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Anatomical features related with pollination of Neottia ovata (L.) Bluff & Fingerh. (Orchidaceae)
Flora 255 (2019) 24–33
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Anatomical features related with pollination of Neottia ovata (L.) Bluff & Fingerh. (Orchidaceae)
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Agnieszka K. Kowalkowskaa, , Agnieszka T. Krawczyńskab a b
Department of Plant Cytology and Embryology, University of Gdańsk, Wita Stwosza 59, 80-308 Gdańsk, Poland Warsaw University of Technology, Faculty of Materials Science and Engineering, Wołoska 141, 02-507 Warsaw, Poland
A R T I C LE I N FO
A B S T R A C T
Edited by: Favio Gonzalez
The survey of anatomical features of Neottia ovata confirmed that the lip base and the groove function as a nectary. The secretory tissue was present in buds about 1,3 mm width. The sweet scent comes from the aromatic constituents of the nectar and also is released by the lip lobes and margins of all tepals. Additionally, the stomata present on the keels on the abaxial surface of sepals and large substomatal cavities may be connected with fragrance emission. The secretory function was not observed on the stem trichomes. The groove is formed by a single-layered epidermis, few layers of subepidermal cells and underlying parenchyma. The abundant starch grains present in buds, absent in sepals and lip epidermis in larger buds and almost absent in open flowers (visible in plastids in TEM) suggest gradual reduction during floral development and nectar secretion. The nectar was gathered under the cuticle, which was visible in the groove and lip base. The main mode of nectar release is by diffusion through micro-channels. The presence of irregular plasmalemma, dictyosomes, and profiles of ER could also indicate the granulocrine mode of secretion. The vesicles originated from ER or dictyosomes are fused with irregular plasmalemma. The irregular plasmalemma with invaginations (observed in groove) and cell wall protuberances (observed at the lip base) along the outer periclinal wall may function as highly specialized cells transfer cells. Numerous idioblasts, visible in all tepals and in the staminodium, attract pollinators. In the anther, the endothecial cell thickenings were composed of numerous complete rings or helical segments in a closely spaced parallel arrangement (type I), providing the mechanical forces for anther dehiscence and opening and release of pollen grains.
Keywords: Common twayblade Endothecial thickenings in anther Idioblasts in staminodium Lip nectary Osmophore
1. Introduction The Orchidaceae are known for their diverse floral structure, peculiar pollination mechanisms and different types of rewards. The most common feeding reward for visiting pollinators is nectar (van der Pijl and Dodson, 1969; Dressler, 1990). The presence of nectar, an aqueous solution with sugars, is considered to be a trait of plant–animal coevolution. The floral nectar, produced nearby the reproductive organs, facilitates pollen dispersal (Nepi, 2017). The nectar can be produced and exposed in orchids in diverse ways, i.e. in labellar superficial nectaries built by a single-layered epidermis and several layers of subepidermal cells (e.g. in Bulbophyllum Lindl.; Kowalkowska et al., 2015b, 2017; Wiśniewska et al., 2018), in hypochile and knobs of epichile (e.g. in Epipactis helleborine (L.) Crantz.; Kowalkowska et al., 2018; and Epipactis palustris (L.) Crantz.; Kowalkowska et al., 2015a), on labellar papillae (e.g. in Epipogium aphyllum Sw.; Święczkowska and Kowalkowska, 2015; Krawczyk et al., 2016; and in Dactylorhiza Neck.
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ex Nevski; Naczk et al., 2018) or in spurs formed at the base of the labellum (e.g. in Anacamptis pyramidalis f. fumeauxiana Marg. & Kowalk.; Kowalkowska et al., 2010, 2012; and in Epipogium aphyllum Sw.; Święczkowska and Kowalkowska, 2015), or tubular nectaries embedded alongside the ovary (van der Pijl and Dodson, 1969). Shallow, superficial nectaries are also found in Neottia Guett. (= Listera) R. Br. (Dressler, 1990). The genus consists of two species groups which were previously treated as separate genera, Neottia Guett. and Listera R. Br. (Claessens and Kleynen, 2011). Neottia ovata (L.) Bluff & Fingerh. (= Listera ovata (L.) R. Br.; common twayblade) is an inconspicuous perennial plant, entirely green (Claessens and Kleynen, 2011) with two opposite leaves on the hairy stem. It is one of the most common orchids in western Europe and its geographical range reaches as far as eastern Siberia in Asia where it can be found in forests and less frequently in meadows (Delforge, 2006). As the plant has green flowers, it offers no contrast with the surrounding plants (Claessens and Kleynen, 2011). Having no visual cues, the distinct, somewhat sweet
Corresponding author. E-mail address: [email protected] (A.K. Kowalkowska).
https://doi.org/10.1016/j.flora.2019.03.015 Received 3 October 2018; Received in revised form 2 March 2019; Accepted 21 March 2019 Available online 27 March 2019 0367-2530/ © 2019 Elsevier GmbH. All rights reserved.
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Following dehydration in an ethanol series the samples were prepared for scanning electron microscopy (SEM) and subjected to criticalpoint drying using liquid CO2, coated with gold and observed in a Philips XL-30 (Laboratory of Electron Microscopy, University of Gdańsk, Poland). For transmission electron microscopy (TEM), floral material was fixed in 2.5% (v/v) glutaraldehyde (GA) in 0.05 M cacodylate buffer (pH 7.0). The material was then post-fixed overnight in 1% (w/v) OsO4 in cacodylate buffer in a refrigerator and finally rinsed in buffer. After 1 h in a 1% (w/v) aqueous solution of uranyl acetate, the material was dehydrated with acetone and embedded in Spurr’s resin. Ultrathin sections were cut on a Leica EM UC 7 ultramicrotome with a diamond knife. The first part of sections was stained with uranyl acetate and lead citrate (Reynolds, 1963) and the second part of sections was contrasted with the UranyLess and Lead Citrate (the protocol available on http:// uranyless.com). The sections on Fig. 6 b–d were examined in a JEOL JEM 1200EX transmission electron microscope (Warsaw University of Technology) and on Fig. 6a, e–h; Fig. 7a-b were examined in a FEI transmission electron microscope (University of Gdańsk) at an accelerating voltage 120 kV.
scent is the main long-distance attractant, composed mainly by monoand sesquiterpenes, with linalool and trans-b-ocimene as the major components (Nilsson, 1981). Also terpenes have been found, such as the oxygenated and related terpenes perillen and dendrolasin. The hexadecanoic acid isopropyl ester and a constituent with a mass spectrum similar to isobutyl nitrile are present. The nectar is produced in the wide lip base and in less quantity in a central longitudinal groove (Claessens and Kleynen, 2011). After landing, a visiting insect licks the nectar secreted in a groove. Following the nectar trail, the insect is guided to the lip base and the gynostemium (Sprengel, 1793). The staminodium forms a wide petaloid structure behind the anther. The anther contains two pollinia in two pairs. The rostellum tip is transformed into a sticky, semi-fluid viscidium (Szlachetko and Rutkowski, 2000). Depending on the particular size of the insect, after touching the viscidium, the pollinia are expelled and attached to the insect’s head or thorax. The freely accessible nectar facilitates pollination and fruit set (Sprengel, 1793). Although the flowers are visited by a large suite of generalist insect species (Nilsson, 1981; Brys et al., 2008; Kotilínek et al., 2015), the main pollinators are ichneumonids, sawflies and beetles e.g. with 56, 28 and 10% of transported pollinia on the Island of Öland (Sweden) and 28, 54 and 15% of pollinia in Uppland (mainland Sweden), respectively (Nilsson, 1981). Although the flower macromorphology and pollination mechanism of Neottia ovata is well studied (Sprengel, 1793; Delforge, 2006; Claessens and Kleynen, 2011; Tałałaj et al., 2017), our studies are the first one on the anatomy and ultrastructure of N. ovata. The aims of the present research are: a) to examine the anatomy of structures related with pollination efficacy, and also the stem trichomes; b) to verify the presence of secretion in the flower (from bud stage to open flower); and c) to discuss the anatomical results alongside previously published research.
