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.     

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.     

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.     

Validating a Peruvian Scelochilus (Orchidaceae: Oncidiinae)

Scelochilus crucicornibus, which had been mentioned in the literature as a nomen nudum, is described and illustrated.     

Pseudopollen and food-hair diversity in Polystachya Hook. (Orchidaceae)

Labellar food-hairs in Polystachya Hook. exhibit considerable morphological diversity. The commonest type of trichome is uniseriate, two to four-celled, with a clavate or subclavate terminal cell. This type occurs amongst representatives of most sections examined. Other trichomes are bristle-like with tapering or fusiform terminal cells, whereas representatives of section Polystachya have uniseriate, moniliform trichomes that fragment with the formation of rounded or elliptical component cells. Most contain protein and, while some contain starch, lipid is invariably absent. The presence of particular types of labellar trichomes does not coincide with variations in vegetative morphology. Thus, current taxonomic treatment of the genus indicates that trichome types, with perhaps the sole exception of moniliform, pseudopollen-forming hairs found in section Polystachya only, have limited taxonomic value. However, the remarkable similarity between pseudopollen-forming hairs of Polystachya and those of the Neotropical genus Maxillaria in terms of morphology, cellular dimensions and food content indicates that pseudopollen may have arisen several times and evolved in response to similar pollinator 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.     

Elaiophore diversity in three contrasting members of Oncidiinae (Orchidaceae)

The elaiophores of Trichocentrum cavendishianum (Bateman) M.W. Chase & N.H. Williams, Oncidium loefgrenii Cogn., and Gomesa recurva R. Br. display considerable morphological and anatomical diversity. Oil secretion by flowers of T. cavendishianum and O. loefgrenii can be related to the presence of saddle-like, labellar elaiophores and the labellar callus, respectively, whereas, in G. recurva , although oil is present, no obvious structure appears to be involved in its secretion. In the first two species, the secretory tissue consists of palisade-like cells, whereas, in G. recurva , these cells are oval. Many Oncidiinae are thought to mimic members of the Malpighiaceae, and the elaiophores of that family also contain palisade-like cells that may indicate evolutionary convergence. As oils accumulate below the elaiophore cuticle, that of T. cavendishianum becomes distended, whereas that of the other two species does not. Full discharge of oil from the elaiophores of T. cavendishianum probably occurs only after the cuticle is ruptured by a visiting insect, and this may contribute towards pollinator selection.  © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society , 2007, 155 , 135–148.     

Osmophore and elaiophores of Grobya amherstiae (Catasetinae,Orchidaceae) and their relation to pollination

Grobya amherstiae flowers release a honey‐like scent produced by an osmophore, comprising a papillate epidermis. The scent attracts bee pollinators (Paratetrapedia fervida), which collect floral oils produced by elaiophores on the lip apex and column base. The secretory tissue of the elaiophore on the lip apex consists of both palisade‐like epidermal cells and conspicuously elongated unicellular trichomes. From an anatomical point of view, this elaiophore differs in structure from those known in angiosperms to date. The elaiophore on the column base is exclusively composed of short unicellular trichomes. In addition, there is an elaiophore comprising a papillate epidermis on the internal surface of the lip. The elaiophores produce a heterogeneous secretion, composed of fatty acids and mucilage. The elaiophore on the internal surface of the lip produces oil in non‐collectible amounts, but it is enough to maintain the interest of the bees, guiding them to the elaiophore on the column base, a necessary step in pollination. The former elaiophore is here identified as an oil guide and it plays an essential role in ensuring pollination. The presence of three types of elaiophores on the flowers of this species of Orchidaceae is peculiar and noteworthy. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 159 , 408–415.     

Life history evolution and genome size in subtribe Oncidiinae (Orchidaceae)

• Background and Aims Within Oncidiinae, there are severalgroups of species that are effectively annuals, and we wishedto see if these species had smaller genome sizes than averagefor the subtribe. • Methods Fifty-four genome size estimates (50 of whichare new) for species in subtribe Oncidiinae (Orchidaceae) wereexamined for the first time in a phylogenetic context to evaluatehypotheses concerning genome sizes and life history traits. • Results and Conclusions Within the limits of still relativelysparse sampling, the species that are effectively annuals doappear to have smaller genome sizes than average. However, thegenome sizes of their immediate sister group are also small,indicating that changes in genome size preceded the change inlife history traits. Genome sizes and chromosome numbers alsodo not correlate; some slowly growing species have lower chromosomenumbers but large genomes and vice versa. Based on a surveyof the literature on orchids, it is also clear that epiphyticspecies have smaller genome sizes than do terrestrial species,which could be an effect of different water relations or thefact that most terrestrial orchids are geophytic or have distinctgrowth and dormancy phases.     

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.     

Reproductive biology and pollination of southeastern Brazilian Stanhopea Frost ex Hook. (Orchidaceae)

Reproductive biology and pollination of Stanhopea lietzei and Stanhopea insignis were studied in a semi-deciduous mesophytic forest in the Serra do Japi (SJ), and in the coastal plain of Picinguaba, both in the State of São Paulo, Brazil. Floral morphology, pollination, breeding system and fruit set of both species were investigated. S. lietzei and S. insignis are pollinator-specific, being pollinated by male bees of Eufriesea (Apidae, Euglossini), which collect the fragrance produced by pluricellular osmophores at the base of the saccate hypochile. S. lietzei and S. insignis were pollinated by Eufriesea pulchra and Eufriesea purpurata, respectively. Observations using substances present in the floral fragrance of both studied species as chemical baits were also performed. E. purpurata was attracted by benzyl alcohol, the major compound of the perfume of S. insignis, while E. pulchra was attracted by none of the compounds used. Both studied Stanhopea are self-compatible but pollinator dependent. Self-pollination, however, tends to be avoided by floral mechanisms. In experimental self- and cross-pollinations the proportion of fruit abortion was high and related to resource limitation. The reproductive success of S. lietzei and S. insignis was low as a consequence of deficient pollen transference while pollinator scarcity was the main factor.     

