Floral anatomy and ultrastructure in Lepanthes calodictyon group

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The microscopic features of species within Pleurothallidine subtribe is poorly understood. In this study we investigated three Lepanthes species: L. calodictyon , L. saltatrix and L. tentaculata . All of them represent similar flower morphology, distinct from the other representative of the genus. Results : Our analyses revealed presence of features usually associated with myophily. Papillae of lips and petals of the Lepanthes calodictyon group, were rich in lipids and proteins, what was correlated with high metabolic activity. Furthermore, ultrastructural and morphological features are similar to those observed in other fragrance emitting orchid species. Conclusions : Histochemical, ultrastructural and morphological features of the papillae on the surface of the lip and petals of examined taxa indicate that papillae are in fact osmophores. micromorphology Orchidaceae osmophore Pleurothallidinae ultrastructure Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction The subtribe Pleurothallidinae Lindl. is one of the richest and the most diverse group among Orchidaceae. It comprises ca. 40 well-recognized genera with over 5100 species (Luer 1996 ; Jost & Endara 2004 ; Pridgeon 2005 ; Pridgeon et al. 2001 ; Higgins 2009 ; Luer & Thoerle 2012; Karremans 2016 ). Representatives of Pleurothallidinae are usually small, mainly epiphytic, Neotropical orchids with sympodial growth habit and occur predominantly at elevation above 1000 m in the humid rainforest or the páramo formation (Luer 1986 , 1996 ; Salazar & Soto 1996; Pridgeon 2005 ; Luer & Thoerle 2012). The species of Pleurothallidinae are considered together with unrelated Bulbophyllum genus as fly-pollinated orchids (Dressler 1993 ; Cozzolino & Widmer 2005 ; Borba et al. 2011 ; de Melo et al. 2011). The representatives of Pleurothallidinae subtribe exhibit a large diversity of morphological characters associated with myophily. They usually have small brightly coloured flowers. The surface is frequently covered by papillae, motile hairs or trichomes, which attract the pollinators. The nectary production or fragrance emission are regarded as factors increasing pollination effectiveness. However, among ca. 40 recognized and acknowledged genera, only few of them were investigated as far: Acianthera Scheidw. (Borba & Semir 2001 ; de Melo et al. 2010 ), Anathallis Barb. Rodr. (Borba & Semir 2001 ), Dracula Luer (Endara et al. 2010 ), Lepanthes (Blanco & Barboza 2005 ), Masdevallia Ruiz & Pav. (Borba & Semir 2001 ), Octomeria R.Br. (Barbosa et al. 2009 ), Pleurothallis R.Br. (Duque 1993 , Duque-Buitrago et al. 2014 ), Restrepia Kunth, (Pridgeon & Stern 1983 ), Scaphosepalum Pfitzer (Pridgeon & Stern 1985 ), Specklinia Lindl. (Chase 1985 ; Karremans et al. 2015 ), Stelis Sw. (Albores & Sosa 2006) and Zootrophion (Borba & Semir 2001 ), nectary production was proved once (Karremans et al. 2015 ), and the most common the presence of the variable osmophores were detected. Among all Pleurothallidinae, Lepanthes is the most richest in species genus (Karremans 2016 ). It comprises approximately 1100 species distributed from southern part of Florida, through Central America to Bolivia in the south. The greatest diversity is observed in Ecuador and Colombia (Luer 1986 , 1996 ; Salazar & Soto 1996; Pridgeon 2005 ; Luer & Thoerle 2012). Species belonging to the Lepanthes genus are characterized by the presence of the dilated and ciliate sheaths of the ramicaul, the small, often yellow flowers with petals broader than long. Usually they have trilobed lip with the midlobe reduced to a minute appendix, the blades of the lip are above the column which is elongated and embraced by the lip. Although the appendix vary in form, it is most often covered by motile trichomes or hairs and typically founded pressed beneath to the column (Luer 1986 , 1996 ; Salazar & Soto 1996; Pridgeon 2005 ; Luer & Thoerle 2012). Moreover, flowers are scentless, not offering any reward for pollinators, such as nectar or pseudonectar (e.g. Salazar & Soto 1996). The sexual deception was considered by many authors as a pollination syndrome in Lepanthes (Dressler 1993 ; Tremblay & Ackerman 1993 ; Luer 1996 ; Salazar & Soto 1996; Pridgeon 2005 ; Tremblay et al. 2005 ). The first observation of male flies mating with Lepanthes was recorded in Costa Rica by Blanco and Barboza ( 2005 ). They photographed and described males of Bradysia floribunda (Sciaridae) attached by their sexual organs with appendix of the lip of L. glicensteinii Luer. The male flies of the Bradysia floribunda visited the flowers of L. glicensteinii exclusively, which suggests very specific plant-pollinator interactions. Moreover, Blanco and Barboza ( 2005 ) distinguished the three crucial steps in the pollinator behaviour. First step was reception of the “olfactory” signal – flies hover by few seconds around the plant and after landing, they tried to detect the scent produced by the flower. Second step – if flower was already open, the fungus gnats looked for the female and started their mating dance. The last step was reception of the sensory signal. Only after the securing the appendix of the lip, flies started to stake out in the right position and start to “copulate” with the flowers. During the pseudocopulation, pollinia were removed from the column and attached to the abdomen of the fungus gnats. Furthermore, the spermatophore was deposited on the ventral side of the column, near the appendix. Authors also reported that the Bradysia floribunda males pollinated the flowers (Blanco & Barboza 2005 ). The three species, Lepanthes calodictyon , L. tentaculata and L. saltatrix are distinctive in both floral and vegetative if compared to other Lepanthes representatives were investigated anatomically here in detail. The species of this group are characterized by the simultaneous presence of: the reticulated leaves with undulate margins, the short and dense inflorescence on the upper side of the leaf, the bilobed petals contracted in to slender tails at the apex, unlobed or slightly bilobed lip without appendix, the lip hinged to the column by the claw. The petals and lip are bright deep red or crimson-cream, papillate in contrast to the inconspicuous, similar in shape, glabrous, pale yellow-green sepals. Moreover, the sepals embrace the straight ovary, which made the central part of the flower elevated and probably, more easily noticeable for pollinators. It is well known in Orchidaceae, that vivid colours, and various appendages on the surface of the floral segments are frequently connected with the secretory activity. We suspect, that the contrastive to pale sepals, red colouration of the petals and the lip, could implicate the crucial role of these floral segments in the attraction of the pollinators. This hypothesis about very particular role of the central parts of the flower seems to be legitimate, especially if consider the lack of the appendix of the lip. Due to that, the aim of this study were to investigate the morphological, histochemical and ultrastructural features of petals and lip as well as to look for the evidences to the secretory activity of their cells using light, fluorescent and electron microscopy methods. Material and methods Plant source Specimens of Lepanthes calodictyon , L. saltatrix and L. tentaculata were purchased from Ecuagenera Ltd. (Ecuador). Flowers were collected at anthesis from March 2016 to September 2017. The flowers of L. calodictyon, L. saltatrix and L. tentaculata (voucher numbers: UGDA 012573, 012605 and 012614 respectively) were preserved in the KEW mixture (53% ethanol, 37% water, 5% formaldehyde and 5% glycerol) and deposited in the herbarium. Light microscopy Plant material were examined and measured using a SteREO Discovery V12 Zeiss stereomicroscope with the magnification range from 5× to 63× and an ocular graticule 10×/2.3. All measurements were taken according to the central axis of structures. The photographs of the flowers were made by Zeiss Axiocam 512 and Nikon SMZ 1500. The preliminary delimination of secretory active parts of the flower were performed according to Wiśniewska et al. ( 2018 ) and comprise staining with in 0.01% solution of methylene blue or in 0.1% neutral red. Whole flowers and/or parts were used for light microscopy analyses and were preserved in the dark at 4ºC in the phosphate buffered saline (PBS, pH 5.7) containing 2.5% glutaraldehyde (GA) and 4% paraformaldehyde (PFA). After 24h samples were rinsed two times in PBS following by distilled water, dissected, dehydrated by increasing concentration of the ethanol (Hülskamp et al. 2010 ) and embedded in Steedman’s Wax (Krawczyk et al 2016 ). The histochemical tests were performed on 10 µm sections made on HM 360 Microm and comprised: toluidine O for general histology (Federer and O’Brien 1968) Coomasie Brilliant Blue (CBB) for protein detection (Hu and Xu 1990 , modified), Sudan Black B for lipids (Bronner 1975 ) and the periodic acid-Schiff reaction (PAS) which was used for detection of total insoluble polysaccharides (Jensen 1962 ). Catechol-type dixydroxyphenol inclusions were visualized by 10% (w/v) aqueous solution of FeCl 3 (Gahan 1984 ). Microtubules were visualized using rat primary antibody against α-tubulin (Ab6161; Abcam, UK) and a goat anti-rat secondary antibody conjugated with DyLight™ 549 (AS12 2084; Thermo Fisher Scientific). F-actin was detected using monoclonal mouse antibody against actin (MP Biomedicals) and goat anti-mouse secondary antibody conjugated with Alexa Fluor 488 (Life Technologies). Both, primary and secondary antibodies were used in 1:800 dilutions. The chromatin of the nuclei was stained with 7 µl/ml DAPI (Sigma-Aldrich). In negative control experiments, the primary, secondary or both antibodies were omitted. All light microscopy sections were mounted in Mowiol medium and viewed with Leica DM6000 B or Nikon Eclipse E 800. Electron microscopy Samples for scanning electron microscopy (SEM) were fixed overnight at room temperature in the KEW mixture, dehydrated with increasing concentration of the ethanol then coated with gold and were observed on Philips XL-30 scanning electron microscope. Sample preparation for transmission electron microscopy (TEM) analyses, was performed by standard electron microscopy procedure (Kowalkowska et al. 2010 ) followed by post-fixation overnight in 1% OsO 4 . Plant material was embedded in Spurr’s resin, cut to ultrathin sections and examined on FEI Tecnai Spirit BioTWIN transmission electron microscope. Results Among all three species (Figs. 1 – 3 a) the all flower segments except lip and petals were similar in shape. The flowers do not produce any noticeable scent and were born on the congested inflorescence on the top of the reticulated leaves. The data about histochemistry and features of the flowers are summarized in the Table 1 . Table 1 The results of micromorphological, ultrastructural and histochemical analyses of Lepanthes calodictyon, L. saltatrix and L. tentaculata Species/method Lepanthes calodictyon Lepanthes saltatrix Lepanthes tentaculata sepal petal lip sepal petal lip sepal petal lip Osmophores LM: methylene blue, neutral red - + + - + + - + + Secretions SM - - + - - + - + - Proteins LM: CBB + +++ +/+++ + +++ +++ + +++ +++ Starch grains LM: PAS - - - - - - - - - Dihydroxyphenols In plastids/vacuoles LM: FeCl 3 nt + - nt - - nt - - Lipids LM: SBB nt + +++ nt +++ +++ nt ++ ++ Secretions TEM nt + +/++ nt + ++ nt + ++ Cuticle LM: auramin 0 nt s s nt s s nt s s Cytoskeleton (α-tubulin and F-actin) LM + ++ ++ + ++ ++ + ++ ++ +++ - abundant; ++ - rich; + - present; - - absent; nt- not tested; s- smooth. Sepals The sepals were translucent and yellow to yellow green in colour (Figs. 1 – 3 a). SEM studies revealed the surface of dorsal sepal of Lepanthes calodictyon was built by regular epidermal cells with smooth cuticle. That kind of cells were covered evenly whole area. The presence of cubical secretions was observed. On the edge of sepal’s apical part epidermal cells were formed unicellular trichomes. They were covered by the smooth cuticle and few cubical particle. Similar to dorsal sepal the epidermal cells, covering whole surface of both lateral sepal, were regular. Whole area was covered by the smooth cuticle and particle of secretions. Only on the part under the outer edge the epidermal two-celled trichomes were notice. Many cubicle part of secretion was noticed on trichomes and the surface around them. Almost whole length of outer edge of sepals was built by unicellular trichomes with smooth cuticle, which were covered by few particle secretions. The surface of both Lepanthes saltatrix dorsal and lateral sepals was built by regular epidermal cells, with smooth cuticle. Only on the edge, especially at the top, small, unicellular trichomes with slightly undulated cuticle were observed. On the surface of dorsal and lateral sepals the presence of secretion was not notice. Convex area of surface, situated along the both sepal’s edges were observed. This swellings was covered by regular, epidermal cells with smooth cuticle. The surface of dorsal and lateral sepals of Lepanthes tentaculata was built by regular epidermal cells, with smooth cuticle. Only the apical part of sepals was deprived of few two-celled trichomes. On the edge, especially at the sepal’s top were observed small, unicellular trichomes with smooth cuticle. The presence of secretion was not noticed on the surface of dorsal and lateral sepals. Petals The petals were red in L. calodictyon and L. tentaculata or bicoloured in L. saltatrix - crimson in upper lobe and cream on lower. The petals were bilobed with long, slender tails on both apex (Figs. 1 – 3 a). The lobes of the petals as well as the tails found on the apices of them were covered by unicellular papillae, similar in morphology to these observed on the surface of the lip (Figs. 1 – 3 d – e, 4 a – c). The intense colouration after treatment with neutral red and methylene blue was noticed in each