The stem was covered by numerous, 2–3 celled trichomes with a grandular apical cell (Fig. 1b-e). The transverse sections revealed typical monocot anatomy: a single layer of epidermis, cortex, sclerenchymatous layer, and ground tissue with numerous distinct collateral vascular bundles (Fig. 1e). The secretory activity was not observed on the stem trichomes.
2. Materials and methods
3.2. Buds
The plant material was collected in the beginning of June 2013 from young buds to recently open flowers (voucher number AKK 2013−001) from ravine near Staw Wróbla in Gdańsk (Fig. 1a). The fresh flowers were observed under a Nikon SMZ1500 stereomicroscope. For the purpose of localization of osmophores, the whole, fresh flowers were immersed in an aqueous solution of 001%-0,001% (w/v) neutral red for 20 min. Plant material was fixed in 2,5% (v/v) glutaraldehyde (GA) in 0.05 M cacodylate buffer (pH = 7.0). Material for light microscopy (LM), following fixation was rinsed with cacodylate buffer and then dehydrated in an acetone or ethanol series. Subsequently, material dehydrated in acetone was embedded in epoxy resin (Spurr, 1969), whereas material dehydrated in ethanol was embedded in methylmethacrylate-based resin (Technovit 7100, Heraeus Kulzer GmbH). For light microscopy, semi-thin control sections were stained with 0.05% (w/v) aqueous sodium tetraborate solution (Toluidine Blue O, TBO, C.I. 52040) (Feder and O’Brien, 1968; Ruzin, 1999). Aniline Blue Black (ABB, C.I. 20470) was used for the detection of water-insoluble proteins (Jensen, 1962) and a 0.05% (w/v) aqueous Ruthenium Red (C.I. 77800) solution to test for pectic acids/mucilage (Johansen 1940). The Periodic Acid-Schiff reaction (PAS) was used to identify the presence of water-insoluble polysaccharides (Jensen, 1962) and a 0.3% (w/v) ethanolic solution of Sudan Black B (SBB, C.I. 26150) for lipid localization (Bronner, 1975). The preparations were examined and photographed by means of a Nikon Eclipse E 800 light microscope and a Nikon DS–5Mc camera using the Lucia Image software. The Auramine O (C.I. 41000; 0.01% (w/v) solution in 0.05 M buffer Tris/HCl, pH = 7.2) was used to detect the presence of cuticle (Heslop-Harrison, 1977), especially unsaturated acidic waxes and cutin precursors (Gahan, 1984), and the staining reaction was examined by means of a Nikon Eclipse E800 fluorescence microscope equipped with filter: B-2A (EX 450–490 nm, DM 505 nm, BA 520 nm).
The presence of labellar secretory tissue was noted from buds (Fig. 2a, about 1,3 mm width) and developed in larger buds (Fig. 2b–j): in the groove (Fig. 2a–d, f, g, i), the lip base (Fig. 2j) and the lip lobes (Fig. 2a–d, f, g, i, j). The most remarkable feature was the occurrence of substomatal cavities (Fig. 2a-b) in the sepals, which widen (Fig. 2c, d, f, g, i, j) almost over the whole sepal width during development (Fig. 2i). The idioblasts with raphides were scattered in all tepals (Fig. 2a-b) and even filled the entire tissue in parts of the staminodium (Fig. 2g–i). Type I endothecial cell thickenings were observed in a wall layer of anthers (Fig. 2d-g, i, j).
3. Results 3.1. Stem
3.3. Open flower 3.3.1. Sepals and petals The sepals and petals surround the gynostemium like a helmet (Fig. 3a). The sepals (one dorsal and two laterals) were keeled (Fig. 3ab). The margins of petals (two lateral, inner whorl) were folded (Fig. 3c). The both surfaces of the sepals and petals were built by the irregularly shaped cells, sometimes rectangular, on the inner surface with striated cuticle (Fig. 3d). Anomocytic stomata were visible on both surfaces, but in higher numbers on the external keels (Fig. 3e). Twocelled trichomes were noted at the base of dorsal (on both surfaces) (Fig. 3f) and lateral sepals (only on external suface). 3.4. Lip The lip was divided into two lobes (Fig. 4a). A longitudinal, median groove was slightly raised, especially at the end (Fig. 4b). A basal, shallow cavity in the lip base and the groove alongside the lip was covered by nectar (Fig. 4c). The test with neutral red showed the accumulation of stain in the lip base and in the groove, as well as on the lip lobes and the margins of all tepals (Fig. 4d-e), which indicated the 25
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Fig. 1. a. Neottia ovata - plant; b. stem with trichomes (LM); c. trichomes (SEM); d. detail of c, 3-celled trichome with grandular top cell; e. transverse section of stem with a single layer of epidermis (e) with 2–3-celled trichomes, cortex (co), sclerenchymatous layer (sc), ground tissue with numerous distinct collateral vascular bundles (vb)(TBO).
was covered by cuticle (Fig. 6a, c–e), swollen on some cells (Fig. 6a). The micro-channels were visible in the cuticle, and a few secretory material on its surface (Fig. 6c–e). In the cell wall sometimes there was a material - the same as that, which built the cuticle (Fig. 6e). The dense parietal cytoplasm also contained chloroplasts with starch grains (Fig. 6f), lipid droplets (Fig. 6f), dictyosomes (Fig. 6g), profiles of SER and RER (Fig. 6g-h), ribosomes (Fig. 6g), mitochondria (Fig. 6f, h). The plasmalemma was irregularly formed with invaginations (Fig. 6b, f–h), sometimes with vesicles (Fig. 6f–h). TEM studies of lip basal cavity also proved the secretory epidermis with striated cell wall covered by cuticle with micro-channels and the remnants of secreted material on its surface (Fig. 7a-b). The irregular plasmalemma sometimes had cell wall protuberances (Fig. 7b).
