Labellum Features and Chemical Composition of Floral Scent in Bulbophyllum carunculatum Garay, Hamer & Siegrist (Section Lepidorhiza Schltr., Bulbophyllinae Schltr., Orchidaceae Juss.).

The vast majority of fly-pollinated Bulbophyllum species use a combination of visual and olfactory clues to mimic food sources and brood/oviposition sites of pollinators. The aims of the present work were to characterize the floral secretory tissue and the floral scent and compare them with those previously described in B. echinolabium. Based on the histochemical results, the labellar secretion in B. carunculatum is the protein-rich mucilage. The adaxial epidermal cells of the labellum showed typical features of secretory activity. Plastids contained plastoglobuli, which are thought to be the places for scent production in osmophores. Juxtaposed with FeCl3 staining, the presence of dihydroxyphenolic globules in the cytoplasm of the epidermis and sub-epidermis was confirmed. Phenolic derivatives were also described with GC/MS analysis of the floral scent. The number of aromatic compounds and hydrocarbons was indicated in the floral scent of B. carunculatum. Moreover, pregnane-3,20-dione, occurring in the highest percentage in the floral fragrance of B. carunculatum, is a biologically active, 5-alpha-reduced metabolite of plasma progesterone. Progesterone is a mammalian gonadal hormone, but, like other steroid hormones, has been found in plants as intermediates in different biosynthetic pathways. The research on biosynthesis and functions of progesterone and its derivatives in flowers is still lacking.

Active Compounds with Medicinal Potential Found in Maxillariinae Benth. (Orchidaceae Juss.) Representatives-A Review.

Orchids are widely used in traditional medicine for the treatment of a whole range of different health conditions, and representatives of the Neotropical subtribe Maxillariinae are not an exception. They are utilized, for instance, for their spasmolytic and anti-inflammatory activities. In this work, we analyze the literature concerning the chemical composition of the plant extracts and secretions of this subtribe's representatives published between 1991 and 2022. Maxillariinae is one of the biggest taxa within the orchid family; however, to date, only 19 species have been investigated in this regard and, as we report, they produce 62 semiochemicals of medical potential. The presented review is the first summary of biologically active compounds found in Maxillariinae.

Floral attractants in the black orchid Brasiliorchis schunkeana (Orchidaceae, Maxillariinae): clues for presumed sapromyophily and potential antimicrobial activity.

BACKGROUND: Orchids have evolved various strategies that aim to ensure their reproduction success. These may include the production of rewards for pollinators, or on the contrary, deception. Specific sets of features such as flower morphology, color, nectar, and odor presence (or lack thereof) are considered to determine suitability for pollination by different groups of animals. Stingless bees are thought to be the primary pollinators of the orchids of the Neotropical subtribe Maxillariinae. However, almost black flowered Brasiliorchis schunkeana at first glance presents floral adaptations that may suggest another pollination syndrome-sapromyophily. RESULTS: A few traces of secretion were noticed on the glabrous lip callus and lip apex built by conical to villiform papillae (SEM analysis). Histochemical studies revealed huge amounts of lipids in the epidermis, subepidermis, and some parenchyma cells (SBB test) with various stages of lipids accumulation between cells. Further TEM analysis showed a heterogeneous (lipoid and phenolic) nature of secretion. The dense osmiophilic cytoplasm contained organelles (RER, free ribosomes, dictyosomes, plastids with plastoglobuli, nucleus) and vesicles migrating to plasmalemma. The vesicles, osmiophilic globules, and flocculent material were visible in periplasmic space. The central vacuole possessed osmiophilic phenolic content and flocculent material. GC-MS analysis revealed in floral extract the presence of 7,9-di-tert-butyl-1-oxaspiro(4,5)deca-6,9-diene-2,8-dione (77.06%) and 2,5-di-tert-butyl-1,4-benzoquinone (16.65%). Both compounds are known for their biological activity. CONCLUSIONS: The juxtaposition of results led us to the conclusion that the labellar tissue produces lipoid and phenolic material, which is responsible for the glossiness and rotten herring scent. This type of secretion could be classified as a phenolic resin. The chemical analysis revealed the presence of five semiochemicals that are known to be attractants for some Diptera, which together with the rest of the results constitutes a strong premise that representatives of this order could be potential pollinators of B. schunkeana. Field observations however are still needed to confirm this pollination syndrome.

Ornithophily in the subtribe Maxillariinae (Orchidaceae) proven with a case study of Ornithidium fulgens in Guatemala.