species (Figs. 1 – 3 f – k). The cuticle of all examined petals’ species was consistent when stained by Auramin O, lacking cracks (Fig. 4 j). Nevertheless ultrastructural studies revealed the small abruptions of the cuticle in case of L. saltatrix and L. tentaculata (Figs. 5 d, 5 f – g) when in L. calodictyon no disruptions of the cuticle were not noticed (Fig. 5 a). The surface of petals of Lepanthes calodictyon was covered by two kind of cells: regular, isometric epidermal cells and papillae cells (Figs. 1 d – e). Bottle – shaped, unicellular papillae were formed by epidermal cells on the edge of petal. The cuticle of papillae was smooth without any secretion (Fig. 1 e). Similar papillae on the surface of the lengthened, thread- shaped petal’s part were visible. The middle part was built by regular epidermal cells with smooth cuticle. Petal is composed of two different component: rounded middle part and lengthened, two, thread-like side part. Lepanthes saltatrix The surface of petal’ middle part was covered by bottle – shaped, unicellular papillae which filled densely whole area (Fig. 2 d). Similar papillae cells were visible on the whole surface of side part. Their cuticle was smooth, but any secretion was not observed (Fig. 2 , e). Petal of Lepanthes tentaculata was composed of rounded middle part and lengthened, two, thread-like side part. The surface of petal’ middle part was covered by papillose cells (Fig. 3 d. Bottle-shaped, unicellular papillae were filled densely whole area, only on the edge were located lengthened, unicellular trichomes (Fig. 3 e). Similar papillae cells were visible on the whole surface of side part. As well their cuticle was smooth, but any secretion was not observed (Fig. 3 e). Histochemical studies revealed that petals’ papillae of three investigated species were rich in proteins and lipids (Table 1 ; Figs. 5 a – g). The TEM studies revealed the presence of the features associated with secretory activity and residues of secretion was detected on the surface of the petals’ papillae of L. calodictyon and L. saltatrix , and no secretion was found in L. tentaculata (Figs. 5 a – i). Ultrastructure of the petals’ papillae vacuole occupied the main part of the cells, the cytoplasm was dense with numerous organelles crowded on the perimeter of the cell (Figs. 5 a – i). The small vesicles connected with plasmalemma were also observed between irregular plasmalemma and cell wall (Fig. 5 f). The SBB treatment indicated lipids deposited inside the cells especially large amount of lipid bodies was found in Lepanthes saltatrix’s trichomes (Fig. 4 e and Fig. 5 d). Lipid bodies are often accompanied with plastids (Figs. 5 b, d, h and i). Within plastids numerous osmiophilic plastoglobuli were observed but no starch grains noted (Figs. 5 a – i). This observations are comparative for all examined species with the negative results of the PAS reaction for detection of starch grains (Figs. 4 h – i). Few, small dihdroxyphenols inclusions were observed only in cytoplasm of Lepanthes calodictyon’s petal (Fig. 4 l), other staining remained inconclusive (Fig. 4 k and m). The cytoskeleton of the petals’ papillae was well developed in all three species, cortical hoop-like microtubules and delicate network of microfilaments were noted (Figs. 4 n – o). Lip The morphology of the lip was the most diverse feature and vary in shape between the species, however always hinged on the claw and never trilobed, in contrast to the most of the Lepanthes . The surface of the lip of each examined species was covered by unicellular papillae, which were conical with rounded tips (Figs. 6 a – c). No ornamentation of the cuticle of the labellar papillae was observed. The only difference between species was presence of longer and shorter form of the papillae on the margin of the Lepanthes calodictyon lip. The intense colouration of the lip was noticed when stained by neutral red and methylene blue solutions. The whole surface of the lip of Lepanthes calodictyon were covered by unicellular, bottle-shaped papillae, which were formed by epidermal cells. Papillae were covered by the smooth cuticle and presence of any particle of secreted substance were not observed (Figs. 1 b – c).Only a few lengthened trichomes on the corner of lip were notice (Fig. 1 b). The cuticle of trichomes were smooth and any secreted substances was not observed. The ray-like lip of Lepanthes saltatrix was built by regular epidermal cell, forming papillae. Whole area of lip (Figs. 2 b – c) was covered by this kind of cells, whereas longer unicellular trichomes on the corner of edges were observed (Fig. 2 b). The cuticle of papillae and trichomes was smooth. The ray-like lip of Lepanthes tentaculata was built by regular epidermal cell, forming papillae. Whole middle area of lip (Fig. 3 b) was covered by this kind of cells, whereas longer unicellular trichomes on the edges were observed. The cuticle of papillae and trichomes was smooth, however any particle of secretion were not presented (Fig. 3 c). The cuticle was smooth and intact when stained by Auramin O (Figs. 6 k – l) but unlikely to petals examination, only few small abruption of the cuticle were noticed in Lepanthes tentaculata lip (Fig. 9 b, black arrows). The CBB staining showed that the papillae were generally rich in proteins in each species (Figs. 6 d – e), but the longer papillae found on the margin of the Lepanthes calodictyon stained more intensively if compared to shorter papillae (Fig. 6 d). Treatment with SBB indicated high amount of lipids in all examined species (Figs. 6 h – j). The PAS for starch grains (Figs. 6 f – g) and staining for dihydroxyphenoles were negative (Fig. 6 m). The TEM studies revealed the presence of the features associated with secretory activity but no traces of secretion was detected on the surface of the papillae. The main part of papillae of the lip was occupied by vacuole and the cellular structures were crowded on the perimeter (Figs. 5 c, f and i). The cytoplasm was dense with plenty of rough or smooth endoplasmic reticulum (RER/SER) (Figs. 7 – 9 ), mitochondria with well developed cristae (Figs. 8 b and 9 d), numerous vacuoles and dictyosomes (Figs. 7 – 9 ), plastids with plastoglobuli (Figs. 7 a and c – d, 8d and 9 a). The small vesicles were observed between irregular plasmalemma and cell wall. Some of the vesicles were connected with plasmalemma (Figs. 7 a, 8 a and 9 c – d; black arrows). The cytoskeleton of the lip was well developed in all three species, comparatively to petals’ trichomes (Figs. 6 n – o). Discussion Features which may be connected with pseudocopulatory are the three dimensional flowers’ form, contrastive colouration, presence of narrow, unicellular trichomes with frequently pointed tips and in many cases reflective surfaces. Various combinations of these features were frequently reported for diverse plants such like orchids ( Mormolyca (Davies and Stpiczyńska 2006), representatives of Ophrys (Servettaz et al. 1994 ; Ascensao et al. 2005) and Trigonidium (Singer 2002 , Whitten and Blanco 2011 )) or bladderworts (Płachno et al. 2016 ). Moreover, the flowers of all three examined species exhibit the features of fly pollinated orchids such as colours, motile lip hinged to the claw and the presence of the osmophores which lure the pollinators as it is in Bulbophyllum (Kowalkowska et al. 2017 ). The investigation of anatomy of the flowers revealed the presence of the osmophores on the surface of the lip and petals, and sepals are rather inactive in secretion process. The cytoplasm of the labellar and petals’ cells is dense with large amount of organelles like numerous mitochondria, lipid bodies, vacuoles and vesicles, the plastoglobuli are surrounded by well-developed endoplasmatic reticulum and cytoskeleton network. The anatomy of the cells suggest the high metabolic activity of the cell like in osmophoric tissues (Pridgeon and Stern 1983 , Stpiczyńska et al. 2005 , Kowalkowska et al. 2017 ). The osmophores are found on the epidermis and subepidermal layers (e.g. Vogel 1990 ; Curry et al. 1991 ; Stpiczyńska 2003 ; Płachno et al. 2010 ; Antoń et al. 2012 ). The subsecretory parenchyma is characterized by the presence of numerous starch grains and by hydrolysis of polysaccharides they provide energy for secretory activity. In the osmophores, starch grains occur frequently, but not exclusively, together with lipid bodies (Vogel 1990 ). The osmophores without starch was reported previously for various orchid species e.g. Anacamptis pyramidalis f. fumeuxiana (Kowalkowska et al. 2012 ), Bulbophyllum wendelianum (Kowalkowska et al. 2015 ), Cyclopogon elatus (Wiemer et al. 2008 ), Cypripedium (Swanson et al. 1980) or Gymnadenia conopsea (Stpiczyńska 2001 ). Nevertheless, in orchid Grobya amherstiae Lindl. (Pansarin et al. 2009) and carnivorous bladderworts U. cornigera and U. nelumbifolia , only the epidermis was identified as highly active physiologically. The conclusion about physiological condition was made based on ultrastructure and histochemistry evidences. In case of Lepanthes calodictyon group, the papillae on the surface of the lip and petals could emit the fragrance. Synthesis of the volatile compounds could occur in plastids with plastoglobuli as it was found in osmophores and nectaries (Stpiczyńska 1997 , 2001 ). The proximity of the endoplasmatic reticulum to plastoglobuli is frequently interpreted as feature connected with fragrance. The fragrant compounds could be produced in plastoglobuli, then transported via ER to plasmalemma or transported as lipophilic substances directly in cytoplasm (Stern et al. 1987 , Pais and Figueiredo 1994 , Stpiczyńska 1997 , Kowalkowska et al. 2012 , 2017 ). The endoplasmatic reticulum is in connection with plasmalemma as observed in other orchids genera such Restepia (Pridgeon and Stern 1983 ) or Epipactis (Kowlakowska et al. 2015). Ultrastructural and histochemical studies revealed the spherical structures, which were identified as lipid bodies inside the lips’ and petals’ cells and were noted in all examined species. The possibility of fragrance synthesis was suggested by the cell contained large lipid bodies and presence of elongated plastids surrounded by well developed ER (Lange and Turner 2013 , Kowalkowska et al. 2017 ). The lipids in the cytoplasm are considered as the equivalents of fragrance production (Pridgeon and Stern 1983 , Curry et al. 1988 ). Starch accumulation is typical of osmophore cells (Antoń et al. 2012 ) may have been used in mitochondria as a source of energy for the fragrance production. The histochemical studies and ultrathin sections revealed lack of the starch grains inside the cytoplasm. On the other side, numerous plastoglobuli and lipid bodies in the cytoplasm suggest that the starch was utilised before anthesis (Kowalkowska et al. 2015 ). Transport of secreted substances through the plasmalemma is frequently connected with the presence of the ingrowths of the cell wall. The granulocrine secretion is suggested by both the numerous vesicles and relatively large ingrowths of the cell wall. The fusion of the vesicles with plasmalemma is interpreted as granulocrine secretion (Paiva 2016) also found in orchids (Kowalkowska et al. 2012 ). The vesicles close to plasmalemma were found in other orchid genera e.g. Anacamptis (Kowalkowska et al. 2012 ), Bulbophyllum (Kowalkowska et al. 2014), Epipactis (Kowalkowska et al. 2015 ) and Gymnadenia (Stpiczyńska and Matusiewicz 2001 ). The cuticular ruptures were reported as a way to exudation of the substances to the outside of the secretory cells (Curry et al. 1991 ) and was proved also in orchids e.g. Stanhopea oculata (Stern et al., 1987 ), Bulbophyllum (Kowalkowska et al. 2017 ), Platanthera chlorantha (Stpiczyńska 2003 ) as well as in other plants such as Passiflora suberosa (Garcıa and Teresa 2008), Prunus persica (Radice and Galati, 2003 ) or Orbea variegata (Płachno 2010). The epidermal disruptions found in all specimens from L. calodictyon group seems to support the possible granulocrine secretion. On the other side no residues of the secreted substances was found on the surface of the cells. This could be explained by the periodical production and release of the fragrances without accumulation on the surface of the cells (Vogel 1990 ). The fragrance volatilization was described previously in other orchids (Vogel 1990 , Curry et al. 1988 , Stpiczyńska 1993 , 2001 ). Papillae without starch grains, with lipid bodies and plastoglobuli and no traces of secreted substances as it is found in representatives of L. calodictyon group, were reported previously e.g. B. saltatorium. Furthermore, the anatomy of the papillae in both cases differs from elaiophore, frequently reported in orchids (Pacek et al. 2012 ; Blanco et al. 2013). Conclusions Morphological and anatomical features of the papillae are similar to those observed in different orchid taxa. These structures were identified as osmophores. Additionally, papillae of the Lepanthes calodictyon group, were rich in lipids and proteins, what was correlated with high metabolic activity. Due to the similarities in structures we suspect that the papillae on the surface of the lip and petals of examined taxa could emit the fragrance. Abbreviations CBB Coomasie Brilliant Blue DAPI 2–[4–(Aminoiminomethyl)phenyl]–1 H –Indole–6–carboximidamide hydrochloride LM light microscopy PAS periodic acid–Schiff reaction PBS phosphate buffered saline PFA paraformaldehyde RER rough endoplasmic reticulum SBB Sudan Black B SEM scanning electron microscopy SER smooth endoplasmic reticulum TEM transmission electron microscopy UGDA Herbarium Universitatis Gedanensis, Wita Stwosza 59, Gdansk Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and materials The original microphotographs used and/or analysed during the current study are available from the corresponding author on reasonable request. Reference plant material has been deposited in UGDA Herbarium. Competing interests The authors declare that they have no competing interests Funding This study received support from the SYNTHESYS Project (http://www.synthesys.info/), which is financed by the European Community Research Infrastructure Action within the framework of the FP6 ‘Structuring the European Research Area’ Programme GB-TAF-6914 and NL-TAF-6915. Authors’ contributions MR and MK identified research problem and designed the experiments. MK, MN, EB and MR prepared plant material for analyses. NM and DŁ performed electron microscopy analyses. MK and ED performed plant histochemistry analyses. MR, KM and MN interpreted the data and compared results with available literature. MR obtained funds for research. MR, MK and DLSz prepared draft of the manuscript. All authors contributed in and reviewed final manuscript. Acknowledgments Not applicable. Authors' information Brzezicka Emilia ORCID 0000-0002-3619-1238 Kapusta Małgorzata ORCID 0000-0003-4103-594X Narajczyk Magdalena ORCID 0000-0001-9806-8844 Rykaczewski Max ORCID 0000-0003-3457-9690 Szlachetko Dariusz L. ORCID 0000-0002-3210-7537 References Albores Ortiz O, Sosa V.Polinización de dos especies simpátricas de Stelis (Pleurothallidinae, Orchidaceae). Acta Bot Mex. 2014;74:155-575. Antoń S, Kamińska M, Stpiczyńska M (2012) Comparative structure of the osmophores in the flower of Stanhopea graveolens Lindley and Cycnoches chlorochilon Klotzsch (Orchidaceae). Acta Agrobot. 