secretory activity: the nectary in the lip base and groove and the osmophoric activity on the lip lobes and on the margins of all tepals. The micromorphological studies proved the occurrence of shallow lip cavity and the groove on the adaxial surface (Fig. 4f). The cuticle striation was more folded on the epidermal cells of groove, sometimes swelled, protruding, not appearing on each cell at the same time (Fig. 4g). On the lip lobes elongated ellipsoidal cells were observed (Fig. 4h). The lip tissue consisted of a single layer of epidermal cells, few layers of subepidermal cells, and parenchyma with collateral vascular bundles (Fig. 5a). The epidermal cells exhibit a distended cuticle (Fig. 5b-c). The Sudan Black B test (SBB) displayed highly vacuolated cells with parietal cytoplasm having several lipid droplets per cell (Fig. 5d). The test for the presence of proteins (ABB) and the test of ruthenium red showed no proteinaceous material (not shown) and no mucilage (Fig. 5e), respectively. The starch grains were observed in buds (PAS) (Fig. 5f-g), but their distribution was restricted: they were not found in the external epidermis of the sepals in bigger buds (Fig. 5f), totally absent in tissue of open flower. Such occurrence of starch grains could indicate the utilization of starch grains from the external epidermis of the sepals (Fig. 5f), then from the lip epidermis (Fig. 5g) and finally parenchyma cells in open flowers. Ultrastructural analysis of the lip groove showed epidermal cells with large nucleus (Fig. 6a) and one to few vacuoles per cell, sometimes contained flocculent material (Fig. 6a-b). The thick, striated cell wall
3.5. Gynostemium The gynostemium was short, with the column articulated with the lip (Fig. 7c). The staminodium formed a wide petaloid structure with raphides (Fig. 7c-d, also in Fig. 2g-i, 4a, d, f). The anther had two pollinia formed by two halves containing loosely connected pollen grains (Fig. 7d-e). The stigma was ventral, elliptic (Fig. 7f). The test with neutral red (see Fig. 4d-e) showed the accumulation of stain in the stigma, rostellum and viscidium, which were covered by fluid, also slightly in the anther, which was withered in open flowers (Fig. 4d). 26
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Fig. 2. Transverse sections of buds: from about 1,3 mm width (a) to the bud before anthesis (j): labellar secretory tissue in the groove (a–d, f–g, i), lip base (j) and lip lobes (a–d, f–g, i–j); substomatal cavities (asterisks) below the external epidermis of sepals (a-d, f–g, i–j), widened in bigger buds (i); secondary cell wall thickenings in endothecium - type I (d, e - detail of d, f–g, i–j, black arrowsheads); numerous raphides in idioblasts in all floral parts (a-b), also in the staminodium (g–i, h - detail of g). ds – dorsal sepal, gr - lip groove, i - idioblast with raphides, lb - lip lobes, lba - lip base, ls - lateral sepal, po - pollinia, pt – petal, s - stoma, sc, asterisk - substomatal cavity; st - stigma, vb - vascular bundle.
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Fig. 3. a. Flower – side view, sepals and petals surround the gynostemium like a helmet, sepals with abaxial keel (LM); b. abaxial (outer) surface of the lateral sepal with keel alongside the sepal (SEM); c. abaxial (outer) surface of the petal with folded margins (SEM); d. irregularly shaped cells, sometimes rectangular, with striated cuticle on the inner surface of petal (SEM); e. keel of lateral sepal with anomocytic stomata (SEM); f. abaxial (outer) surface of the dorsal sepal base with twocelled trichomes and anomocytic stomata (SEM). ds – dorsal sepal, ls - lateral sepal, pt – petal.
4. Discussion
Cymbidium lowianum Rchb.f. and C. devonianum Paxton (Davies et al., 2006). Additionally, the stomata present on the keels on the abaxial surface of sepals and large substomatal cavities, as described in Epipactis palustris (Kowalkowska et al., 2015a) and Bulbophyllum levanae (Wiśniewska et al., 2018) may be connected with fragrance emission, as in Acianthera (De Melo et al., 2010). They could function as osmophores, releasing fragrance from the bud stage, which could signalize the presence of developing flower for pollinators. The flowers are totally green, so we hypothesized that at first fragrance from sepals of buds could attract pollinators. The secretory function was not observed on the stem trichomes. They are only providing a good grip for pollinators (Claessens and Kleynen, 2011). The groove of N. ovata is formed by a single-layered epidermis, few layers of subepidermal cells and underlying parenchyma, described previously in species with superficial nectaries, such as Bulbophyllum wendlandianum (Kowalkowska et al., 2015b), in hypochile of E. palustris
The inconspicuous flowers of Neottia ovata secrete nectar onto the labellum surface and have a distinct and somewhat sweet scent that attracts many insect species (Brys et al., 2008). The survey of anatomical features of N. ovata confirmed that the lip base and the groove function as a nectary (the secretory tissue was present in buds about 1,3 mm), noted in previous ecological observations (Dressler, 1990; Claessens and Kleynen, 2011). The sweet scent comes from the aromatic constituents of the nectar and also scent is released by the lip lobes and margins of all tepals, which were stained in the test of neutral red. Such test was used to identify osmophores (Vogel, 1990; Kowalkowska et al., 2017). Because of the cytotoxicity of secretory material, the fragrance is produced and released periodically in osmophores (Stern et al., 1987; Vogel, 1990). The striation of cuticle may play a role as visual cues for pollinators reflecting light, noted in 28
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Fig. 4. a. Flower – frontal view, the outer whorl built by dorsal sepal and two lateral sepals, the inner whorl formed by two lateral petals and lip divided into two lobes, with groove and shallow lip base, gynostemium built by staminodium, anther, stigma (LM); b. longitudinal, median groove - slightly raised, especially at the end, older flower (LM); c. the nectar visible in a basal, shallow cavity in the lip base and the groove alongside the lip (LM); d. flower after staining with neutral red; note accumulation of stain in the tissue of the lip base and the groove (nectary); and on anther, rostellum, viscidium, stigma; and the lib lobes and the margins of all tepals (osmophores) (LM); e. detail of d (LM); f. flower with the shallow lip cavity and the groove on the adaxial surface (SEM); g. folded cuticle striation on the epidermal cells of groove, sometimes swelled, protruding (white arrowheads) (SEM); h. elongated, ellipsoidal cells on lip lobes (SEM); an - anther, co - column, ds – dorsal sepal, g – gynostemium, gr - lip groove, lb - lip lobes, lba - lip base, ls – lateral sepal, po - pollinnia, pt – petal, ro – rostellum, sm - staminodium, st – stigma, vi viscidium.
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Fig. 5. Histochemical tests: a. lip groove consisted of a single layer of epidermal cells, few layers of subepidermal cells, and parenchyma with collateral vascular bundles (TBO); b. detail of a, cells of the groove with distended cuticle (black arrowheads), secretory cells with large nucleus (TBO); c. distended cuticle in the groove (white arrowheads) (Auramine O); d. highly vacuolated cells with parietal cytoplasm having several lipid droplets per cell (SBB); e. lack of mucilage in the groove (Ruthenium Red); f. starch grains present in the parenchyma (black arrows), a few or not visible in the external epidermis of the sepals in bigger buds (PAS); g. starch grains (black arrows) noted in parenchyma of lip base, not present in the epidermis of the lip and sepals (PAS). ab - abaxial (outer) surface, ad - adaxial (inner) surface, ds - dorsal sepal, ee - external (outer) epidermis, gr - lip groove, i - idioblast with raphides, ie - inner (adaxial) epidermis, l - lipid droplet, lb - lip lobes, lba - lip base, ls - lateral sepal, n - nucleus, pa – parenchyma, pt - petal, s - stoma, sc - substomatal cavity, se - secretory epidermis, sm - staminodium, t - trichome, va - vacuole, vb - vascular bundle.