Ornithophily has been long speculated to occur in the subtribe Maxillariinae (Orchidaceae), relying either solely on micromorphological analyses or scarce field observations of undefined species. In Guatemala we were able to observe regular visits of the azure-crowned hummingbirds feeding on flowers of Ornithidium fulgens. These observations have led us to investigation of floral attractants by means of scanning and transmission microscopy, histochemical and chemical analyses (GC-MS). Conducted investigation revealed that the epidermis of basal protuberance of column-foot has features proving the secretory activity and that secreted nectar is sucrose-dominant. Slight secretion on the middle part of the lip is puzzling. The presence of other potential pollinators has not been reported. Based on the results of this study, we confirmed that the flowers of O. fulgens meet all criteria of ornithophily and thus that the hypothesis about bird pollination in the subtribe Maxillariinae is proven. The presented results confirm that the previously described floral features predicting the bird pollination in this group are justified. This strengthens the theory about floral adaptations to different pollinators and gives valid reasons to consider species with flowers with a certain set of traits as ornithophilous, even in the absence of the pollination observation.

Labellum structure of Bulbophyllum echinolabium J.J. Sm. (section Lepidorhiza Schltr., Bulbophyllinae Schltr., Orchidaceae Juss.).

This micromorphological, chemical and ultrastructural study is a continuation of research conducted on the section Lepidorhiza. The Bulbophyllum echinolabium flowers comprised features that characterize a sapromyophilous syndrome, having large, glistening parts that emit an intense scent of rotten meat. The secretory activity was described in the hypochile (nectary in longitudinal groove and in the prickles) and the epichile (putative osmophore). The ultrastructural studies revealed a dense cytoplasm in the epidermis and subepidermal tissue with large nuclei and numerous mitochondria, the profiles of SER and RER, and dictyosomes. Large amounts of heterogeneous residues of secreted material (possibly phenolic) were present on the cuticle surface, similar to the unusual prominent periplasmic space with flocculent secretory material. The chemical analysis (GC/MS) of the scent profile of lips comprised carbohydrates and their derivatives (29.55% of all compounds), amino acids (1.66%), lipids (8.04%) and other organic compounds (60.73%). A great number of identified compounds are Diptera attractants (mainly Milichiidae, Tephritidae, Drosophilidae, Muscidae, Sarcophagidae, Tachinidae). The examination of visual and olfactory features indicates correlation between colour of flowers and the type of olfactory mimicry, where a dark colour labellum emits strong smell of rotten waste.

Floral nectary and osmophore of Epipactis helleborine (L.) Crantz (Orchidaceae).

The analysis of flowers collected at different stages of anthesis provides strong evidence to conclude that the shell-shaped hypochile and the knobs of epichile form a nectary. The scent comes from the aromatic constituents of nectar and the epichile tissue and the apices of all tepals (osmophores). The comparison between pollinated and unpollinated flowers revealed that the anthesis of unpollinated flowers lasted up to the 16th day. The nectariferous secretory cells formed single-layered epidermis and several layers of underlying parenchyma built by small, isodiametric cells with thin walls and dense cytoplasm, relatively large nuclei, supplied by collateral vascular bundles. During the floral lifespan, the residues of secreted material were higher on the hypochile cells. The lipoid-carbohydrate material and lipid globules in the cell walls and in the cytoplasm were localised. The abundance of starch grains was observed at the beginning of anthesis and their gradual reduction during the flower lifespan. At the end of anthesis in unpollinated flowers, the lipoid-carbohydrate-phenolic materials have been demonstrated. The phenolic material was the same as in plastoglobuli. The features such as irregular plasmalemma, the secretory vesicles that fuse with it, fully developed dictyosomes, numerous profiles of ER indicate vesicle-mediated process of secretion. The substances could be transported by vesicles to the periplasmic space via granulocrine secretion and then to the external surface. Both micro-channels and slightly developed periplasmic space were visible in the hypochile epidermis. This is the first time for anatomical survey of secretory tissue in pollinated and unpollinated flowers of E. helleborine.

Floral features of two species of Bulbophyllum section Lepidorhiza Schltr.: B. levanae Ames and B. nymphopolitanum Kraenzl. (Bulbophyllinae Schltr., Orchidaceae).

Two representatives of section Lepidorhiza, previously sometimes considered conspecific, Bulbophyllum levanae and Bulbophyllum nymphopolitanum, demonstrated both similarities and differences in floral features. There were significant differences in the length of sepals and micromorphological features of the labellum. In both species, osmophores are located on the extended apices of sepals and possibly on petals. An abundance of proteins in tepals is probably associated with the unpleasant scent of the flowers, whereas the thin wax layers on the epidermis are probably involved in the maintenance of the brilliance of floral tepals, which strongly attracts flies. In all tepals of both species, we noted the presence of dihydroxyphenolic globules in the cytoplasm after staining with FeCl3. Comparison with ultrastructure results revealed that they were associated with plastids containing plastoglobuli. The most remarkable feature was the presence of a prominent periplasmic space in the epidermal cells of both investigated species. Furthermore, in the labellum of B. levanae, the cuticle contained microchannels. The combination of periplasmic space and microchannels has not previously been recorded.