2012; 65: 11–22. Ascensão L, Francisco A, Cotrim H, Pais MS. Comparative structure of the labellum in Ophrys fusca and O. lutea (Orchidaceae). Am J Bot.; 92: 1059–1067. Barbosa AR, de Melo MC, Borba EL. Self-incompatibility and myophily in Octomeria (Orchidaceae, Pleurothallidinae) species. Plant Syst Evol. 2009; 283:1-8. Blanco MA, Barboza G. Pseudocopulatory pollination in Lepanthes (Pleurothallidinae, Orchidaceae) by fungus gnats. Ann Bot-London. 2005; 95:763-772. Borba EL, Barbosa AR, de Melo MC, Gontijo SL, de Oliveira HO. Mating systems in Pleurothallidinae (Orchidaceae): Evolutionary and systematic implications. Lankesteriana. 2011;11(3): 207-221. Borba EL, Semir J. Pollinator specificity and convergence in fly-pollinated Pleurothallis (Orchidaceae) species: a multiple population approach. Ann Bot-London. 2001; 88:75-88. Bronner R (1975) Simultaneous demonstration of lipid and starch in plant tissues. Stain Technol. 1975; 50(1):1–4. Chase MW. Pollination of Pleurothallis endotrachys . American Society Bulletin 1985; 54:431-434. van der Cingel NA. An Atlas of Orchid Pollination: European Orchids: Orchids of America, Africa, Asia and Australia. CRC Press, Rotterdam; 2001. Ciotek L, Giorgis P, Benitez-Vieyra S, Cocucci AA. First confirmed case of pseudocopulation in terrestrial orchids of South America: pollination of Geoblasta pennicillata (Orchidaceae) by Campsomeris bistrimacula (Hymenoptera, Scoliidae). Flora. 2006; 201:365-369. Cozzolino S, Widmer A. Orchid diversity: an evolutionary consequence of deception? Trends Ecol Evol. 2005; 20:487–494. Curry KJ, Stern WL, McDowell LM. Osmophore development in Stanhopea anfracta and S. pulla (Orchidaceae). Lindleyana. 1988; 3:212. Curry KJ, McDowell LM, Judd WS, Stern WL. Osmophores, floral features, and systematics of Stanhopea (Orchidaceae). Am J Bot. 1991; 78:610. Davies KL, Stpiczyńska M. Comparative floral micromorphology and the ultrastructural basis of fragrance production in pseudocopulatory Mormolyca s.s. and non-pseudocopulatory Maxillaria section Rufescens s.s. (Orchidaceae). Bot. J. Linn. Soc. 2017; 185:81-112. Dod DD. Afidos y trípidos polinizan orquídeas en las Pleurothallidinae (Orchidaceae). Moscosoa. 1986;4:200–202. Dressler RL. The Orchids. Natural History and Classification. Washington: Smithsonian Institution; 1981. Duque O. Polinización de Pleurothallis . Orquideología. 1993; 19:55-69. Dressler RL. Phylogeny and Classification of Orchid Family. Cambridge University Press; 1993 Duque-Buitrago CA, Alzate-Quitntero NF, Otero JT. Nocturnal pollination by fungus gnats of the Colombian endemic species Pleurothallis marthae (Orchidaceae: Pleurothallidinae) Lankesteriana. 2014; 13:407-417. Ellis GA, Johnson SD. Floral mimicry enhances pollen export: The Evolution of pollination by sexual deceit outside of the Orchidaceae. Am Nat. 2010; 276(5):143-151. Endara L, Grimaldi DA, Roy BA. Lord of the flies: pollination of Dracula orchids. Lankesteriana. 2010; 10:1-11. Feder N, O’Brien TP. Plant microtechnique: some principles and new methods. American Journal of Botany. 1968; 55: 123–142. Gahan PB. Plant histochemistry and cytochemistry: an introduction. Academic Press, London; 1984. Garcia A, Teresa M. Breeding system and related floral features under natural and experimental conditions of Passiflora suberosa (Passifloraceae). Bol. Soc. Argent. Bot. 2008; 43 (1-2): 83 – 93. Givnish TJ et al. Orchid phylogenomics and multiple drivers of their extraordinary diversification. Proc. R. Soc. B. 2015; 282:20151553. Higgins W. Pleurothallidinae: How Many Genera? 19th World Orchid Conference; 2009. Hu S, Xu L. A Cytochemical Technique for Demonstration of Lipids, Polysaccharides and Protein Bodies in Thick Resin Sections. J Integr Plant Biol. 1990; 32. Hülskamp M, Schwab B, Grini P, Schwarz H. Transmission Electron Microscopy (TEM) of Plant Tissues. Cold Spring Harbor Laboratory Press 7 2010; doi:10.1101/pdb.prot495 Jensen W. Botanical histochemistry. Freeman, San Francisco; 1962. Jersáková J, Johnson SD, Kindlman P. Mechanisms and evolution of deceptive pollination in orchids. Biol Rev. 2006;81:219-235. Jost L, Endara L. Analysis of the DNA-Based Reclassification of the Pleurothallidinae. 2004. http://www.loujost.com/DNAFrameset/DNAanalysisDocument.htm. Accessed 30 April 2025. Karremans AP. Genera Pleurothallidinarum: An updated phylogenetic overview of Pleurothallidinae. Lankesteriana 2016;16(2):219-241. Karremans AP, Pupulin F, Grimaldi D, Beentjes K, Butot R, Fazzi GE, Kaspers K, Kruizinga J, Roessingh P, Smets EF, Gravendeel B. Pollination of Specklinia by nectar-feeding Drosophila : The first reported case of a deceptive syndrome employing aggregation pheromones in Orchidaceae. Ann Bot-London. 2015; 116(3):437-455. Kowalkowska AK, Kozieradzka-Kiszkurno M, Turzyński S. Morphological, histological and ultrastructural features of osmophores and nectary of Bulbophyllum wendlandianum (Kraenzl.) Dammer (B. section Cirrhopetalum Lindl., Bulbophyllinae Schltr., Orchidaceae) Plant Syst Evol. 2015; 301:609–622. Kowalkowska AK, Turzyński S, Kozieradzka-Kiszkurno M, Wiśniewska N. Floral structure of two species of Bulbophyllum section Cirrhopetalum Lindl.: B. weberi Ames and B. cumingii (Lindl.) Rchb. f. (Bulbophyllinae Schltr., Orchidaceae) Protoplasma. 2017; 254(3):1431–1449. Kowalkowska AK, Margońska HB, Kozieradzka-Kiszkurno M. Comparative anatomy of the lip spur and additional lateral sepal spur in a three-spurred form (f. fumeauxiana ) of Anacamptis pyramidalis . Acta Biol Cracov Ser Bot. 2010; 52:13–18. Kowalkowska AK, Margońska HB, Kozieradzka-Kiszkurno M, Bohdanowicz J. Studies on the ultrastructure of a three-spurred fumeauxiana form of Anacamptis pyramidalis. Plant Syst Evol. 2012; 298:1025-1035. Krawczyk E, Rojek J, Kowalkowska AK, Kapusta M, Znaniecka J, Minasiewicz J. Evidence for mixed sexual and asexual reproduction in the rare European mycoheterotrophic orchid Epipogium aphyllum, Orchidaceae (ghost orchid). Ann Bot. 2016; 118:159–172. Lange BM, Turner GW. Terpenoid biosynthesis in trichomes current status and future opportunities. Plant Biotechnol J. 2013: 11:2–22. Luer CA. Icones Pleurothallidinarum I. Systematics of the Pleurothallidinae (Orchidaceae). Monog Syst Botan, St. Louis. 15:1-81; 1986. Luer CA. Icones Pleurothallidinarum XIV. Systematics of Draconanthes, Lepanthes subgenus Marsipanthes and subgenus Lepanthes from Ecuador (Orchidaceae). Monog Syst Botan, St. Louis. 61:1-255; 1996. Luer CA, Thoerle L. Icones Pleurothallidinarum Vol XXXII. Lepanthes of Colombia. Monog Syst Botan, St. Louis. 123:1-300; 2012. de Melo MC, Borba EL, Paiva EAS. Morphological and histological characterization of the osmophores and nectaries of four species of Acianthera (Orchidaceae: Pleurothallidinae) Plant Syst Evol. 2010; 286:141-151. O’Brien TP, Feder N, McCully ME. Polychromatic staining of plant cell walls by toluidine blue O. Protoplasma. 1965; 59: 368–373. O’Brien TP, McCully ME. The study of plant structure: principles and selected methods. Melbourne: Termarcarphi Pty. Ltd; 1981. Pacek A, Stpiczyńska M, Davies KL, Szymczak G. Floral elaiophore structure in four representatives of the Ornithocephalus clade (Orchidaceae: Oncidiinae). Annals of Botany. 2012; 110:809-820. Pais MS, Figueiredo ACS. Floral nectaries from Limodorum abortivum (L.) Sw. and Epipactis atropurpurea rafin. (Orchidaceae); ultrastructural changes in plastids during the secretory process. Apidologie. 1994; 25:615–626. van der Pijl L, Dodson CH. Orchid flowers: their pollination and evolution. Coral Gables University of Miami Press; 1966. Płachno BJ, Świątek P, Szymczak G. Can a stench be beautiful?—osmophores in stem-succulent stapeliads (Apocynaceae-Asclepiadoideae-Ceropegieae-Stapeliinae) Flora. 2010;205:101–105. Płachno BJ, Stpiczyńska M, Świątek P, Davies KL. Floral micromorphology of the Australian carnivorous bladderwort Utricularia dunlopii, a putative pseudocopulatory species. Protoplasma. 2016; 253:1463–1473. Pridgeon AM. Pleurothallidinae In: Pridgeon AM, Cribb P, Chase MW, Rasmussen P eds. Genera Orchidacearum Volume 4 –Epidendroidae (Part One). Oxford: Oxford University Press; 2005. p. 319-422. Pridgeon AM, Solano R, Chase MW. Phylogenetic relationships in Pleurothallidinae (Orchidaceae): combined evidence from nuclear and plastid DNA sequences. Am J Bot. 2001; 88:2286-2308. Pridgeon AM, Stern WL. Ultrastructure of Osmophores in Restrepia (Orchidaceae). Am J Bot. 1983; 70(8):1233-1243. Pridgeon AM, Stern WL. Osmophores of Scaphosepalum (Orchidaceae). Bot Gaz. 1985; 146(1):115-123. Radice S, Galati B. Floral nectary ultrastructure of Prunus persica (L.) Batch cv. Forastero (Newcomer), an Argentine peach. Plant Syst Evol. 2003; 238 , 23–32. Salazar-Cháves GA, Soto-Arenas MA. El género Lepanthes Sw. en México. Orquídea (México). 1996; 14:1-231. Scopece G, Cozzolino S, Johnson SD, Schiestl FP. Pollination efficiency and the evolution of specialized deceptive pollination systems. Am Nat. 2010;175:98-105. Servettaz O, Maleci LB, Grünanger P. Labellum micromorphology in the Ophrys bertolonii agg. and some related taxa (Orchidaceae). Plant Syst Evol. 1994; 189: 123–131. Singer RB. The Pollination Mechanism in Trigonidium obtusum Lindl (Orchidaceae: Maxillariinae): Sexual Mimicry and Trap flowers. Ann Bot. 2002; 89:157-163. Smithson A. Pollinator limitation and inbreeding depression in orchid species with and without nectar rewards. New Phytol. 2006; 169:419-430. Stern WL, Curry KJ, Pridgeon AM. Osmophores of Stanhopea (Orchidaceae) Am J Bot. 1987;74:1323–1331. Stpiczyńska M. Anatomy and ultrastructure of osmophores of Cymbidium tracyanum Rolfe (Orchidaceae). Acta Soc Bot Pol. 1993; 62:5-9. Stpiczyńska M. The structure of nectary of Platanthera bifolia L. Orchidaceae. Acta Soc Bot Pol. 1997; 1:5–7. Stpiczyńska M. Osmophores of the fragrant orchid Gymnadenia conopsea L. (Orchidaceae). Acta Soc Bot Pol. 2001; 70(2):91–96. Stpiczyńska M. Nectar resorption in the spur of Platanthera chlorantha (Custer) Rchb. Orchidaceae structural and microautoradiographic study. Plant Syst Evol 2003; 238:119–12. Stpiczyńska M, Matusiewicz J. Anatomy and ultrastructure of spur nectary of Gymnadenia conopsea (L.) Orchidaceae. Acta Soc Bot Pol 2001; 4:267–272. Stpiczyńska M, Davies KL, Gregg A. Comparative account of nectary structure in Hexisea imbricata (Lindl.) Rchb.f. (Orchidaceae). Ann Bot. 2005; 95(5):749–756. Teixeira SP Borba EL, Semir J. Lip anatomy and its implications for pollination mechanism of Bulbophyllum species (Orchidaceae). Ann Bot-London. 2004; 93:499-505. Tremblay RL & Ackerman JD. A new species of Lepanthes (Orchidaceae) from Puerto Rico. Brittonia. 1993; 45:339−342. Tremblay RL, Ackerman JD, Zimmerman JK, Calvo RN. Variation in sexual reproduction in orchids and its evolutionary consequences: a journey to diversification. Biol J Linn Soc. 2005; 84:1-54. Vogel S, Renner S. The role of scent glands in pollination: On the structure and function of osmophores. Washington, D.C: Smithsonian Institution Libraries and National Science Foundation; 1990. Whitten M, Blanco M (2011) Defining generic limits in Maxillaria: a return to the orchidaceous mine. Orchids 80:104–113. Wiemer AP, More M, Benitez-Vieyra S, Cocucci AA, Raguso RA, Sersic AN. A simple floral fragrance and unusual osmophore structure in Cyclopogon elatus (Orchidaceae). Plant Biol (Stuttg). 2008; 11(4):506–514. Wiśniewska N, Kowalkowska A, Kozieradzka-Kiszkurno M, Krawczyńska A, Bohdanowicz J. Floral features of two species of Bulbophyllum section Lepidorhiza Schltr.: B. levanae Ames and B. nymphopolitanum Kraenzl. (Bulbophyllinae Schltr., Orchidaceae). Protoplasma. 2018; 255:485–499. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 30 Sep, 2025 Read the published version in BMC Plant Biology → Version 1 posted Editorial decision: Revision requested 26 Jun, 2025 Reviews received at journal 25 Jun, 2025 Reviews received at journal 23 Jun, 2025 Reviewers agreed at journal 29 May, 2025 Reviewers agreed at journal 26 May, 2025 Reviewers invited by journal 23 May, 2025 Editor assigned by journal 23 May, 2025 Editor invited by journal 22 May, 2025 Submission checks completed at journal 20 May, 2025 First submitted to journal 20 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Szlachetko","email":"","orcid":"","institution":"University of Gdańsk","correspondingAuthor":false,"prefix":"","firstName":"Dariusz","middleName":"L.","lastName":"Szlachetko","suffix":""}],"badges":[],"createdAt":"2025-05-05 13:38:32","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6594964/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6594964/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12870-025-07141-1","type":"published","date":"2025-09-30T15:56:58+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":83611895,"identity":"a32fb3e7-944c-4b9f-b827-c27f35c9a0bb","added_by":"auto","created_at":"2025-05-29 12:27:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":29789943,"visible":true,"origin":"","legend":"\u003cp\u003eLepanthes calodictyon. a flower overview; SEM results: b – c lip, visible papillae, d – e petal, visible papillae; results of neutral red (g and j) and methylene blue (h and k) staining of labellar (f – h) and petal’s papillae (i – k) with comparison to not stained ones (f and i). Bars: b – 100 µm; c – 10 µm; d – 200 nm, e – 20 nm; f-k – 50 µm\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6594964/v1/511fd691369c64b365313712.png"},{"id":83610999,"identity":"55798b0c-4138-485f-b48f-04c88b485362","added_by":"auto","created_at":"2025-05-29 12:19:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":28509556,"visible":true,"origin":"","legend":"\u003cp\u003eLepanthes saltatrix. a flower overview; SEM results: b – c lip, visible papillae, d – e petal, visible papillae; results of neutral red (g and j) and methylene blue (h and k) staining of labellar (f – h) and petal’s papillae (i - k) with comparison to not stained ones (f and i). Bars: b – 500 µm; c – 50 nm; d – 50 nm, e – 20 nm; f-k – 50 µm\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6594964/v1/d65725c73ac4af7bf61571f5.png"},{"id":83611001,"identity":"2fdf1d20-bdcd-4cc6-afb5-fb802b0a71c1","added_by":"auto","created_at":"2025-05-29 12:19:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":26873919,"visible":true,"origin":"","legend":"\u003cp\u003eLepanthes tentaculata. a flower overview; SEM results: b – c lip, visible papillae, d – e petal, visible papillae; results of neutral red (g and j) and methylene blue (h and k) staining of labellar (f – h) and petal’s papillae (i – k) with comparison to not stained ones (f and i). Bars: b – 200 µm; c – 20 µm; d – 500 nm, e – 50 nm; f-k – 50 µm\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6594964/v1/32d5db1d333b8543fb79172a.png"},{"id":83611894,"identity":"c32b2b12-5582-415a-9c82-ded4f763cbaa","added_by":"auto","created_at":"2025-05-29 12:27:34","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":32915897,"visible":true,"origin":"","legend":"\u003cp\u003eHistochemical features of petals of L. calodictyon (c and d, g, j, l, n), L. saltatrix (a, e and f, h, k, o) and L. tentaculata (b, i, m). a - c Transverse section (TBO), d and e abundant lipid droplets in trichomes (SBB), f and g lack of starch grains, j intact cuticle without visible\u0026nbsp; abruptions (Auramine O), k - m\u0026nbsp; FeCl\u003csub\u003e3\u003c/sub\u003e staining, dihydroxyphenol inclusions only visible in l, n – o cortical hoop like microtubules (red fluorescence), delicate microfilamets network (green fluorescence) and chromatin staining (blue fluorescence) in epidermal cells. Bars: a, b, and c – 100 µm; d, g, j, and k, - 50 µm; e, f, and m – 20 µm; h, l, n, and o – 10 µm\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6594964/v1/58955f0b57f87a777d7a4cb8.png"},{"id":83610998,"identity":"ebb35372-37b0-4f53-a66b-dd7ad809829e","added_by":"auto","created_at":"2025-05-29 12:19:34","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":9079319,"visible":true,"origin":"","legend":"\u003cp\u003eUltrastructural features of L. calodictyon petals (a – c), L. saltatrix (d – f) and L. tentaculata (g – i). Dense cytoplasm of epidermal cells with numerous plastids with plastoglobuli and internal membranes (a – i), profiles of ER (c, h), fully developed dictyosomes (g), lipid droplets (a and b, d, f, h and i). Cuticle with visible abruptions (b, d, f, g) and vesicles connected with plasmalemma (f, asteriks). Some epidermal cells with collapsed proplasts (a, i, arrow). c cuticle, cw cell wall, ER endoplasmic reticulum, l lipid droplets, n nuclei, p plastid, v vesicles, v vacuole. Bars: a – 2 µ; b, c and i – 1 µm; d-h – 500 nm\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6594964/v1/bddc7e5bf8229e56497dba23.png"},{"id":83611003,"identity":"84d0aeda-da5e-42b9-aa5b-b77ab0e08ad7","added_by":"auto","created_at":"2025-05-29 12:19:34","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":27725696,"visible":true,"origin":"","legend":"\u003cp\u003eHistochemical features of lip of L. calodictyon (a, d, f, h, k, n), L. saltatrix\u0026nbsp; (b, i, l, o) and L. tentaculata (c, e, g, j, m). a – c transverse section (TBO), d\u0026nbsp; and e proteins in the epidermal cells (CBB), f and g lack of starch grains in epidermal and subepidermal cells (PAS), h – j lipid droplets in epidermal cells (SBB), k and l smooth and intact cuticle (Auramine O), m lack of dihydroxyphenols inclusions in cytoplasm of epidermal cells (FeCl\u003csub\u003e3\u003c/sub\u003e), n and o dense network of microtubules (red fluorescence) and microfilaments (green fluorescence) chromatin of nuclei of epidermal cells (blue staining). Bars: a, b, and c – 100 µm; d, e, f, h, i, j, and k, - 50 µm; l – 20 µm; g, m, n, and o – 10 µm\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6594964/v1/59a22caf58483cda37bfb884.png"},{"id":83611893,"identity":"a0f1fcfe-6257-4f52-8832-0b433c445b19","added_by":"auto","created_at":"2025-05-29 12:27:34","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3987387,"visible":true,"origin":"","legend":"\u003cp\u003eUltrastructural features of L. calodictyon lip. Some of vesicles connected with plasmalemma (a and b, black arrow). Cuticle is smooth without abruptions (a – d). In peripheral dense cytoplasm visible abundant ER (a – c), accompanied with plastids with numerous plastoglobuli and internal membranes (a – d), lipid droplets (a, d), vesicles (b, d) while the centre is occupied with large vacuole (a – d). c cuticle, cw cell wall, ER endoplasmic reticulum, l lipid droplets, p plastid, v vesicles, v vacuole. Bars: 500 nm\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6594964/v1/6b44245bb0560986ccf0f7f5.png"},{"id":83610996,"identity":"6d8ea348-5cd3-4e3d-a21c-0dc91ea26760","added_by":"auto","created_at":"2025-05-29 12:19:34","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":4219945,"visible":true,"origin":"","legend":"\u003cp\u003eUltrastructural features of L. saltatrix lip. Dense, peripheral cytoplasm of trichomes with abundant SER and vesicles (a – d), lipid droplets (b – d) and plastids with plastoglobuli and internal membranes (b, d). Note the invaginations of plasmalemma (a – c). Cuticle lacking abruptions (a – d). c cuticle, cw cell wall, SER smooth endoplasmic reticulum, l lipid droplets, p plastid, v vesicles, v vacuole. Bars: a and c - 1µm; b and d – 500 nm\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6594964/v1/4a5977c1e8036da900feb286.png"},{"id":83610995,"identity":"0ddcf463-da0b-446c-8d44-07796d1287ca","added_by":"auto","created_at":"2025-05-29 12:19:34","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":4145123,"visible":true,"origin":"","legend":"\u003cp\u003eUltrastructural features of L. tenatculata lip. Epidermal cells with numerous SER, vesicles, well developed dictyosomes and mitochondria (a – d). Plastids with plastoglobuli (a). Vesicles building into plasmalemma (b – d) and some lipid droplets (c) observed in dense cytoplasm. Cuticle with visible abruptions (b). c cuticle, cw cell wall, d dictyosomes, ER endoplasmic reticulum, l lipid droplets, m mitochondria, p plastid, v vesicles, v vacuole.\u003c/p\u003e\n\u003cp\u003eBars: a, c, and d – 500 nm; b – 200 nm.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-6594964/v1/5b14c367b2078176a056eb16.png"},{"id":92883901,"identity":"56b93480-9efd-4d67-8bb6-99236ef10c02","added_by":"auto","created_at":"2025-10-06 16:10:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":156151813,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6594964/v1/673b5360-ee1d-4c3e-ba71-e6d662b91973.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Floral anatomy and ultrastructure in Lepanthes calodictyon group","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe subtribe Pleurothallidinae Lindl. is one of the richest and the most diverse group among Orchidaceae. It comprises ca. 40 well-recognized genera with over 5100 species (Luer \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Jost \u0026amp; Endara \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Pridgeon \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Pridgeon et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Higgins \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Luer \u0026amp; Thoerle 2012; Karremans \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Representatives of Pleurothallidinae are usually small, mainly epiphytic, Neotropical orchids with sympodial growth habit and occur predominantly at elevation above 1000 m in the humid rainforest or the p\u0026aacute;ramo formation (Luer \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1986\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Salazar \u0026amp; Soto 1996; Pridgeon \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Luer \u0026amp; Thoerle 2012). The species of Pleurothallidinae are considered together with unrelated \u003cem\u003eBulbophyllum\u003c/em\u003e genus as fly-pollinated orchids (Dressler \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Cozzolino \u0026amp; Widmer \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Borba et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; de Melo et al. 2011). The representatives of Pleurothallidinae subtribe exhibit a large diversity of morphological characters associated with myophily. They usually have small brightly coloured flowers. The surface is frequently covered by papillae, motile hairs or trichomes, which attract the pollinators. The nectary production or fragrance emission are regarded as factors increasing pollination effectiveness. However, among ca. 40 recognized and acknowledged genera, only few of them were investigated as far: \u003cem\u003eAcianthera\u003c/em\u003e Scheidw. (Borba \u0026amp; Semir \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; de Melo et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), \u003cem\u003eAnathallis\u003c/em\u003e Barb. Rodr. (Borba \u0026amp; Semir \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), \u003cem\u003eDracula\u003c/em\u003e Luer (Endara et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), \u003cem\u003eLepanthes\u003c/em\u003e (Blanco \u0026amp; Barboza \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), \u003cem\u003eMasdevallia\u003c/em\u003e Ruiz \u0026amp; Pav. (Borba \u0026amp; Semir \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), \u003cem\u003eOctomeria\u003c/em\u003e R.Br. (Barbosa et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), \u003cem\u003ePleurothallis\u003c/em\u003e R.Br. (Duque \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1993\u003c/span\u003e, Duque-Buitrago et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), \u003cem\u003eRestrepia\u003c/em\u003e Kunth, (Pridgeon \u0026amp; Stern \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e1983\u003c/span\u003e), \u003cem\u003eScaphosepalum\u003c/em\u003e Pfitzer (Pridgeon \u0026amp; Stern \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e1985\u003c/span\u003e), \u003cem\u003eSpecklinia\u003c/em\u003e Lindl. (Chase \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1985\u003c/span\u003e; Karremans et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), \u003cem\u003eStelis\u003c/em\u003e Sw. (Albores \u0026amp; Sosa 2006) and \u003cem\u003eZootrophion\u003c/em\u003e (Borba \u0026amp; Semir \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), nectary production was proved once (Karremans et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), and the most common the presence of the variable osmophores were detected.\u003c/p\u003e \u003cp\u003eAmong all Pleurothallidinae, \u003cem\u003eLepanthes\u003c/em\u003e is the most richest in species genus (Karremans \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). It comprises approximately 1100 species distributed from southern part of Florida, through Central America to Bolivia in the south. The greatest diversity is observed in Ecuador and Colombia (Luer \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1986\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Salazar \u0026amp; Soto 1996; Pridgeon \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Luer \u0026amp; Thoerle 2012). Species belonging to the \u003cem\u003eLepanthes\u003c/em\u003e genus are characterized by the presence of the dilated and ciliate sheaths of the ramicaul, the small, often yellow flowers with petals broader than long. Usually they have trilobed lip with the midlobe reduced to a minute appendix, the blades of the lip are above the column which is elongated and embraced by the lip. Although the appendix vary in form, it is most often covered by motile trichomes or hairs and typically founded pressed beneath to the column (Luer \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1986\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Salazar \u0026amp; Soto 1996; Pridgeon \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Luer \u0026amp; Thoerle 2012). Moreover, flowers are scentless, not offering any reward for pollinators, such as nectar or pseudonectar (e.g. Salazar \u0026amp; Soto 1996).