Kowalkowska et al., 2018; Naczk et al., 2018). Nectar can be accumulated under the cuticle and released by its rupture under the growing pressure outside the cell (Durkee, 1983; Curry et al., 1991), which was observed in Epipactis (Kowalkowska et al., 2015a, 2018). The presence of irregular plasmalemma, dictyosomes, and profiles of ER could also indicate the granulocrine mode of secretion, proposed by Fahn (1979). The pre-nectar (substances transported into nectary tissue to be transformed into nectar by the nectary parenchyma or epidermal cells; definition after Nepi, 2007) is transported through the symplast of the secretory parenchyma, then loaded onto ER or dictyosomes. The vesicles originated from ER or dictyosomes are fused with irregular plasmalemma and the nectar is released outside the cells. Such a process has frequently been reported in orchid secretory tissues (i.e. Kowalkowska et al., 2012, 2015b, 2018). The irregular plasmalemma with invaginations (observed in groove) and cell wall protuberances (observed in lip base) along the outer periclinal wall may function highly specialized cells - transfer cells, where an intensive transport of solutes is high through the plasmalemma (Gunning and Pate, 1974; Schnepf and Christ, 1980). The wall ingrowths, with the presence of cuticular micro-channels or pores, play a role in the nectar resorption.
(Kowalkowska et al., 2015a) and E. helleborine (Kowalkowska et al., 2018). In nectaries of about 12.6% of angiosperms (Frei, 1955), the presence of collateral vascular bundles are frequently noted, e.g. in E. palustris (Kowalkowska et al., 2015a), in B. wendlandianum (Kowalkowska et al., 2015b) and in Epipogium aphyllum (Święczkowska and Kowalkowska, 2015). At first, the carbohydrates of nectar are deposited in cells as starch and then is hydrolyzed as a source of energy during production and secretion of nectar and scent (Vogel, 1990; Pacini and Nepi, 2007; Nepi, 2007), which is a common feature of nectaries and osmophores (Stern et al., 1987; Pacini and Nepi, 2007; De Melo et al., 2010). The abundant starch grains present in buds, absent in sepals and lip epidermis in larger buds and almost absent in open flowers (visible in plastids in TEM) of N. ovata suggests gradual reduction during floral development and nectar secretion, which was also noted in E. helleborine (Kowalkowska et al., 2018). The nectar was gathered under the cuticle, which was visible in the groove and lip base, as in previously described Epipactis (Kowalkowska et al., 2015a, 2018). The main mode of nectar release is by diffusion through micro-channels, which is often observed in orchids (i.a. 30
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Fig. 6. Ultrastructural analysis of lip groove (TEM): a. epidermal cells with large nucleus, one to few vacuoles per cell, thick striated cell wall with cuticle, swollen on some cells (black arrowheads); b. vacuole with flocculent material, irregular plasmalemma; c. micro-channels in the cuticle and the secretory material on its surface (black arrowheads), irregular plasmalemma; d. details of c, micro-channels (white arrowheads), a few secretory material (black arrowheads); e. detail of a, micro-channels (white arrowheads), a material in the cell wall (black arrows); note that it is the same material as in cuticle; f–h. the dense parietal cytoplasm contained chloroplasts with starch grains, lipid droplets, mitochondria, irregular plasmalemma with vesicles, dictyosomes, profiles of SER and RER, ribosomes, mitochondria. c - cuticle, cw - cell wall, d - dictyosome, fl flocculent material, l - lipid droplet, m mitochondrion, n - nucleus, pl - plasmalemma, RER - rough endoplasmic reticulum, ri – ribosomes, SER - smooth endoplasmic reticulum, sg - starch grains, va - vacuole, ve - vesicles.
(Schnepf and Pross, 1976) and Strelitzia (Kronestedt and Robards, 1987), and in the lip nectary of Bulbophyllum cumingii (Kowalkowska et al., 2017). Lipids are frequently recorded in the nectary cells of orchids
Resorbed nectar can be translocated along the symplast and/or apoplast, and then is transported across vascular bundles to the reservoir of assimilates (Stpiczyńska et al., 2012). Cell wall ingrowths were found in septal nectaries of Tillandsia (Fiordi and Palandri, 1982), Gasteria, Aloe 31
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Fig. 7. TEM micrographs of lip basal cavity with the secretory epidermis: a. striated cell wall covered by cuticle, with the remnants of secreted material (black arrowheads); b. micro-channels in cuticle (white arrowheads) and some remnants of secreted material (black arrowheads), irregular plasmalemma with cell wall protuberances (black arrow); c. short gynostemium with column, articulated with the lip (white arrows), staminodium formed a wide petaloid structure with raphides (white arrowheads), anther without pollinia, pollinium attached to the stigma; d. gynostemium, anther with pollinia; e. two pollinia of two halves; f. ventral, elliptic stigma with rostellum (SEM). an - anther, co - column, po - pollinnia, ro – rostellum, sm - staminodium, st – stigma.
and in the "neottioid" genera, including some groups in the Spiranthoideae, Orchidoideae (Diurideae, Tropidia, Sarcoglottis, Disperis) and in the Epidendroideae (Vanilleae, Triphoreae) (Freudenstein, 1991). The endothecium secondary thickenings play a role for providing the mechanical forces for anther dehiscence and opening and release of pollen grains (Keijzer, 1987; Bonner and Dickinson, 1989; Scott et al., 2004, after Wilson et al., 2011). Althogether, these results of floral anatomy and ultrastructure will give more insight into pollination biology of N. ovata and will be useful for future projects of plant conservation planning.