Floral structure of two species of Bulbophyllum section Cirrhopetalum Lindl.: B. weberi Ames and B. cumingii (Lindl.) Rchb. f. (Bulbophyllinae Schltr., Orchidaceae).

Flowers of Bulbophyllum weberi and B. cumingii are characterized by fly-pollinated features. The secretory activity was described in dorsal sepals in both species (putative osmophores), petals in B. weberi (possible osmophores) and adaxial surface of lips in both species. In the cells of dorsal sepals and petals of B. weberi proteins, dihydroxyphenols, lipids and starch grains were detected, in lateral sepals-lipids. Whereas in dorsal sepal of B. cumingii only lipids and starch grains were noted, in lateral sepals-proteins and dihydroxyphenols and in petals-proteins and starch grains. The lips in both species differed histochemically and ultrastructurally. The epidermal cells of lip groove in B. weberi contained lipids, proteins, starch grains in cytoplasm, dihydroxyphenols in vacuoles and pectic acids/mucilage on surface. Whereas in B. cumingii-few lipids, starch grains, no proteins, no dihydroxyphenols and no mucilage were noted. Ultrastructurally, in B. weberi, the secretory material was present on surface and vesicles building into plasmalemma, while in B. cumingii-cell wall ingrowths and microchannels in cuticle. The osmiophilic irregular materials and globular, osmiophilic globules in B. weberi are probably tannin-like materials. For the first time, we described the cell wall ingrowths in Bulbophyllum species: in lip of B. cumingii and petals of B. weberi.

Evidence for mixed sexual and asexual reproduction in the rare European mycoheterotrophic orchid Epipogium aphyllum, Orchidaceae (ghost orchid).

BACKGROUND AND AIMS: Despite their significant capacity to propagate vegetatively, most orchids reproduce via seeds. Sexual reproduction via seed is commonly reported, in contrast to apomixis, whereby seeds are clones of the mother. Although insect pollination and autonomous self-pollination exist in mycoheterotrophic plants, the reproductive embryology of these plants remains under-studied. This paper provides evidence for the co-occurrence of both sexual and apomictic reproduction in a population of mycoheterotrophic plants - Epipogium aphyllum We investigated seed formation via open pollination, induced autogamy, autogamy sensu stricto and autonomous apomixis. METHODS: The study was performed on a population of E. aphyllum located in northern Poland. The research included studies of the micromorphology, histochemistry and embryology of four types of reproductive systems. Scanning, fluorescence and light microscopy accompanied by graphical and statistical analyses were employed. KEY RESULTS: We observed gametophyte development, from the one-nucleate stage to maturity, in unpollinated flower buds. The lack of zygotes in flower buds indicated that fertilization did not occur at this stage. Manual self-pollination led to a zygote, followed by embryo formation. Fertilization and embryo development derived from embryogenesis via open pollination is delayed compared with hand pollination. Isolation from external pollination resulted only in structures resembling zygotes that may originate either sexually or independent of fertilization. Parthenogenetic structures that resembled zygotes were observed in flowers that were emasculated and isolated from pollination. Zygotes formed at significantly higher frequencies via open pollination and induced autogamy in comparison to the parthenogenetic structures formed in other treatments. CONCLUSIONS: We showed the absence of pre-zygotic barriers for autogamy in E. aphyllum Self-pollination and self-fertilization are possible; however, natural self-pollination is unlikely or rare due to the position of the pollinia. Incidental parthenogenesis in E. aphyllum is very likely, given the biology of ovule development of this mycoheterotrophic orchid. This species therefore has the potential to produce seeds via both sexual and asexual means, although the contribution of apomixis to this process appears largely negligible.

Floral nectary anatomy and ultrastructure in mycoheterotrophic plant, Epipogium aphyllum Sw. (Orchidaceae).

Epipogium aphyllum is a European-Asian obligatory mycoheterotrophic orchid containing no chlorophyll. Flowers are not resupinate with a sack-shape spur and cordate lip, which is divided into two parts: the basal (hypochile) and distal one (epichile). The floral analysis provides strong evidence to conclude that nectar is secreted on the upper surface of pink-coloured papillate ridges and epidermal (adaxial) cells at different place in spur, especially at the apex. The exudation on papillae has been observed through the entire anthesis and it has been stained on polysaccharides, proteins, and lipids. The dense cytoplasm of papillae contains profuse endoplasmic reticulum, plentiful vesicles (bigger ones with tannin-like materials), numerous mitochondria, sometimes dictyosomes, starch grains, and plastids with tubular structures. The large electron-dense bodies in cell walls are structurally the same as tannin-like materials from vesicles that are in contact with plasmalemma. The rupture of thin layer of swelled cuticle is caused by pressure of gathered substances exuded due to granulocrine secretion. The idioblasts with raphides occur mainly in tepals tissue. The dynamic changes of the nectar exudation, released through endocrine secretion, have been noticeable during the anthesis: both on the lip and inside the spur. The nectar secretion is not dependent on the colour form of E. aphyllum blooming shoots. The floral biology and ultrastructure differ from mycoheterotrophic plants known up to date.

