profiles or cavities. This has also been since, unlike the labyrinth-like wall ingrowths observed for a observed for G. radicans, where dissolution of the middle range of secretory and transfer cells (Offler et al. 2002), the lamella of the radial walls of secretory epidermal cells results former protuberances do not significantly enlarge the surface Stpiczyn´ska et al. — Floral elaiophore structure in the Gomesa and Oncidium clades 853 area of the plasmalemma, and are therefore unlikely to be ACKNOWLEDGEMENTS involved in transport. The authors are grateful to Alan Gregg, Swansea Botanical Depending on the species under investigation, avariable quan- Complex, Swansea, UK for help in preparing this paper. tity of starch was observed for epithelial and sub-epithelial cells. The sub-epidermal parenchyma of G. ranifera contains particularly large amyloplasts with starch grains. Heavy starch deposits also occur in G. echinata, but in O. amazonicum and O. oxyceras, LITERATURE CITED starch occurs only as small, individual grains. By contrast, starch Aliscioni SS, Torretta JP, Bello ME, Galati BG. 2009. Elaiophores in Gomesa was absent from the epithelial elaiophore cells of many previously bifolia (Sims) M.W. Chase & N.H. Williams (Oncidiinae: Cymbidieae: studied Oncidiinae species, but was present in the intermediate Orchidaceae): structure and oil secretion. Annals of Botany 104: elaiophore of Ornithocephalus gladiatus Hook. and the tricho- 1141–1149. Brummitt RK, Powell CE. 1992. Authors of plant names. 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