(Figueiredo and Pais, 1992; Paiva, 2009). Several lipid droplets found in the plastid neighbourhood, as in previously examined orchids (Kowalkowska et al., 2017, 2018), suggest their participation in the production and exudation of substances and their subsequent transport by ER profiles or vesicles. They are likely constituents of nectar, as the nectar could also contain lipids, amino or organic acids, enzymes, antioxidants, vitamins, mineral ions, and secondary metabolites, next to the main components: sugars (glucose, fructose, sucrose) (Baker and Baker, 1975; Galetto et al., 1998; Lüttge and Schnepf, 1976). Lipids are also regarded as physical equivalents of fragrances (Swanson et al., 1980; Pridgeon and Stern, 1983; Curry et al., 1988; Kowalkowska et al., 2012). Numerous idioblasts were visible in all tepals, which could cause the light reflection and directing the pollinators attention to the centre of flowers (van der Cingel, 2001; Franceschi, 2001). However, their quantity in part of the staminodium was astonishing. Such idioblasts, with raphides of calcium oxalate crystals, are often described in orchid tissues (i.e. Kowalkowska and Magońska, 2009; Kowalkowska et al., 2015a; 2015b, 2018; Naczk et al., 2018; Wiśniewska et al., 2018), frequently accompanied by the secretory cells (nectaries, resin glands, elaiophores) (Davies and Stpiczyńska, 2012). They possibly help to prevent herbivory (Prychid and Rudall, 1999) or might be related to the exclusion of additional calcium from the cytosol (Paiva and Machado, 2008). In the anther, the endothecial cell thickenings were composed of numerous complete rings or helical segments in a closely spaced parallel arrangement. In Neottia the bars are occasionally half-open. This thickening variety was described as type I in Apostasia and Neuwiedia,
Acknowledgment This work was supported by University of Gdańsk [DS/530-L160D243-17]. References Baker, H.G., Baker, I., 1975. Studies of nectar-constituents and pollinator-plant coevolution. In: Gilbert, L.E., Raven, P.H. (Eds.), Coevolution of Plants and Animals. University Press of Texas, Austin. Bonner, L., Dickinson, H., 1989. Anther dehiscence in Lycopersicon esculentum Mill. New Phytol. 113, 97–115. Bronner, R., 1975. Simultaneous demonstration of lipids and starch in plant tissues. Stain Technol. 50 (1), 1–4. https://doi.org/10.3109/10520297509117023. Brys, R., Jacquemyn, H., Hermy, M., 2008. Pollination efficiency and reproductive patterns in relation to local plant density, population size and floral display in the rewarding Listera ovata (Orchidaceae). Bot. J. Linn. Soc. 157, 713–721. https://doi.org/ 10.1111/j.1095-8339.2008.00830.x. J. Claessens, J. Kleynen. The Flower of the European Orchid. Form and Function, 2011, Schrijen-Lippertz, Druk Voerendaal.
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mycoheterotrophic orchid Epipogium aphyllum, Orchidaceae (ghost orchid). Ann. Bot. 118 (1), 159–172. https://doi.org/10.1093/aob/mcw084. Kronestedt, E.C., Robards, A.W., 1987. Sugar secretion from the nectary of Strelitzia. Protoplasma 137, 168–182. https://doi.org/10.1007/BF01281152. Lüttge, U., Schnepf, E., 1976. Organic substances. In: Lüttge, U., Pitman, M.G. (Eds.), Transport in Plants. II B. Tissues and Organs. Springer-Verlag, Berlin, Heidelberg, New York, pp. 244–277. Naczk, A.M., Kowalkowska, A.K., Wiśniewska, N., Haliński, Ł.P., Kapusta, M., Czerwicka, M., 2018. Floral anatomy, ultrastructure and chemical analysis in Dactylorhiza incarnata/maculata complex (Orchidaceae). Bot. J. Linn. Soc. 187 (3), 512–536. https://doi.org/10.1093/botlinnean/boy027. Nepi, M., 2007. Nectary structure and ultrastructure. In: Nicolson, S.W., Nepi, M., Pacini, E. (Eds.), Nectaries and Nectar. Springer, Netherlands, pp. 129–166. Nepi, M., 2017. New perspectives in nectar evolution and ecology: simple alimentary reward or a complex multiorganism interaction? Acta Agrobot. 70 (1), 1704. https:// doi.org/10.5586/aa.1704. Nilsson, L.A., 1981. The pollination ecology of Listera ovata (Orchidaceae). Nord. J. Bot. 1, 461–480. Pacini, E., Nepi, M., 2007. Nectar production and presentation. In: Nicolson, S.W., Nepi, M., Pacini, E. (Eds.), Nectaries and Nectar. Springer, Rotterdam, pp. 167–214. Paiva, E.A.S., 2009. Ultrastructure and post-floral secretion of the pericarpial nectaries of Erythrina speciosa. Ann. Bot. 104, 937–944. https://doi.org/10.1093/aob/mcp175. Paiva, E.A.S., Machado, S.R., 2008. The floral nectary of Hymenaea stigonocarpa (Fabaceae, Caesalpinioideae): structural aspects during floral development. Ann. Bot. 101, 125–133. Pridgeon, A.M., Stern, W.L., 1983. Ultrastructure of osmophores in Restrepia (Orchidaceae). Am. J. Bot. 70 (8), 1233–1243. https://doi.org/10.2307/2443293. Prychid, C.J., Rudall, P.J., 1999. Calcium oxalate crystals in monocotyledons: a review of their structure and systematics. Ann. Bot. 84, 725–739. https://doi.org/10.1006/ anbo.1999.0975. Reynolds, E.S., 1963. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17 (1), 208–212. https://www.ncbi.nlm.nih.gov/ pmc/articles/PMC2106263/pdf/208.pdf. Ruzin, S.E., 1999. Plant Microtechnique and Microscopy. Oxford University Press, New York. Schnepf, E., Christ, P., 1980. Unusual transfer cells in the epithelium of the nectaries of Asclepias curassavica L. Protoplasma 105, 135–148. Schnepf, E., Pross, E., 1976. Differentiation and redifferentiation of a transfer cell: development of septal nectaries of Aloe and Gasteria. Protoplasma 89, 105–115. https:// doi.org/10.1007/BF01279332. Scott, R.J., Spielman, M., Dickinson, H.G., 2004. Stamen structure and function. Plant Cell 16 (Suppl. 1), S46–S60. Sprengel, C.K., 1793. Das entdeckte Geheimnis der Natur im Bau und in der Befruchtung der Blumen. Friedrich Vieweg dem aeltern, Berlin. https://doi.org/10.5962/bhl.title. 50179. Spurr, A.R., 1969. A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26, 31–43. Stern, W.L., Curry, K.J., Pridgeon, A.M., 1987. Osmophores of Stanhopea (Orchidaceae). Am. J. Bot. 74, 1323–1331. https://doi.org/10.2307/2444310. Stpiczyńska, M., Nepi, M., Zych, M., 2012. Secretion and composition of nectar and the structure of perigonal nectaries in Fritillaria meleagris. Plant Syst. Evol. 298, 997–1013. https://doi.org/10.1007/s00606012-0609-5. Swanson, E.S., Cunningham, W.P., Holman, R.T., 1980. Ultrastructure of glandular ovarian trichomes of Cypripedium caleolus and C. reginae (Orchidaceae). Am. J. Bot. 67, 784–789. https://doi.org/10.1007/s00606-012-0611-y. Szlachetko, D.L., Rutkowski, P., 2000. Gynostemia Orchidalium. Vol. 1. Apostasiaceae, Cypripediaceae, Orchidaceae (Thelymitroideae to Vanilloideae). Acta Bot. Fenn. 169, 1–380. Święczkowska, E., Kowalkowska, A.K., 2015. Floral nectary anatomy and ultrastructure in mycoheterotrophic plant, Epipogium aphyllum Sw. (Orchidaceae). Sci. World J. https://doi.org/10.1155/2015/201702. https://www.hindawi.com/journals/tswj/ 2015/201702/. Tałałaj, I., Ostrowiecka, B., Włostowska, E., Rutkowska, A., Brzosko, E., 2017. The ability of spontaneous autogamy in four orchid species: Cephalanthera rubra, Neottia ovata, Gymnadenia conopsea, and Platanthera bifolia. Acta Biol. Cracov. Bot. 59 (2), 51–61. https://doi.org/10.1515/abcsb-2017-0006. van der Cingel, N.A., 2001. An Atlas of Orchid Pollination: European Orchids. CRC Press, Brookfield. van der Pijl, L., Dodson, C.H., 1969. Orchid Flowers: Their Pollination and Evolution. University of Miami Press, Coral Gables, Florida. Vogel, S., 1990. The Role of Scent Glands in Pollination: On the Structure and Function of Osmophores. Amerind, New Delhi. Wilson, Z.A., Song, J., Taylor, B., Yang, C., 2011. The final split: the regulation of anther dehiscence. J. Exp. Bot. 62 (5), 1633–1649. https://doi.org/10.1093/jxb/err014. Wiśniewska, N., Kowalkowska, A.K., Kozieradzka-Kiszkurno, M., Krawczyńska, A.T., Bohdanowicz, J., 2018. Floral features of two species of Bulbophyllum section Lepidorhiza Schltr.: B. levanae Ames and B. nymphopolitanum Kraenzl. (Bulbophyllinae Schltr., Orchidaceae). Protoplasma 255 (2), 485–499. https://doi.org/10.1007/ s00709-017-1156-2.