Studies on floral nectary, tepals' structure, and gynostemium morphology of Epipactis palustris (L.) Crantz (Orchidaceae).

The lip of Epipactis palustris consists of two movably joined parts: the basal part (hypochile) with central broad isthmus and epichile with callus. The analysis of flowers provides strong evidence to conclude that the whole surface of lip callus and abaxial side of isthmus are secretory. The exudation at first appears on callus, at early stages, later on isthmus. It could be a strategy to prolong the emission of volatile substances and nectar, and this means to prolong luring pollinators. The results from transmission electron microscopy (TEM) support this conclusion. The plastids noted in callus were without starch, whereas the isthmus' cells contained partly hydrolyzed starch. Some plastids, noted in callus, had polymorphic shapes, which were often related to a starch reduction. During the depletion of starch in callus cells, the number of plastoglobuli within the plastids increased, and also lipid bodies appeared in the cytoplasm whereas, in isthmus cells, proplastids with phytoferritin were noted. The endoplasmic reticulum was in contact with plasmalemma, and the vesicles were fusing with plasmalemma in secretory cells of callus and isthmus, which is a way of granulocrine secretion. The cross-sections of sepals revealed that abaxial epidermis was tomentose, with stomata at the top of substomatal cavities. The pollen grains adhering to the rostellum-viscidium prove previous ecological observations that the rostellum-viscidium is not a barrier preventing self-pollination.

Taxonomic position and phylogeny of the genus Vargasiella (Orchidaceae, Vandoideae) based on molecular and morphological evidence.

Since the description of the Neotropical genus Vargasiella in 1952, its taxonomic position has remained unclear, mainly due to a lack of sufficient data. In this study, the taxonomic position of Vargasiella was revised based on the outcomes of macro- and micromorphological studies, analyses of selected molecular markers and ecological methods of niche distribution modeling. The phylogenetic relationships were inferred using three DNA markers: matK, trnL-F and ITS sequences. The morphological studies included the analysis of macromorphological features of herbarium specimens as well as micromorphological examination of preserved flowers. The ecological niche modeling was applied to identify the distribution of the suitable niches of the studied taxa. The relationships between Vargasiella and most similar taxa remain unresolved based on the molecular analysis. The outcomes from the morphological studies indicated significant differences between Vargasiella, Warrea and Warreopsis. Moreover, a niche shift in response to changing climate after the last glacial maximum is observed in Vargasiella, while no substantial changes in the occupied habitats were identified in the other related taxa. The clocktree of the Zygopetaleae estimated from the matK gene indicated that the most recent common ancestors of Vargasiella, Warrea and Warreopsis originated in the Miocene, while the divergence time for Vargasiella and Warrea was assessed at approximately 5.4 Ma ago. Vargasiella seems to be an outshoot of the main branch of evolution of the Zygopetaleae. It is noteworthy that the Vargasiella-Warrea dichotomy could have taken place later than the divergence of Warreopsis from the mutual lineage. The molecular analysis and morphological data suggest that Vargasiella and Warrea could have evolved from a common ancestor. Accumulation of morphological differences and acceleration of the evolution of Vargasiella were more intensive than in other Warreinae and this could probably be synchronized with adaptation to different climatic conditions.

Taxonomic placement of Paphiopedilum canhii (Cypripedioideae; Orchidaceae) based on cytological, molecular and micromorphological evidence.

Paphiopedilum canhii was discovered in Northern Vietnam. Since its description in 2010, it has caused a stir among taxonomists due to its interesting mixture of morphological features, i.e. marbled, relatively thick leaves, flowers similar to species classified in the section Barbata, and unique, large staminodial shield. On the basis of these features, it is difficult to classify the species to existing infrageneric units. Using cytological data, phylogenetic analyses based on plastid and nuclear genes and the study of the adaxial epidermis of the leaves and gynostemium structure obtained from Scanning Electron Microscopy (SEM) and Light Microscopy (LM), the taxonomic position of P. canhii was determined. These results suggest that P. canhii forms an independent phylogenetic line within the genus Paphiopedilum deserving subgeneric status, already proposed by Braem and Gruss (2011) as Megastaminodium.