\u003c/p\u003e \u003cp\u003eThe sexual deception was considered by many authors as a pollination syndrome in \u003cem\u003eLepanthes\u003c/em\u003e (Dressler \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Tremblay \u0026amp; Ackerman \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Luer \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Salazar \u0026amp; Soto 1996; Pridgeon \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Tremblay et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). The first observation of male flies mating with \u003cem\u003eLepanthes\u003c/em\u003e was recorded in Costa Rica by Blanco and Barboza (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). They photographed and described males of \u003cem\u003eBradysia floribunda\u003c/em\u003e (Sciaridae) attached by their sexual organs with appendix of the lip of \u003cem\u003eL. glicensteinii\u003c/em\u003e Luer. The male flies of the \u003cem\u003eBradysia floribunda\u003c/em\u003e visited the flowers of \u003cem\u003eL. glicensteinii\u003c/em\u003e exclusively, which suggests very specific plant-pollinator interactions. Moreover, Blanco and Barboza (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) distinguished the three crucial steps in the pollinator behaviour. First step was reception of the \u0026ldquo;olfactory\u0026rdquo; signal \u0026ndash; flies hover by few seconds around the plant and after landing, they tried to detect the scent produced by the flower. Second step \u0026ndash; if flower was already open, the fungus gnats looked for the female and started their mating dance. The last step was reception of the sensory signal. Only after the securing the appendix of the lip, flies started to stake out in the right position and start to \u0026ldquo;copulate\u0026rdquo; with the flowers. During the pseudocopulation, pollinia were removed from the column and attached to the abdomen of the fungus gnats. Furthermore, the spermatophore was deposited on the ventral side of the column, near the appendix. Authors also reported that the \u003cem\u003eBradysia floribunda\u003c/em\u003e males pollinated the flowers (Blanco \u0026amp; Barboza \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe three species, \u003cem\u003eLepanthes calodictyon\u003c/em\u003e, \u003cem\u003eL. tentaculata\u003c/em\u003e and \u003cem\u003eL. saltatrix\u003c/em\u003e are distinctive in both floral and vegetative if compared to other \u003cem\u003eLepanthes\u003c/em\u003e representatives were investigated anatomically here in detail. The species of this group are characterized by the simultaneous presence of: the reticulated leaves with undulate margins, the short and dense inflorescence on the upper side of the leaf, the bilobed petals contracted in to slender tails at the apex, unlobed or slightly bilobed lip without appendix, the lip hinged to the column by the claw. The petals and lip are bright deep red or crimson-cream, papillate in contrast to the inconspicuous, similar in shape, glabrous, pale yellow-green sepals. Moreover, the sepals embrace the straight ovary, which made the central part of the flower elevated and probably, more easily noticeable for pollinators.\u003c/p\u003e \u003cp\u003eIt is well known in Orchidaceae, that vivid colours, and various appendages on the surface of the floral segments are frequently connected with the secretory activity. We suspect, that the contrastive to pale sepals, red colouration of the petals and the lip, could implicate the crucial role of these floral segments in the attraction of the pollinators. This hypothesis about very particular role of the central parts of the flower seems to be legitimate, especially if consider the lack of the appendix of the lip. Due to that, the aim of this study were to investigate the morphological, histochemical and ultrastructural features of petals and lip as well as to look for the evidences to the secretory activity of their cells using light, fluorescent and electron microscopy methods.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant source\u003c/h2\u003e \u003cp\u003eSpecimens of \u003cem\u003eLepanthes calodictyon\u003c/em\u003e, \u003cem\u003eL. saltatrix\u003c/em\u003e and \u003cem\u003eL. tentaculata\u003c/em\u003e were purchased from Ecuagenera Ltd. (Ecuador). Flowers were collected at anthesis from March 2016 to September 2017. The flowers of \u003cem\u003eL. calodictyon, L. saltatrix\u003c/em\u003e and \u003cem\u003eL. tentaculata\u003c/em\u003e (voucher numbers: UGDA 012573, 012605 and 012614 respectively) were preserved in the KEW mixture (53% ethanol, 37% water, 5% formaldehyde and 5% glycerol) and deposited in the herbarium.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eLight microscopy\u003c/h3\u003e\n\u003cp\u003ePlant material were examined and measured using a SteREO Discovery V12 Zeiss stereomicroscope with the magnification range from 5\u0026times; to 63\u0026times; and an ocular graticule 10\u0026times;/2.3. All measurements were taken according to the central axis of structures. The photographs of the flowers were made by Zeiss Axiocam 512 and Nikon SMZ 1500. The preliminary delimination of secretory active parts of the flower were performed according to Wiśniewska et al. (\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and comprise staining with in 0.01% solution of methylene blue or in 0.1% neutral red.\u003c/p\u003e \u003cp\u003eWhole flowers and/or parts were used for light microscopy analyses and were preserved in the dark at 4\u0026ordm;C in the phosphate buffered saline (PBS, pH 5.7) containing 2.5% glutaraldehyde (GA) and 4% paraformaldehyde (PFA). After 24h samples were rinsed two times in PBS following by distilled water, dissected, dehydrated by increasing concentration of the ethanol (H\u0026uuml;lskamp et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) and embedded in Steedman\u0026rsquo;s Wax (Krawczyk et al \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The histochemical tests were performed on 10 \u0026micro;m sections made on HM 360 Microm and comprised: toluidine O for general histology (Federer and O\u0026rsquo;Brien 1968) Coomasie Brilliant Blue (CBB) for protein detection (Hu and Xu \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1990\u003c/span\u003e, modified), Sudan Black B for lipids (Bronner \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1975\u003c/span\u003e) and the periodic acid-Schiff reaction (PAS) which was used for detection of total insoluble polysaccharides (Jensen \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1962\u003c/span\u003e). Catechol-type dixydroxyphenol inclusions were visualized by 10% (w/v) aqueous solution of FeCl\u003csub\u003e3\u003c/sub\u003e (Gahan \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1984\u003c/span\u003e). Microtubules were visualized using rat primary antibody against α-tubulin (Ab6161; Abcam, UK) and a goat anti-rat secondary antibody conjugated with DyLight\u0026trade; 549 (AS12 2084; Thermo Fisher Scientific). F-actin was detected using monoclonal mouse antibody against actin (MP Biomedicals) and goat anti-mouse secondary antibody conjugated with Alexa Fluor 488 (Life Technologies). Both, primary and secondary antibodies were used in 1:800 dilutions. The chromatin of the nuclei was stained with 7 \u0026micro;l/ml DAPI (Sigma-Aldrich). In negative control experiments, the primary, secondary or both antibodies were omitted. All light microscopy sections were mounted in Mowiol medium and viewed with Leica DM6000 B or Nikon Eclipse E 800.\u003c/p\u003e\n\u003ch3\u003eElectron microscopy\u003c/h3\u003e\n\u003cp\u003eSamples for scanning electron microscopy (SEM) were fixed overnight at room temperature in the KEW mixture, dehydrated with increasing concentration of the ethanol then coated with gold and were observed on Philips XL-30 scanning electron microscope. Sample preparation for transmission electron microscopy (TEM) analyses, was performed by standard electron microscopy procedure (Kowalkowska et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) followed by post-fixation overnight in 1% OsO\u003csub\u003e4\u003c/sub\u003e. Plant material was embedded in Spurr\u0026rsquo;s resin, cut to ultrathin sections and examined on FEI Tecnai Spirit BioTWIN transmission electron microscope.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eAmong all three species (Figs. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea) the all flower segments except lip and petals were similar in shape. The flowers do not produce any noticeable scent and were born on the congested inflorescence on the top of the reticulated leaves. The data about histochemistry and features of the flowers are summarized in the Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eThe results of micromorphological, ultrastructural and histochemical analyses of \u003cem\u003eLepanthes calodictyon, L. saltatrix\u003c/em\u003e and \u003cem\u003eL. tentaculata\u003c/em\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"10\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eSpecies/method\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e\u003cem\u003eLepanthes calodictyon\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e\u003cem\u003eLepanthes saltatrix\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e\u003cem\u003eLepanthes tentaculata\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003esepal\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003epetal\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003elip\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003esepal\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003epetal\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003elip\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003esepal\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003epetal\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003elip\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOsmophores\u003c/p\u003e\n \u003cp\u003eLM: methylene blue, neutral red\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSecretions\u003c/p\u003e\n \u003cp\u003eSM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eProteins\u003c/p\u003e\n \u003cp\u003eLM: CBB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+/+++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+++\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eStarch grains\u003c/p\u003e\n \u003cp\u003eLM: PAS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDihydroxyphenols\u003c/p\u003e\n \u003cp\u003eIn plastids/vacuoles\u003c/p\u003e\n \u003cp\u003eLM: FeCl\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLipids\u003c/p\u003e\n \u003cp\u003eLM: SBB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e++\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSecretions\u003c/p\u003e\n \u003cp\u003eTEM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+/++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e++\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCuticle\u003c/p\u003e\n \u003cp\u003eLM: auramin 0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003es\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003es\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003es\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003es\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003es\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003es\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCytoskeleton\u003c/p\u003e\n \u003cp\u003e(\u0026alpha;-tubulin\u003c/p\u003e\n \u003cp\u003eand F-actin)\u003c/p\u003e\n \u003cp\u003eLM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e++\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e+++ - abundant; ++ - rich; + - present; - - absent; nt- not tested; s- smooth.\u003c/p\u003e\n\u003ch3\u003eSepals\u003c/h3\u003e\n\u003cp\u003eThe sepals were translucent and yellow to yellow green in colour (Figs. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea). SEM studies revealed the surface of dorsal sepal of \u003cem\u003eLepanthes calodictyon\u003c/em\u003e was built by regular epidermal cells with smooth cuticle. That kind of cells were covered evenly whole area. The presence of cubical secretions was observed. On the edge of sepal\u0026rsquo;s apical part epidermal cells were formed unicellular trichomes. They were covered by the smooth cuticle and few cubical particle. Similar to dorsal sepal the epidermal cells, covering whole surface of both lateral sepal, were regular. Whole area was covered by the smooth cuticle and particle of secretions. Only on the part under the outer edge the epidermal two-celled trichomes were notice. Many cubicle part of secretion was noticed on trichomes and the surface around them. Almost whole length of outer edge of sepals was built by unicellular trichomes with smooth cuticle, which were covered by few particle secretions.\u003c/p\u003e\n\u003cp\u003eThe surface of both \u003cem\u003eLepanthes saltatrix\u003c/em\u003e dorsal and lateral sepals was built by regular epidermal cells, with smooth cuticle. Only on the edge, especially at the top, small, unicellular trichomes with slightly undulated cuticle were observed. On the surface of dorsal and lateral sepals the presence of secretion was not notice. Convex area of surface, situated along the both sepal\u0026rsquo;s edges were observed. This swellings was covered by regular, epidermal cells with smooth cuticle.\u003c/p\u003e\n\u003cp\u003eThe surface of dorsal and lateral sepals of \u003cem\u003eLepanthes tentaculata\u003c/em\u003e was built by regular epidermal cells, with smooth cuticle. Only the apical part of sepals was deprived of few two-celled trichomes. On the edge, especially at the sepal\u0026rsquo;s top were observed small, unicellular trichomes with smooth cuticle. The presence of secretion was not noticed on the surface of dorsal and lateral sepals.\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003ePetals\u003c/h2\u003e\n \u003cp\u003eThe petals were red in \u003cem\u003eL. calodictyon\u003c/em\u003e and \u003cem\u003eL. tentaculata\u003c/em\u003e or bicoloured in \u003cem\u003eL. saltatrix\u003c/em\u003e - crimson in upper lobe and cream on lower. The petals were bilobed with long, slender tails on both apex (Figs. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea). The lobes of the petals as well as the tails found on the apices of them were covered by unicellular papillae, similar in morphology to these observed on the surface of the lip (Figs. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ed \u0026ndash; e, 4 a \u0026ndash; c). The intense colouration after treatment with neutral red and methylene blue was noticed in each species (Figs. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ef \u0026ndash; k). The cuticle of all examined petals\u0026rsquo; species was consistent when stained by Auramin O, lacking cracks (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ej). Nevertheless ultrastructural studies revealed the small abruptions of the cuticle in case of \u003cem\u003eL. saltatrix\u003c/em\u003e and \u003cem\u003eL. tentaculata\u003c/em\u003e (Figs. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ed, \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ef \u0026ndash; g) when in \u003cem\u003eL. calodictyon\u003c/em\u003e no disruptions of the cuticle were not noticed (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea).\u003c/p\u003e\n \u003cp\u003eThe surface of petals of \u003cem\u003eLepanthes calodictyon\u003c/em\u003e was covered by two kind of cells: regular, isometric epidermal cells and papillae cells (Figs. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ed \u0026ndash; e). Bottle \u0026ndash; shaped, unicellular papillae were formed by epidermal cells on the edge of petal. The cuticle of papillae was smooth without any secretion (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ee). Similar papillae on the surface of the lengthened, thread- shaped petal\u0026rsquo;s part were visible. The middle part was built by regular epidermal cells with smooth cuticle. Petal is composed of two different component: rounded middle part and lengthened, two, thread-like side part.\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eLepanthes saltatrix\u003c/em\u003e The surface of petal\u0026rsquo; middle part was covered by bottle \u0026ndash; shaped, unicellular papillae which filled densely whole area (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ed). Similar papillae cells were visible on the whole surface of side part. Their cuticle was smooth, but any secretion was not observed (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, e).\u003c/p\u003e\n \u003cp\u003ePetal of \u003cem\u003eLepanthes tentaculata\u003c/em\u003e was composed of rounded middle part and lengthened, two, thread-like side part. The surface of petal\u0026rsquo; middle part was covered by papillose cells (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ed. Bottle-shaped, unicellular papillae were filled densely whole area, only on the edge were located lengthened, unicellular trichomes (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ee). Similar papillae cells were visible on the whole surface of side part. As well their cuticle was smooth, but any secretion was not observed (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ee).\u003c/p\u003e\n \u003cp\u003eHistochemical studies revealed that petals\u0026rsquo; papillae of three investigated species were rich in proteins and lipids (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e; Figs. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea \u0026ndash; g). The TEM studies revealed the presence of the features associated with secretory activity and residues of secretion was detected on the surface of the petals\u0026rsquo; papillae of \u003cem\u003eL. calodictyon\u003c/em\u003e and \u003cem\u003eL. saltatrix\u003c/em\u003e, and no secretion was found in \u003cem\u003eL. tentaculata\u003c/em\u003e (Figs. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea \u0026ndash; i). Ultrastructure of the petals\u0026rsquo; papillae vacuole occupied the main part of the cells, the cytoplasm was dense with numerous organelles crowded on the perimeter of the cell (Figs. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea \u0026ndash; i). The small vesicles connected with plasmalemma were also observed between irregular plasmalemma and cell wall (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ef).\u003c/p\u003e\n \u003cp\u003eThe SBB treatment indicated lipids deposited inside the cells especially large amount of lipid bodies was found in \u003cem\u003eLepanthes saltatrix\u0026rsquo;s\u003c/em\u003e trichomes (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ee and Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ed). Lipid bodies are often accompanied with plastids (Figs. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eb, d, h and i). Within plastids numerous osmiophilic plastoglobuli were observed but no starch grains noted (Figs. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea \u0026ndash; i). This observations are comparative for all examined species with the negative results of the PAS reaction for detection of starch grains (Figs. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eh \u0026ndash; i). Few, small dihdroxyphenols inclusions were observed only in cytoplasm of \u003cem\u003eLepanthes calodictyon\u0026rsquo;s\u003c/em\u003e petal (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003el), other staining remained inconclusive (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ek and m). The cytoskeleton of the petals\u0026rsquo; papillae was well developed in all three species, cortical hoop-like microtubules and delicate network of microfilaments were noted (Figs. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003en \u0026ndash; o).\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eLip\u003c/h3\u003e\n\u003cp\u003eThe morphology of the lip was the most diverse feature and vary in shape between the species, however always hinged on the claw and never trilobed, in contrast to the most of the \u003cem\u003eLepanthes\u003c/em\u003e. The surface of the lip of each examined species was covered by unicellular papillae, which were conical with rounded tips (Figs. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea \u0026ndash; c). No ornamentation of the cuticle of the labellar papillae was observed. The only difference between species was presence of longer and shorter form of the papillae on the margin of the \u003cem\u003eLepanthes calodictyon\u003c/em\u003e lip. The intense colouration of the lip was noticed when stained by neutral red and methylene blue solutions.\u003c/p\u003e\n\u003cp\u003eThe whole surface of the lip of \u003cem\u003eLepanthes calodictyon\u003c/em\u003e were covered by unicellular, bottle-shaped papillae, which were formed by epidermal cells. Papillae were covered by the smooth cuticle and presence of any particle of secreted substance were not observed (Figs. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb \u0026ndash; c).Only a few lengthened trichomes on the corner of lip were notice (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb). The cuticle of trichomes were smooth and any secreted substances was not observed.\u003c/p\u003e\n\u003cp\u003eThe ray-like lip of \u003cem\u003eLepanthes saltatrix\u003c/em\u003e was built by regular epidermal cell, forming papillae. Whole area of lip (Figs. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb \u0026ndash; c) was covered by this kind of cells, whereas longer unicellular trichomes on the corner of edges were observed (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb). The cuticle of papillae and trichomes was smooth.\u003c/p\u003e\n\u003cp\u003eThe ray-like lip of \u003cem\u003eLepanthes tentaculata\u003c/em\u003e was built by regular epidermal cell, forming papillae. Whole middle area of lip (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb) was covered by this kind of cells, whereas longer unicellular trichomes on the edges were observed. The cuticle of papillae and trichomes was smooth, however any particle of secretion were not presented (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ec). The cuticle was smooth and intact when stained by Auramin O (Figs. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ek \u0026ndash; l) but unlikely to petals examination, only few small abruption of the cuticle were noticed in \u003cem\u003eLepanthes tentaculata\u003c/em\u003e lip (Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eb, black arrows). The CBB staining showed that the papillae were generally rich in proteins in each species (Figs. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ed \u0026ndash; e), but the longer papillae found on the margin of the \u003cem\u003eLepanthes calodictyon\u003c/em\u003e stained more intensively if compared to shorter papillae (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ed). Treatment with SBB indicated high amount of lipids in all examined species (Figs. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eh \u0026ndash; j). The PAS for starch grains (Figs. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ef \u0026ndash; g) and staining for dihydroxyphenoles were negative (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003em). The TEM studies revealed the presence of the features associated with secretory activity but no traces of secretion was detected on the surface of the papillae. The main part of papillae of the lip was occupied by vacuole and the cellular structures were crowded on the perimeter (Figs. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ec, f and i). The cytoplasm was dense with plenty of rough or smooth endoplasmic reticulum (RER/SER) (Figs. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e), mitochondria with well developed cristae (Figs. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eb and \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003ed), numerous vacuoles and dictyosomes (Figs. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e), plastids with plastoglobuli (Figs. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ea and c \u0026ndash; d, 8d and 9 a). The small vesicles were observed between irregular plasmalemma and cell wall. Some of the vesicles were connected with plasmalemma (Figs. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ea, \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003ea and \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003ec \u0026ndash; d; black arrows).\u003c/p\u003e\n\u003cp\u003eThe cytoskeleton of the lip was well developed in all three species, comparatively to petals\u0026rsquo; trichomes (Figs. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003en \u0026ndash; o).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eFeatures which may be connected with pseudocopulatory are the three dimensional flowers\u0026rsquo; form, contrastive colouration, presence of narrow, unicellular trichomes with frequently pointed tips and in many cases reflective surfaces. Various combinations of these features were frequently reported for diverse plants such like orchids (\u003cem\u003eMormolyca\u003c/em\u003e (Davies and Stpiczyńska 2006), representatives of \u003cem\u003eOphrys\u003c/em\u003e (Servettaz et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Ascensao et al. 2005) and \u003cem\u003eTrigonidium\u003c/em\u003e (Singer \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2002\u003c/span\u003e, Whitten and Blanco \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2011\u003c/span\u003e)) or bladderworts (Płachno et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Moreover, the flowers of all three examined species exhibit the features of fly pollinated orchids such as colours, motile lip hinged to the claw and the presence of the osmophores which lure the pollinators as it is in \u003cem\u003eBulbophyllum\u003c/em\u003e (Kowalkowska et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The investigation of anatomy of the flowers revealed the presence of the osmophores on the surface of the lip and petals, and sepals are rather inactive in secretion process. The cytoplasm of the labellar and petals\u0026rsquo; cells is dense with large amount of organelles like numerous mitochondria, lipid bodies, vacuoles and vesicles, the plastoglobuli are surrounded by well-developed endoplasmatic reticulum and cytoskeleton network. The anatomy of the cells suggest the high metabolic activity of the cell like in osmophoric tissues (Pridgeon and Stern \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e1983\u003c/span\u003e, Stpiczyńska et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2005\u003c/span\u003e, Kowalkowska et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe osmophores are found on the epidermis and subepidermal layers (e.g. Vogel \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Curry et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Stpiczyńska \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Płachno et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Antoń et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The subsecretory parenchyma is characterized by the presence of numerous starch grains and by hydrolysis of polysaccharides they provide energy for secretory activity. In the osmophores, starch grains occur frequently, but not exclusively, together with lipid bodies (Vogel \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e1990\u003c/span\u003e). The osmophores without starch was reported previously for various orchid species e.g. \u003cem\u003eAnacamptis pyramidalis f. fumeuxiana\u003c/em\u003e (Kowalkowska et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), \u003cem\u003eBulbophyllum wendelianum\u003c/em\u003e (Kowalkowska et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), \u003cem\u003eCyclopogon elatus\u003c/em\u003e (Wiemer et al. \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), \u003cem\u003eCypripedium\u003c/em\u003e (Swanson et al. 1980) or \u003cem\u003eGymnadenia conopsea\u003c/em\u003e (Stpiczyńska \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Nevertheless, in orchid \u003cem\u003eGrobya amherstiae\u003c/em\u003e Lindl. (Pansarin et al. 2009) and carnivorous bladderworts \u003cem\u003eU. cornigera\u003c/em\u003e and \u003cem\u003eU. nelumbifolia\u003c/em\u003e, only the epidermis was identified as highly active physiologically. The conclusion about physiological condition was made based on ultrastructure and histochemistry evidences.