Curry, K.J., Stern, W.L., McDowell, L.M., 1988. Osmophore development inStanhopea anfracta and S. pulla (Orchidaceae). Lindleyana 3, 212–220. Curry, K.J., McDowell, L.M., Judd, W.S., Stern, W.L., 1991. Osmophores, floral features, and systematics of Stanhopea (Orchidaceae). Am. J. Bot. 78, 610–623. Davies, K.L., Stpiczyńska, M., 2012. Comparative labellar anatomy of resin-secreting and putative resin-mimic species ofMaxillaria s.l. (Orchidaceae: Maxillariinae). Bot. J. Linn. Soc. 170, 405–435. https://doi.org/10.1111/j.1095-8339.2012.01278.x. Davies, K.L., Stpiczyńska, M., Turner, M.P., 2006. A rudimentary labellar speculum inCymbidium lowianum (Rchb.f.) Rchb.f. and Cymbidium devonianum Paxton (Orchidaceae). Ann. Bot. 97, 975–984. https://www.ncbi.nlm.nih.gov/pmc/articles/ PMC2803401/. De Melo, M.C., Borba, E.L., Paiva, E.A.S., 2010. Morphological and histological characterization of the osmophores and nectaries of four species ofAcianthera (Orchidaceae: Pleurothallidinae). Plant Syst. Evol. 286, 141–151. https://doi.org/10. 1007/s00606-010-0294-1. Delforge, P., 2006. Orchids of Europe, North Africa and the Middle East. A. & C. Black, London. Dressler, R.L., 1990. The Orchids - Natural History and Classification. Harvard University Press, London. Durkee, L.T., 1983. The ultrastructure of floral and extrafloral nectaries. In: Bentley, B., Elias, T. (Eds.), The Biology of Nectaries. Columbia University Press, pp. 1–29. Fahn, A., 1979. Ultrastructure of nectaries in relation to nectar secretion. Am. J. Bot. 66 (8), 977–985. https://doi.org/10.2307/2442240. Feder, N., O’Brien, T.P., 1968. Plant microtechnique: some principles and new methods. Am. J. Bot. 55, 123–142. https://doi.org/10.2307/2440500. Figueiredo, A.C.S., Pais, M.S., 1992. Ultrastructural aspects of the nectary spur of Limodorum abortivum (L.) Sw. Orchidaceae. Ann. Bot. 70, 325–331. https://hal. archives-ouvertes.fr/hal-00891238. Fiordi, A.C., Palandri, M.R., 1982. Anatomic and ultrastructural study of the septal nectary in some Tillandsia (Bromeliaceae) species. Caryologia 35, 477–489. Franceschi, V.R., 2001. Calcium oxalate in plants. Trends Plant Sci. 6 331-331. Frei, E., 1955. Die Innervierung der floralen Nektarien dikotyler Pflanzenfamilien. Ber Schweiz Bot Ges 65, 60–114. Freudenstein, J.V., 1991. A systematic study of endothecial thickenings in the Orchidaceae. Am. J. Bot. 78 (6), 766–781. https://www.jstor.org/stable/2445067. Gahan, P.B., 1984. Plant Histochemistry and Cytochemistry. Academic Press, London. Galetto, L., Bernardello, G., Sosa, C.A., 1998. The relationship between floral nectar composition and visitors in Lycium (Solanaceae) from Argentina and Chile: what does it reflect? Flora 193, 303–314. https://doi.org/10.1016/S0367-2530(17)30851-4. Gunning, B.E.S., Pate, J.S., 1974. Transfer cells. In: Robards, A.W. (Ed.), Dynamic Aspects of Plant Ultrastructure. McGraw-Hill, London, pp. 441–480. Heslop-Harrison, Y., 1977. The pollen-stigma interaction: pollen-tube penetration in Crocus. Ann. Bot. 41, 913–922. https://www.jstor.org/stable/42756464. Jensen, D.A., 1962. Botanical Histochemistry: Principles and Practice. Freeman, San Francisco. Johansen, D.A., 1940. Plant Microtechnique. McGraw-Hill Book Company, New York. Keijzer, C., 1987. The processes of anther dehiscence and pollen dispersal. I. The opening mechanism of longitudinally dehiscing anthers. New Phytol. 105, 487–489. Kotilínek, M., Těšitelová, T., Jersáková, J., 2015. Biological flora of the British Isles: Neottia ovata. J. Ecol. 103, 1354–1366. Kowalkowska, A.K., Margońska, H.B., 2009. Diversity of labellar micromorphological structures in selected species of Malaxidinae (Orchidales). Acta Soc. Bot. Pol. 78, 141–150. Kowalkowska, A.K., Margońska, H.B., Kozieradzka-Kiszkurno, M., 2010. Comparative anatomy of the lip spur and additional lateral sepal spurs in a three-spurred form (f. fumeauxiana) of Anacamptis pyramidalis. Acta Biol. Cracov. Bot. 52 (1), 13–18. http:// www2.ib.uj.edu.pl/abc/pdf/52_1/02_kowalkowska.pdf. Kowalkowska, A.K., Margońska, H.B., Kozieradzka-Kiszkurno, M., Bohdanowicz, J., 2012. Studies on the ultrastructure of a three-spurred fumeauxiana form of Anacamptis pyramidalis. Plant Syst. Evol. 298, 1025–1035. https://doi.org/10.1007/s00606-0120611-y. Kowalkowska, A.K., Kostelecka, J., Bohdanowicz, J., Kapusta, M., Rojek, J., 2015a. Studies on floral nectary, tepals’ structure, and gynostemium morphology of Epipactis palustris (L.) Crantz (Orchidaceae). Protoplasma 252, 321–333. https://doi.org/10. 1007/s00709-014-0668-2. Kowalkowska, A.K., Kozieradzka-Kiszkurno, M., Turzyński, S., 2015b. Morphological, histological and ultrastructural features of osmophores and nectary ofBulbophyllum wendlandianum (Kraenzl.) Dammer (B. section Cirrhopetalum Lindl., Bulbophyllinae Schltr., Orchidaceae). Plant Syst. Evol. 301 (2), 609–622. https://doi.org/10.1007/ s00606-014-1100-2. Kowalkowska, A.K., Turzyński, S., Kozieradzka-Kiszkurno, M., Wiśniewska, N., 2017. Floral structure of two species of Bulbophyllum section Cirrhopetalum Lindl.:B. weberi Ames and B. cumingii (Lindl.) Rchb. f. (Bulbophyllinae Schltr., Orchidaceae). Protoplasma 254, 1431–1449. https://doi.org/10.1007/s00709-016-1034-3. Kowalkowska, A.K., Pawłowicz, M., Guzanek, P., Krawczyńska, A.T., 2018. Floral nectary and osmophore of Epipactis helleborine (L.) Crantz (Orchidaceae). Protoplasma 255 (6), 1811–1825. https://doi.org/10.1007/s00709-018-1274-5. Krawczyk, E., Rojek, J., Kowalkowska, A.K., Kapusta, M., Znaniecka, J., Minasiewicz, J., 2016. Evidence for mixed sexual and asexual reproduction in the rare European
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Anatomical features related with pollination of Neottia ovata (L.) Bluff & Fingerh. (Orchidaceae)
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Agnieszka K. Kowalkowskaa, , Agnieszka T. Krawczyńskab a b
