Agnieszka K. Kowalkowska

Morphological, histological and ultrastructural features of osmophores and nectary of Bulbophyllum wendlandianum (Kraenzl.) Dammer (B. section Cirrhopetalum Lindl., Bulbophyllinae Schltr., Orchidaceae)

The species from Bulbophyllinae are generally regarded as fly-pollinated: myophilous or sapromyophilous. The section Cirrhopetalum is characterized by umbellate inflorescence and elongated lateral sepals. The aim of the floral anatomical investigation (micromorphology, histochemistry, ultrastructure) in Bulbophyllum wendlandianum (section Cirrhopetalum) was the detection of secretory activity. The appendages of dorsal sepal and petals function as osmophores. The exudation is transported inside vesicles via granulocrine secretion. The cuticle stretches and forms swellings on the entire cell surface. Such swellings of cuticle on the appendages of dorsal sepal and petals were not previously described in Bulbophyllum species. The nectary is located in the central groove on the adaxial lip surface. It comprises epidermal epithelial cells and few subepidermal layers.

Studies on the ultrastructure of a three-spurred fumeauxiana form of Anacamptis pyramidalis

Floral spurs are regarded as features affecting pollinator behaviour. Anacamptis pyramidalis is regarded as a deceitful, non-rewarding orchid species. In the form fumeauxiana, additional spurs occur on the lateral sepals. In this study we analyse micromorphological and ultrastructural floral features and suggest the mechanism of deception in A. pyramidalis and A. pyramidalis f. fumeauxiana. In f. fumeauxiana, the adaxial surface of the lip, the lip calli, the tips of the lateral sepals, the abaxial and adaxial epidermises of the lip spur, and the lateral sepal spur have a secretory function. Numerous stomata were observed on the abaxial surfaces of spurs and sepals. The characteristic features of the ultrastructure of osmophore cells were noted: dense cytoplasm with numerous profiles of ER, mitochondria, plastids with plastoglobuli and tubular structures, a large nucleus, lipid droplets, and vesicles fusing with the plasmalemma. The similarity of the floral morphology and anatomy, the flowering period, and pollinators of A. pyramidalis, A. pyramidalis f. fumeauxiana and Gymnadeniaconopsea suggest a possible food-deceptive mechanism—imitation of nectar presence produced in the spurs of Gymnadenia.

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.

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 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.

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.

Taxonomic Revision of Dienia (Malaxidinae, Orchidaceae)

A taxonomic revision of the genus Dienia (Malaxidinae, Orchidaceae) is completed. Determination keys for closely similar Asian Malaxidinae genera and Dienia species, as well as descriptions and illustrations of the two accepted species are provided.

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.

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.