\u003c/p\u003e \u003cp\u003eIn case of \u003cem\u003eLepanthes calodictyon\u003c/em\u003e group, the papillae on the surface of the lip and petals could emit the fragrance. Synthesis of the volatile compounds could occur in plastids with plastoglobuli as it was found in osmophores and nectaries (Stpiczyńska \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e1997\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). The proximity of the endoplasmatic reticulum to plastoglobuli is frequently interpreted as feature connected with fragrance. The fragrant compounds could be produced in plastoglobuli, then transported via ER to plasmalemma or transported as lipophilic substances directly in cytoplasm (Stern et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e1987\u003c/span\u003e, Pais and Figueiredo \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1994\u003c/span\u003e, Stpiczyńska \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e1997\u003c/span\u003e, Kowalkowska et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2012\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The endoplasmatic reticulum is in connection with plasmalemma as observed in other orchids genera such Restepia (Pridgeon and Stern \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e1983\u003c/span\u003e) or Epipactis (Kowlakowska et al. 2015). Ultrastructural and histochemical studies revealed the spherical structures, which were identified as lipid bodies inside the lips\u0026rsquo; and petals\u0026rsquo; cells and were noted in all examined species. The possibility of fragrance synthesis was suggested by the cell contained large lipid bodies and presence of elongated plastids surrounded by well developed ER (Lange and Turner \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, Kowalkowska et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The lipids in the cytoplasm are considered as the equivalents of fragrance production (Pridgeon and Stern \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e1983\u003c/span\u003e, Curry et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1988\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eStarch accumulation is typical of osmophore cells (Antoń et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) may have been used in mitochondria as a source of energy for the fragrance production. The histochemical studies and ultrathin sections revealed lack of the starch grains inside the cytoplasm. On the other side, numerous plastoglobuli and lipid bodies in the cytoplasm suggest that the starch was utilised before anthesis (Kowalkowska et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTransport of secreted substances through the plasmalemma is frequently connected with the presence of the ingrowths of the cell wall. The granulocrine secretion is suggested by both the numerous vesicles and relatively large ingrowths of the cell wall. The fusion of the vesicles with plasmalemma is interpreted as granulocrine secretion (Paiva 2016) also found in orchids (Kowalkowska et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The vesicles close to plasmalemma were found in other orchid genera e.g. \u003cem\u003eAnacamptis\u003c/em\u003e (Kowalkowska et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), \u003cem\u003eBulbophyllum\u003c/em\u003e (Kowalkowska et al. 2014), \u003cem\u003eEpipactis\u003c/em\u003e (Kowalkowska et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) and \u003cem\u003eGymnadenia\u003c/em\u003e (Stpiczyńska and Matusiewicz \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). The cuticular ruptures were reported as a way to exudation of the substances to the outside of the secretory cells (Curry et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1991\u003c/span\u003e) and was proved also in orchids e.g. \u003cem\u003eStanhopea oculata\u003c/em\u003e (Stern et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e1987\u003c/span\u003e), \u003cem\u003eBulbophyllum\u003c/em\u003e (Kowalkowska et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), \u003cem\u003ePlatanthera chlorantha\u003c/em\u003e (Stpiczyńska \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2003\u003c/span\u003e) as well as in other plants such as \u003cem\u003ePassiflora suberosa\u003c/em\u003e (Garcıa and Teresa 2008), \u003cem\u003ePrunus persica\u003c/em\u003e (Radice and Galati, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2003\u003c/span\u003e) or \u003cem\u003eOrbea variegata\u003c/em\u003e (Płachno 2010). The epidermal disruptions found in all specimens from \u003cem\u003eL. calodictyon\u003c/em\u003e group seems to support the possible granulocrine secretion.\u003c/p\u003e \u003cp\u003eOn the other side no residues of the secreted substances was found on the surface of the cells. This could be explained by the periodical production and release of the fragrances without accumulation on the surface of the cells (Vogel \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e1990\u003c/span\u003e). The fragrance volatilization was described previously in other orchids (Vogel \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e1990\u003c/span\u003e, Curry et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1988\u003c/span\u003e, Stpiczyńska \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e1993\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePapillae without starch grains, with lipid bodies and plastoglobuli and no traces of secreted substances as it is found in representatives of \u003cem\u003eL. calodictyon\u003c/em\u003e group, were reported previously e.g. \u003cem\u003eB. saltatorium.\u003c/em\u003e Furthermore, the anatomy of the papillae in both cases differs from elaiophore, frequently reported in orchids (Pacek et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Blanco et al. 2013).\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eMorphological and anatomical features of the papillae are similar to those observed in different orchid taxa. These structures were identified as osmophores. Additionally, papillae of the \u003cem\u003eLepanthes calodictyon\u003c/em\u003e group, were rich in lipids and proteins, what was correlated with high metabolic activity. Due to the similarities in structures we suspect that the papillae on the surface of the lip and petals of examined taxa could emit the fragrance.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCBB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCoomasie Brilliant Blue\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDAPI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e2\u0026ndash;[4\u0026ndash;(Aminoiminomethyl)phenyl]\u0026ndash;1\u003cem\u003eH\u003c/em\u003e\u0026ndash;Indole\u0026ndash;6\u0026ndash;carboximidamide hydrochloride\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003elight microscopy\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePAS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eperiodic acid\u0026ndash;Schiff reaction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ephosphate buffered saline\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePFA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eparaformaldehyde\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRER\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003erough endoplasmic reticulum\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSBB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSudan Black B\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSEM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003escanning electron microscopy\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSER\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esmooth endoplasmic reticulum\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTEM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etransmission electron microscopy\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eUGDA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHerbarium Universitatis Gedanensis, Wita Stwosza 59, Gdansk\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe original microphotographs used and/or analysed during the current study are available\u0026nbsp;from the corresponding author on reasonable request. Reference plant material has been deposited in UGDA Herbarium.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study received support from the SYNTHESYS Project (http://www.synthesys.info/), which is financed by the European Community Research Infrastructure Action within the framework of the FP6 ‘Structuring the European Research Area’ Programme GB-TAF-6914 and NL-TAF-6915.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMR and MK identified research problem and designed the experiments. MK, MN, EB and MR prepared plant material for analyses. NM and DŁ performed electron microscopy analyses. MK and ED performed plant histochemistry analyses. MR, KM and MN interpreted the data and compared results with available literature. MR obtained funds for research. MR, MK and DLSz prepared draft of the manuscript. All authors contributed in and reviewed final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBrzezicka Emilia ORCID 0000-0002-3619-1238\u003c/p\u003e\n\u003cp\u003eKapusta Małgorzata ORCID\u0026nbsp;0000-0003-4103-594X\u003c/p\u003e\n\u003cp\u003eNarajczyk Magdalena ORCID\u0026nbsp;0000-0001-9806-8844\u003c/p\u003e\n\u003cp\u003eRykaczewski Max ORCID\u0026nbsp;0000-0003-3457-9690\u003c/p\u003e\n\u003cp\u003eSzlachetko Dariusz L. ORCID\u0026nbsp;0000-0002-3210-7537\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAlbores Ortiz O, Sosa V.Polinizaci\u0026oacute;n de dos especies simp\u0026aacute;tricas de \u003cem\u003eStelis\u003c/em\u003e (Pleurothallidinae, Orchidaceae). Acta Bot Mex. 2014;74:155-575.\u003c/li\u003e\n \u003cli\u003eAntoń S, Kamińska M, Stpiczyńska M (2012) Comparative structure of the osmophores in the flower of Stanhopea graveolens Lindley and Cycnoches chlorochilon Klotzsch (Orchidaceae). Acta Agrobot. 2012; 65: 11\u0026ndash;22.\u003c/li\u003e\n \u003cli\u003eAscens\u0026atilde;o L, Francisco A, Cotrim H, Pais MS. Comparative structure of the labellum in Ophrys fusca and O. lutea (Orchidaceae). Am J Bot.; 92: 1059\u0026ndash;1067.\u003c/li\u003e\n \u003cli\u003eBarbosa AR, de Melo MC, Borba EL. Self-incompatibility and myophily in \u003cem\u003eOctomeria\u003c/em\u003e (Orchidaceae, Pleurothallidinae) species. Plant Syst Evol. 2009; 283:1-8.\u003c/li\u003e\n \u003cli\u003eBlanco MA, Barboza G. Pseudocopulatory pollination in \u003cem\u003eLepanthes\u003c/em\u003e (Pleurothallidinae, Orchidaceae) by fungus gnats. Ann Bot-London. 2005; 95:763-772.\u003c/li\u003e\n \u003cli\u003eBorba EL, Barbosa AR, de Melo MC, Gontijo SL, de Oliveira HO. Mating systems in Pleurothallidinae (Orchidaceae): Evolutionary and systematic implications. Lankesteriana. 2011;11(3): 207-221.\u003c/li\u003e\n \u003cli\u003eBorba EL, Semir J. Pollinator specificity and convergence in fly-pollinated \u003cem\u003ePleurothallis\u003c/em\u003e (Orchidaceae) species: a multiple population approach. Ann Bot-London. 2001; 88:75-88.\u003c/li\u003e\n \u003cli\u003eBronner R (1975) Simultaneous demonstration of lipid and starch in plant tissues. Stain Technol. 1975; 50(1):1\u0026ndash;4.\u003c/li\u003e\n \u003cli\u003eChase MW. Pollination of \u003cem\u003ePleurothallis\u003c/em\u003e \u003cem\u003eendotrachys\u003c/em\u003e. American Society Bulletin 1985; 54:431-434.\u003c/li\u003e\n \u003cli\u003evan der Cingel NA. An Atlas of Orchid Pollination: European Orchids: Orchids of America, Africa, Asia and Australia. CRC Press, Rotterdam; 2001.\u003c/li\u003e\n \u003cli\u003eCiotek L, Giorgis P, Benitez-Vieyra S, Cocucci AA. First confirmed case of pseudocopulation in terrestrial orchids of South America: pollination of \u003cem\u003eGeoblasta\u003c/em\u003e \u003cem\u003epennicillata\u003c/em\u003e (Orchidaceae) by \u003cem\u003eCampsomeris\u003c/em\u003e \u003cem\u003ebistrimacula\u003c/em\u003e (Hymenoptera, Scoliidae). Flora. 2006; 201:365-369.\u003c/li\u003e\n \u003cli\u003eCozzolino S, Widmer A. Orchid diversity: an evolutionary consequence of deception? Trends Ecol Evol. 2005; 20:487\u0026ndash;494.\u003c/li\u003e\n \u003cli\u003eCurry KJ, Stern WL, McDowell LM. Osmophore development in Stanhopea anfracta and S. pulla (Orchidaceae). Lindleyana. 1988; 3:212.\u003c/li\u003e\n \u003cli\u003eCurry KJ, McDowell LM, Judd WS, Stern WL. Osmophores, floral features, and systematics of Stanhopea (Orchidaceae). Am J Bot. 1991; 78:610.\u003c/li\u003e\n \u003cli\u003eDavies KL, Stpiczyńska M. Comparative floral micromorphology and the ultrastructural basis of fragrance production in pseudocopulatory Mormolyca s.s. and non-pseudocopulatory Maxillaria section Rufescens s.s. (Orchidaceae). Bot. J. Linn. Soc. 2017; 185:81-112.\u003c/li\u003e\n \u003cli\u003eDod DD. Afidos y tr\u0026iacute;pidos polinizan orqu\u0026iacute;deas en las Pleurothallidinae (Orchidaceae). Moscosoa. 1986;4:200\u0026ndash;202.\u003c/li\u003e\n \u003cli\u003eDressler RL. The Orchids. Natural History and Classification. Washington: Smithsonian Institution; 1981.\u003c/li\u003e\n \u003cli\u003eDuque O. Polinizaci\u0026oacute;n de \u003cem\u003ePleurothallis\u003c/em\u003e. Orquideolog\u0026iacute;a. 1993; 19:55-69.\u003c/li\u003e\n \u003cli\u003eDressler RL. Phylogeny and Classification of Orchid Family. Cambridge University Press; 1993\u003c/li\u003e\n \u003cli\u003eDuque-Buitrago CA, Alzate-Quitntero NF, Otero JT. Nocturnal pollination by fungus gnats of the Colombian endemic species \u003cem\u003ePleurothallis\u003c/em\u003e \u003cem\u003emarthae\u003c/em\u003e (Orchidaceae: Pleurothallidinae) Lankesteriana. 2014; 13:407-417.\u003c/li\u003e\n \u003cli\u003eEllis GA, Johnson SD. Floral mimicry enhances pollen export: The Evolution of pollination by sexual deceit outside of the Orchidaceae. \u003ccite\u003eAm Nat. 2010; 276(5):143-151.\u003c/cite\u003e\u003c/li\u003e\n \u003cli\u003eEndara L, Grimaldi DA, Roy BA. Lord of the flies: pollination of \u003cem\u003eDracula\u003c/em\u003e orchids. Lankesteriana. 2010; 10:1-11.\u003c/li\u003e\n \u003cli\u003eFeder N, O\u0026rsquo;Brien TP. Plant microtechnique: some principles and new methods. American Journal of Botany. 1968; 55: 123\u0026ndash;142.\u003c/li\u003e\n \u003cli\u003eGahan PB. Plant histochemistry and cytochemistry: an introduction. Academic Press, London; 1984.