Department of Plant Cytology and Embryology, University of Gdańsk, Wita Stwosza 59, 80-308 Gdańsk, Poland Warsaw University of Technology, Faculty of Materials Science and Engineering, Wołoska 141, 02-507 Warsaw, Poland
A R T I C LE I N FO
A B S T R A C T
Edited by: Favio Gonzalez
The survey of anatomical features of Neottia ovata confirmed that the lip base and the groove function as a nectary. The secretory tissue was present in buds about 1,3 mm width. The sweet scent comes from the aromatic constituents of the nectar and also is released by the lip lobes and margins of all tepals. Additionally, the stomata present on the keels on the abaxial surface of sepals and large substomatal cavities may be connected with fragrance emission. The secretory function was not observed on the stem trichomes. The groove is formed by a single-layered epidermis, few layers of subepidermal cells and underlying parenchyma. The abundant starch grains present in buds, absent in sepals and lip epidermis in larger buds and almost absent in open flowers (visible in plastids in TEM) suggest gradual reduction during floral development and nectar secretion. The nectar was gathered under the cuticle, which was visible in the groove and lip base. The main mode of nectar release is by diffusion through micro-channels. The presence of irregular plasmalemma, dictyosomes, and profiles of ER could also indicate the granulocrine mode of secretion. The vesicles originated from ER or dictyosomes are fused with irregular plasmalemma. The irregular plasmalemma with invaginations (observed in groove) and cell wall protuberances (observed at the lip base) along the outer periclinal wall may function as highly specialized cells transfer cells. Numerous idioblasts, visible in all tepals and in the staminodium, attract pollinators. In the anther, the endothecial cell thickenings were composed of numerous complete rings or helical segments in a closely spaced parallel arrangement (type I), providing the mechanical forces for anther dehiscence and opening and release of pollen grains.
Keywords: Common twayblade Endothecial thickenings in anther Idioblasts in staminodium Lip nectary Osmophore
1. Introduction The Orchidaceae are known for their diverse floral structure, peculiar pollination mechanisms and different types of rewards. The most common feeding reward for visiting pollinators is nectar (van der Pijl and Dodson, 1969; Dressler, 1990). The presence of nectar, an aqueous solution with sugars, is considered to be a trait of plant–animal coevolution. The floral nectar, produced nearby the reproductive organs, facilitates pollen dispersal (Nepi, 2017). The nectar can be produced and exposed in orchids in diverse ways, i.e. in labellar superficial nectaries built by a single-layered epidermis and several layers of subepidermal cells (e.g. in Bulbophyllum Lindl.; Kowalkowska et al., 2015b, 2017; Wiśniewska et al., 2018), in hypochile and knobs of epichile (e.g. in Epipactis helleborine (L.) Crantz.; Kowalkowska et al., 2018; and Epipactis palustris (L.) Crantz.; Kowalkowska et al., 2015a), on labellar papillae (e.g. in Epipogium aphyllum Sw.; Święczkowska and Kowalkowska, 2015; Krawczyk et al., 2016; and in Dactylorhiza Neck.
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ex Nevski; Naczk et al., 2018) or in spurs formed at the base of the labellum (e.g. in Anacamptis pyramidalis f. fumeauxiana Marg. & Kowalk.; Kowalkowska et al., 2010, 2012; and in Epipogium aphyllum Sw.; Święczkowska and Kowalkowska, 2015), or tubular nectaries embedded alongside the ovary (van der Pijl and Dodson, 1969). Shallow, superficial nectaries are also found in Neottia Guett. (= Listera) R. Br. (Dressler, 1990). The genus consists of two species groups which were previously treated as separate genera, Neottia Guett. and Listera R. Br. (Claessens and Kleynen, 2011). Neottia ovata (L.) Bluff & Fingerh. (= Listera ovata (L.) R. Br.; common twayblade) is an inconspicuous perennial plant, entirely green (Claessens and Kleynen, 2011) with two opposite leaves on the hairy stem. It is one of the most common orchids in western Europe and its geographical range reaches as far as eastern Siberia in Asia where it can be found in forests and less frequently in meadows (Delforge, 2006). As the plant has green flowers, it offers no contrast with the surrounding plants (Claessens and Kleynen, 2011). Having no visual cues, the distinct, somewhat sweet
Corresponding author. E-mail address: [email protected] (A.K. Kowalkowska).
https://doi.org/10.1016/j.flora.2019.03.015 Received 3 October 2018; Received in revised form 2 March 2019; Accepted 21 March 2019 Available online 27 March 2019 0367-2530/ © 2019 Elsevier GmbH. All rights reserved.
Flora 255 (2019) 24–33
A.K. Kowalkowska and A.T. Krawczyńska
Following dehydration in an ethanol series the samples were prepared for scanning electron microscopy (SEM) and subjected to criticalpoint drying using liquid CO2, coated with gold and observed in a Philips XL-30 (Laboratory of Electron Microscopy, University of Gdańsk, Poland). For transmission electron microscopy (TEM), floral material was fixed in 2.5% (v/v) glutaraldehyde (GA) in 0.05 M cacodylate buffer (pH 7.0). The material was then post-fixed overnight in 1% (w/v) OsO4 in cacodylate buffer in a refrigerator and finally rinsed in buffer. After 1 h in a 1% (w/v) aqueous solution of uranyl acetate, the material was dehydrated with acetone and embedded in Spurr’s resin. Ultrathin sections were cut on a Leica EM UC 7 ultramicrotome with a diamond knife. The first part of sections was stained with uranyl acetate and lead citrate (Reynolds, 1963) and the second part of sections was contrasted with the UranyLess and Lead Citrate (the protocol available on http:// uranyless.com). The sections on Fig. 6 b–d were examined in a JEOL JEM 1200EX transmission electron microscope (Warsaw University of Technology) and on Fig. 6a, e–h; Fig. 7a-b were examined in a FEI transmission electron microscope (University of Gdańsk) at an accelerating voltage 120 kV.