Floral longevity and nectar secretion of Platanthera chlorantha (Custer) Rchb. (Orchidaceae)

Flowering and nectar secretion were studied in Platanthera chlorantha in two years. Nectar was secreted and accumulated in this orchid's spur, originating from part of the labellum. The nectary spur was, on average, 32 mm long. It produced 6.86 micro l nectar in 1999 and 7.84 micro l in 2000. The number of flowers per inflorescence and the volume of nectar secreted per flower were not correlated. Nectar secretion and flower longevity differed depending on pollination and flower position in the inflorescence. Among pairs of pollinated and unpollinated flowers there was no difference in the volume of nectar produced; however, the life span of pollinated flowers was shorter than that of unpollinated ones. Within an inflorescence, the lowest-positioned flowers had the largest nectar production and the longest life compared with flowers positioned higher up.     

A new species of Ornithocephalus (Orchidaceae) from Panama

Ornithocephalus aristatus, a new species from Panama, is described and illustrated. Among the species of the genusOrnithocephalus, it can be distinguished by the nonresupinate flowers, the sepals each provided with a flexuous awn half as long as the sepal itself, the porrect petals with revolute margins, the hastate lip with triangular-ovate, crose lateral lobes, the linear, acute midlobe, conduplicate and subreflexed at apex, and the dise with a bilobed, obreniform callus provided with a conical tuft of stiff hairs. A key to the species ofOrnithocephalus from Panama is provided.     

Minority cytotypes in European populations of the Gymnadenia conopsea complex (Orchidaceae) greatly increase intraspecific and intrapopulation diversity

Background and Aims

Patterns of ploidy variation among and within populations can provide valuable insights into the evolutionary mechanisms shaping the dynamics of plant systems showing ploidy diversity. Whereas data on majority ploidies are, by definition, often sufficiently extensive, much less is known about the incidence and evolutionary role of minority cytotypes.

Methods

Ploidy and proportions of endoreplicated genome were determined using DAPI (4'',6-diamidino-2-phenylindole) flow cytometry in 6150 Gymnadenia plants (fragrant orchids) collected from 141 populations in 17 European countries. All widely recognized European species, and several taxa of less certain taxonomic status were sampled within Gymnadenia conopsea sensu lato.

Key Results

Most Gymnadenia populations were taxonomically and/or ploidy heterogeneous. Two majority (2x and 4x) and three minority (3x, 5x and 6x) cytotypes were identified. Evolution largely proceeded at the diploid level, whereas tetraploids were much more geographically and taxonomically restricted. Although minority ploidies constituted <2 % of the individuals sampled, they were found in 35 % of populations across the entire area investigated. The amount of nuclear DNA, together with the level of progressively partial endoreplication, separated all Gymnadenia species currently widely recognized in Europe.

Conclusions

Despite their low frequency, minority cytotypes substantially increase intraspecific and intrapopulation ploidy diversity estimates for fragrant orchids. The cytogenetic structure of Gymnadenia populations is remarkably dynamic and shaped by multiple evolutionary mechanisms, including both the ongoing production of unreduced gametes and heteroploid hybridization. Overall, it is likely that the level of ploidy heterogeneity experienced by most plant species/populations is currently underestimated; intensive sampling is necessary to obtain a holistic picture.     

Mycoheterotrophy evolved from mixotrophic ancestors: evidence in Cymbidium (Orchidaceae)

Background and Aims

Nutritional changes associated with the evolution of achlorophyllous, mycoheterotrophic plants have not previously been inferred with robust phylogenetic hypotheses. Variations in heterotrophy in accordance with the evolution of leaflessness were examined using a chlorophyllous–achlorophyllous species pair in Cymbidium (Orchidaceae), within a well studied phylogenetic background.

Methods

To estimate the level of mycoheterotrophy in chlorophyllous and achlorophyllous Cymbidium, natural ¹³C and ¹⁵N contents (a proxy for the level of heterotrophy) were measured in four Cymbidium species and co-existing autotrophic and mycoheterotrophic plants and ectomycorrhizal fungi from two Japanese sites.

Key Results

δ¹³C and δ¹⁵N values of the achlorophyllous C. macrorhizon and C. aberrans indicated that they are full mycoheterotrophs. δ¹³C and δ¹⁵N values of the chlorophyllous C. lancifolium and C. goeringii were intermediate between those of reference autotrophic and mycoheterotrophic plants; thus, they probably gain 30–50 % of their carbon resources from fungi. These data suggest that some chlorophyllous Cymbidium exhibit partial mycoheterotrophy (= mixotrophy).

Conclusions

It is demonstrated for the first time that mycoheterotrophy evolved after the establishment of mixotrophy rather than through direct shifts from autotrophy to mycoheterotrophy. This may be one of the principal patterns in the evolution of mycoheterotrophy. The results also suggest that the establishment of symbiosis with ectomycorrhizal fungi in the lineage leading to mixotrophic Cymbidium served as pre-adaptation to the evolution of the mycoheterotrophic species. Similar processes of nutritional innovations probably occurred in several independent orchid groups, allowing niche expansion and radiation in Orchidaceae, probably the largest plant family.