\u003c/li\u003e\n \u003cli\u003eGarcia A, Teresa M. Breeding system and related floral features under natural and experimental conditions of Passiflora suberosa (Passifloraceae). Bol. Soc. Argent. Bot. 2008; 43 (1-2): 83 \u0026ndash; 93.\u003c/li\u003e\n \u003cli\u003eGivnish TJ et al. Orchid phylogenomics and multiple drivers of their extraordinary diversification. Proc. R. Soc. B. 2015; 282:20151553.\u003c/li\u003e\n \u003cli\u003eHiggins W. Pleurothallidinae: How Many Genera? 19th World Orchid Conference; 2009.\u003c/li\u003e\n \u003cli\u003eHu S, Xu L. A Cytochemical Technique for Demonstration of Lipids, Polysaccharides and Protein Bodies in Thick Resin Sections. J Integr Plant Biol. 1990; 32.\u003c/li\u003e\n \u003cli\u003eH\u0026uuml;lskamp M, Schwab B, Grini P, Schwarz H. Transmission Electron Microscopy (TEM) of Plant Tissues. Cold Spring Harbor Laboratory Press 7 2010; doi:10.1101/pdb.prot495\u003c/li\u003e\n \u003cli\u003eJensen W. Botanical histochemistry. Freeman, San Francisco; 1962.\u003c/li\u003e\n \u003cli\u003eJers\u0026aacute;kov\u0026aacute; J, Johnson SD, Kindlman P. Mechanisms and evolution of deceptive pollination in orchids. Biol Rev. 2006;81:219-235.\u003c/li\u003e\n \u003cli\u003eJost L, Endara L. Analysis of the DNA-Based Reclassification of the Pleurothallidinae. 2004. http://www.loujost.com/DNAFrameset/DNAanalysisDocument.htm. Accessed 30 April 2025.\u003c/li\u003e\n \u003cli\u003eKarremans AP. Genera Pleurothallidinarum: An updated phylogenetic overview of Pleurothallidinae. Lankesteriana 2016;16(2):219-241.\u003c/li\u003e\n \u003cli\u003eKarremans AP, Pupulin F, Grimaldi D, Beentjes K, Butot R, Fazzi GE, Kaspers K, Kruizinga J, Roessingh P, Smets EF, Gravendeel B. Pollination of \u003cem\u003eSpecklinia\u003c/em\u003e by nectar-feeding \u003cem\u003eDrosophila\u003c/em\u003e: The first reported case of a deceptive syndrome employing aggregation pheromones in Orchidaceae. Ann Bot-London. 2015; 116(3):437-455.\u003c/li\u003e\n \u003cli\u003eKowalkowska AK, Kozieradzka-Kiszkurno M, Turzyński S. Morphological, histological and ultrastructural features of osmophores and nectary of Bulbophyllum wendlandianum (Kraenzl.) Dammer (B. section Cirrhopetalum Lindl., Bulbophyllinae Schltr., Orchidaceae) Plant Syst Evol. 2015; 301:609\u0026ndash;622.\u003c/li\u003e\n \u003cli\u003eKowalkowska AK, Turzyński S, Kozieradzka-Kiszkurno M, Wiśniewska N. Floral structure of two species of Bulbophyllum section Cirrhopetalum Lindl.: B. weberi Ames and B. cumingii (Lindl.) Rchb. f. (Bulbophyllinae Schltr., Orchidaceae) Protoplasma. 2017; 254(3):1431\u0026ndash;1449.\u003c/li\u003e\n \u003cli\u003eKowalkowska AK, Margońska HB, Kozieradzka-Kiszkurno M. Comparative anatomy of the lip spur and additional lateral sepal spur in a three-spurred form (f. \u003cem\u003efumeauxiana\u003c/em\u003e) of \u003cem\u003eAnacamptis pyramidalis\u003c/em\u003e. Acta Biol Cracov Ser Bot. 2010; 52:13\u0026ndash;18.\u003c/li\u003e\n \u003cli\u003eKowalkowska AK, Margońska HB, Kozieradzka-Kiszkurno M, Bohdanowicz J. Studies on the ultrastructure of a three-spurred fumeauxiana form of Anacamptis pyramidalis. Plant Syst Evol. 2012; 298:1025-1035.\u003c/li\u003e\n \u003cli\u003eKrawczyk E, Rojek J, Kowalkowska AK, Kapusta M, Znaniecka J, Minasiewicz J. Evidence for mixed sexual and asexual reproduction in the rare European mycoheterotrophic orchid Epipogium aphyllum, Orchidaceae (ghost orchid). Ann Bot. 2016; 118:159\u0026ndash;172.\u003c/li\u003e\n \u003cli\u003eLange BM, Turner GW. Terpenoid biosynthesis in trichomes current status and future opportunities. Plant Biotechnol J. 2013: 11:2\u0026ndash;22.\u003c/li\u003e\n \u003cli\u003eLuer CA. Icones Pleurothallidinarum I. Systematics of the Pleurothallidinae (Orchidaceae). Monog Syst Botan, St. Louis. 15:1-81; 1986.\u003c/li\u003e\n \u003cli\u003eLuer CA. Icones Pleurothallidinarum XIV. Systematics of Draconanthes, Lepanthes subgenus Marsipanthes and subgenus Lepanthes from Ecuador (Orchidaceae). Monog Syst Botan, St. Louis. 61:1-255; 1996.\u003c/li\u003e\n \u003cli\u003eLuer CA, Thoerle L. Icones Pleurothallidinarum Vol XXXII. Lepanthes of Colombia. Monog Syst Botan, St. Louis. 123:1-300; 2012.\u003c/li\u003e\n \u003cli\u003ede Melo MC, Borba EL, Paiva EAS. Morphological and histological characterization of the osmophores and nectaries of four species of \u003cem\u003eAcianthera\u003c/em\u003e (Orchidaceae: Pleurothallidinae) Plant Syst Evol. 2010; 286:141-151.\u003c/li\u003e\n \u003cli\u003eO\u0026rsquo;Brien TP, Feder N, McCully ME. Polychromatic staining of plant cell walls by toluidine blue O. Protoplasma. 1965; 59: 368\u0026ndash;373.\u003c/li\u003e\n \u003cli\u003eO\u0026rsquo;Brien TP, McCully ME. The study of plant structure: principles and selected methods. Melbourne: Termarcarphi Pty. Ltd; 1981.\u003c/li\u003e\n \u003cli\u003ePacek A, Stpiczyńska M, Davies KL, Szymczak G. Floral elaiophore structure in four representatives of the Ornithocephalus clade (Orchidaceae: Oncidiinae). Annals of Botany. 2012; 110:809-820.\u003c/li\u003e\n \u003cli\u003ePais MS, Figueiredo ACS. Floral nectaries from Limodorum abortivum (L.) Sw. and Epipactis atropurpurea rafin. (Orchidaceae); ultrastructural changes in plastids during the secretory process. Apidologie. 1994; 25:615\u0026ndash;626.\u003c/li\u003e\n \u003cli\u003evan der Pijl L, Dodson CH. Orchid flowers: their pollination and evolution. Coral Gables University of Miami Press; 1966.\u003c/li\u003e\n \u003cli\u003ePłachno BJ, Świątek P, Szymczak G. Can a stench be beautiful?\u0026mdash;osmophores in stem-succulent stapeliads (Apocynaceae-Asclepiadoideae-Ceropegieae-Stapeliinae) Flora. 2010;205:101\u0026ndash;105.\u003c/li\u003e\n \u003cli\u003ePłachno BJ, Stpiczyńska M, Świątek P, Davies KL. Floral micromorphology of the Australian carnivorous bladderwort Utricularia dunlopii, a putative pseudocopulatory species. Protoplasma. 2016; 253:1463\u0026ndash;1473.\u003c/li\u003e\n \u003cli\u003ePridgeon AM. Pleurothallidinae In: Pridgeon AM, Cribb P, Chase MW, Rasmussen P eds. \u003cem\u003eGenera\u003c/em\u003e \u003cem\u003eOrchidacearum\u003c/em\u003e Volume 4 \u0026ndash;Epidendroidae (Part One). Oxford: Oxford University Press; 2005. p. 319-422.\u003c/li\u003e\n \u003cli\u003ePridgeon AM, Solano R, Chase MW. Phylogenetic relationships in Pleurothallidinae (Orchidaceae): combined evidence from nuclear and plastid DNA sequences. Am J Bot. 2001; 88:2286-2308.\u003c/li\u003e\n \u003cli\u003ePridgeon AM, Stern WL. Ultrastructure of Osmophores in \u003cem\u003eRestrepia\u003c/em\u003e (Orchidaceae). Am J Bot. 1983; 70(8):1233-1243.\u003c/li\u003e\n \u003cli\u003ePridgeon AM, Stern WL. Osmophores of \u003cem\u003eScaphosepalum\u003c/em\u003e (Orchidaceae). Bot Gaz. 1985; 146(1):115-123.\u003c/li\u003e\n \u003cli\u003eRadice S, Galati B. Floral nectary ultrastructure of Prunus persica (L.) Batch cv. Forastero (Newcomer), an Argentine peach. Plant Syst Evol. 2003; \u003cstrong\u003e238\u003c/strong\u003e, 23\u0026ndash;32.\u003c/li\u003e\n \u003cli\u003eSalazar-Ch\u0026aacute;ves GA, Soto-Arenas MA. El g\u0026eacute;nero \u003cem\u003eLepanthes\u003c/em\u003e Sw. en M\u0026eacute;xico. Orqu\u0026iacute;dea (M\u0026eacute;xico). 1996; 14:1-231.\u003c/li\u003e\n \u003cli\u003eScopece G, Cozzolino S, Johnson SD, Schiestl FP. Pollination efficiency and the evolution of specialized deceptive pollination systems. Am Nat. 2010;175:98-105.\u003c/li\u003e\n \u003cli\u003eServettaz O, Maleci LB, Gr\u0026uuml;nanger P. Labellum micromorphology in the Ophrys bertolonii agg. and some related taxa (Orchidaceae). Plant Syst Evol. 1994; 189: 123\u0026ndash;131.\u003c/li\u003e\n \u003cli\u003eSinger RB. The Pollination Mechanism in Trigonidium obtusum Lindl (Orchidaceae: Maxillariinae): Sexual Mimicry and Trap flowers. Ann Bot. 2002; 89:157-163.\u003c/li\u003e\n \u003cli\u003eSmithson A. Pollinator limitation and inbreeding depression in orchid species with and without nectar rewards. New Phytol. 2006; 169:419-430.\u003c/li\u003e\n \u003cli\u003eStern WL, Curry KJ, Pridgeon AM. Osmophores of Stanhopea (Orchidaceae) Am J Bot. 1987;74:1323\u0026ndash;1331.\u003c/li\u003e\n \u003cli\u003eStpiczyńska M. Anatomy and ultrastructure of osmophores of Cymbidium tracyanum Rolfe (Orchidaceae). Acta Soc Bot Pol. 1993; 62:5-9.\u003c/li\u003e\n \u003cli\u003eStpiczyńska M. The structure of nectary of Platanthera bifolia L. Orchidaceae. Acta Soc Bot Pol. 1997; 1:5\u0026ndash;7.\u003c/li\u003e\n \u003cli\u003eStpiczyńska M. Osmophores of the fragrant orchid Gymnadenia conopsea L. (Orchidaceae). Acta Soc Bot Pol. 2001; 70(2):91\u0026ndash;96.\u003c/li\u003e\n \u003cli\u003eStpiczyńska M. Nectar resorption in the spur of Platanthera chlorantha (Custer) Rchb. Orchidaceae structural and microautoradiographic study. Plant Syst Evol 2003; 238:119\u0026ndash;12.\u003c/li\u003e\n \u003cli\u003eStpiczyńska M, Matusiewicz J. Anatomy and ultrastructure of spur nectary of Gymnadenia conopsea (L.) Orchidaceae. Acta Soc Bot Pol 2001; 4:267\u0026ndash;272.\u003c/li\u003e\n \u003cli\u003eStpiczyńska M, Davies KL, Gregg A. Comparative account of nectary structure in Hexisea imbricata (Lindl.) Rchb.f. (Orchidaceae). Ann Bot. 2005; 95(5):749\u0026ndash;756.\u003c/li\u003e\n \u003cli\u003eTeixeira SP Borba EL, Semir J. Lip anatomy and its implications for pollination mechanism of \u003cem\u003eBulbophyllum\u003c/em\u003e species (Orchidaceae). Ann Bot-London. 2004; 93:499-505.\u003c/li\u003e\n \u003cli\u003eTremblay RL \u0026amp; Ackerman JD. A new species of Lepanthes (Orchidaceae) from Puerto Rico. Brittonia. 1993; 45:339\u0026minus;342.\u003c/li\u003e\n \u003cli\u003eTremblay RL, Ackerman JD, Zimmerman JK, Calvo RN. Variation in sexual reproduction in orchids and its evolutionary consequences: a journey to diversification. Biol J Linn Soc. 2005; 84:1-54.\u003c/li\u003e\n \u003cli\u003eVogel S, Renner S. The role of scent glands in pollination: On the structure and function of osmophores. Washington, D.C: Smithsonian Institution Libraries and National Science Foundation; 1990.\u003c/li\u003e\n \u003cli\u003eWhitten M, Blanco M (2011) Defining generic limits in Maxillaria: a return to the orchidaceous mine. Orchids 80:104\u0026ndash;113.\u003c/li\u003e\n \u003cli\u003eWiemer AP, More M, Benitez-Vieyra S, Cocucci AA, Raguso RA, Sersic AN. A simple floral fragrance and unusual osmophore structure in Cyclopogon elatus (Orchidaceae). Plant Biol (Stuttg). 2008; 11(4):506\u0026ndash;514.\u003c/li\u003e\n \u003cli\u003eWiśniewska N, Kowalkowska A, Kozieradzka-Kiszkurno M, Krawczyńska A, Bohdanowicz J. Floral features of two species of Bulbophyllum section Lepidorhiza Schltr.: B. levanae Ames and B. nymphopolitanum Kraenzl. (Bulbophyllinae Schltr., Orchidaceae). Protoplasma. 2018; 255:485\u0026ndash;499.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"micromorphology, Orchidaceae, osmophore, Pleurothallidinae, ultrastructure","lastPublishedDoi":"10.21203/rs.3.rs-6594964/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6594964/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: Previous studies revealed that ultrastructure and histochemistry of the flowers give insight to pollination mechanism of the orchid species. The microscopic features of species within Pleurothallidine subtribe is poorly understood. In this study we investigated three \u003cem\u003eLepanthes\u003c/em\u003e species: \u003cem\u003eL. calodictyon\u003c/em\u003e, \u003cem\u003eL. saltatrix\u003c/em\u003e and \u003cem\u003eL. tentaculata\u003c/em\u003e. All of them represent similar flower morphology, distinct from the other representative of the genus.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: Our analyses revealed presence of features usually associated with myophily. Papillae of lips and petals of the \u003cem\u003eLepanthes\u003c/em\u003e \u003cem\u003ecalodictyon\u003c/em\u003egroup, were rich in lipids and proteins, what was correlated with high metabolic activity. Furthermore, ultrastructural and morphological features are similar to those observed in other fragrance emitting orchid species.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e: Histochemical, ultrastructural and morphological features of the papillae on the surface of the lip and petals of examined taxa indicate that papillae are in fact osmophores.\u003c/p\u003e","manuscriptTitle":"Floral anatomy and ultrastructure in Lepanthes calodictyon group","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-29 12:19:29","doi":"10.21203/rs.3.rs-6594964/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-26T11:55:01+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-25T17:59:46+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-23T23:59:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"234055011777835582767863836336647909474","date":"2025-05-29T15:44:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"176666892801470887606866248187574812095","date":"2025-05-26T14:13:50+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-23T10:35:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-23T10:34:52+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-05-22T08:39:17+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-20T09:18:54+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2025-05-20T09:17:47+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1a3e1b79-87a7-43b3-a6fe-9caa6d4479e6","owner":[],"postedDate":"May 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-10-06T16:02:36+00:00","versionOfRecord":{"articleIdentity":"rs-6594964","link":"https://doi.org/10.1186/s12870-025-07141-1","journal":{"identity":"bmc-plant-biology","isVorOnly":false,"title":"BMC Plant Biology"},"publishedOn":"2025-09-30 15:56:58","publishedOnDateReadable":"September 30th, 2025"},"versionCreatedAt":"2025-05-29 12:19:29","video":"","vorDoi":"10.1186/s12870-025-07141-1","vorDoiUrl":"https://doi.org/10.1186/s12870-025-07141-1","workflowStages":[]},"version":"v1","identity":"rs-6594964","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6594964","identity":"rs-6594964","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}