scent is the main long-distance attractant, composed mainly by monoand sesquiterpenes, with linalool and trans-b-ocimene as the major components (Nilsson, 1981). Also terpenes have been found, such as the oxygenated and related terpenes perillen and dendrolasin. The hexadecanoic acid isopropyl ester and a constituent with a mass spectrum similar to isobutyl nitrile are present. The nectar is produced in the wide lip base and in less quantity in a central longitudinal groove (Claessens and Kleynen, 2011). After landing, a visiting insect licks the nectar secreted in a groove. Following the nectar trail, the insect is guided to the lip base and the gynostemium (Sprengel, 1793). The staminodium forms a wide petaloid structure behind the anther. The anther contains two pollinia in two pairs. The rostellum tip is transformed into a sticky, semi-fluid viscidium (Szlachetko and Rutkowski, 2000). Depending on the particular size of the insect, after touching the viscidium, the pollinia are expelled and attached to the insect’s head or thorax. The freely accessible nectar facilitates pollination and fruit set (Sprengel, 1793). Although the flowers are visited by a large suite of generalist insect species (Nilsson, 1981; Brys et al., 2008; Kotilínek et al., 2015), the main pollinators are ichneumonids, sawflies and beetles e.g. with 56, 28 and 10% of transported pollinia on the Island of Öland (Sweden) and 28, 54 and 15% of pollinia in Uppland (mainland Sweden), respectively (Nilsson, 1981). Although the flower macromorphology and pollination mechanism of Neottia ovata is well studied (Sprengel, 1793; Delforge, 2006; Claessens and Kleynen, 2011; Tałałaj et al., 2017), our studies are the first one on the anatomy and ultrastructure of N. ovata. The aims of the present research are: a) to examine the anatomy of structures related with pollination efficacy, and also the stem trichomes; b) to verify the presence of secretion in the flower (from bud stage to open flower); and c) to discuss the anatomical results alongside previously published research.
The stem was covered by numerous, 2–3 celled trichomes with a grandular apical cell (Fig. 1b-e). The transverse sections revealed typical monocot anatomy: a single layer of epidermis, cortex, sclerenchymatous layer, and ground tissue with numerous distinct collateral vascular bundles (Fig. 1e). The secretory activity was not observed on the stem trichomes.
2. Materials and methods
3.2. Buds
The plant material was collected in the beginning of June 2013 from young buds to recently open flowers (voucher number AKK 2013−001) from ravine near Staw Wróbla in Gdańsk (Fig. 1a). The fresh flowers were observed under a Nikon SMZ1500 stereomicroscope. For the purpose of localization of osmophores, the whole, fresh flowers were immersed in an aqueous solution of 001%-0,001% (w/v) neutral red for 20 min. Plant material was fixed in 2,5% (v/v) glutaraldehyde (GA) in 0.05 M cacodylate buffer (pH = 7.0). Material for light microscopy (LM), following fixation was rinsed with cacodylate buffer and then dehydrated in an acetone or ethanol series. Subsequently, material dehydrated in acetone was embedded in epoxy resin (Spurr, 1969), whereas material dehydrated in ethanol was embedded in methylmethacrylate-based resin (Technovit 7100, Heraeus Kulzer GmbH). For light microscopy, semi-thin control sections were stained with 0.05% (w/v) aqueous sodium tetraborate solution (Toluidine Blue O, TBO, C.I. 52040) (Feder and O’Brien, 1968; Ruzin, 1999). Aniline Blue Black (ABB, C.I. 20470) was used for the detection of water-insoluble proteins (Jensen, 1962) and a 0.05% (w/v) aqueous Ruthenium Red (C.I. 77800) solution to test for pectic acids/mucilage (Johansen 1940). The Periodic Acid-Schiff reaction (PAS) was used to identify the presence of water-insoluble polysaccharides (Jensen, 1962) and a 0.3% (w/v) ethanolic solution of Sudan Black B (SBB, C.I. 26150) for lipid localization (Bronner, 1975). The preparations were examined and photographed by means of a Nikon Eclipse E 800 light microscope and a Nikon DS–5Mc camera using the Lucia Image software. The Auramine O (C.I. 41000; 0.01% (w/v) solution in 0.05 M buffer Tris/HCl, pH = 7.2) was used to detect the presence of cuticle (Heslop-Harrison, 1977), especially unsaturated acidic waxes and cutin precursors (Gahan, 1984), and the staining reaction was examined by means of a Nikon Eclipse E800 fluorescence microscope equipped with filter: B-2A (EX 450–490 nm, DM 505 nm, BA 520 nm).
The presence of labellar secretory tissue was noted from buds (Fig. 2a, about 1,3 mm width) and developed in larger buds (Fig. 2b–j): in the groove (Fig. 2a–d, f, g, i), the lip base (Fig. 2j) and the lip lobes (Fig. 2a–d, f, g, i, j). The most remarkable feature was the occurrence of substomatal cavities (Fig. 2a-b) in the sepals, which widen (Fig. 2c, d, f, g, i, j) almost over the whole sepal width during development (Fig. 2i). The idioblasts with raphides were scattered in all tepals (Fig. 2a-b) and even filled the entire tissue in parts of the staminodium (Fig. 2g–i). Type I endothecial cell thickenings were observed in a wall layer of anthers (Fig. 2d-g, i, j).
3. Results 3.1. Stem
3.3. Open flower 3.3.1. Sepals and petals The sepals and petals surround the gynostemium like a helmet (Fig. 3a). The sepals (one dorsal and two laterals) were keeled (Fig. 3ab). The margins of petals (two lateral, inner whorl) were folded (Fig. 3c). The both surfaces of the sepals and petals were built by the irregularly shaped cells, sometimes rectangular, on the inner surface with striated cuticle (Fig. 3d). Anomocytic stomata were visible on both surfaces, but in higher numbers on the external keels (Fig. 3e). Twocelled trichomes were noted at the base of dorsal (on both surfaces) (Fig. 3f) and lateral sepals (only on external suface). 3.4. Lip The lip was divided into two lobes (Fig. 4a). A longitudinal, median groove was slightly raised, especially at the end (Fig. 4b). A basal, shallow cavity in the lip base and the groove alongside the lip was covered by nectar (Fig. 4c). The test with neutral red showed the accumulation of stain in the lip base and in the groove, as well as on the lip lobes and the margins of all tepals (Fig. 4d-e), which indicated the 25
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Fig. 1. a. Neottia ovata - plant; b. stem with trichomes (LM); c. trichomes (SEM); d. detail of c, 3-celled trichome with grandular top cell; e. transverse section of stem with a single layer of epidermis (e) with 2–3-celled trichomes, cortex (co), sclerenchymatous layer (sc), ground tissue with numerous distinct collateral vascular bundles (vb)(TBO).
was covered by cuticle (Fig. 6a, c–e), swollen on some cells (Fig. 6a). The micro-channels were visible in the cuticle, and a few secretory material on its surface (Fig. 6c–e). In the cell wall sometimes there was a material - the same as that, which built the cuticle (Fig. 6e). The dense parietal cytoplasm also contained chloroplasts with starch grains (Fig. 6f), lipid droplets (Fig. 6f), dictyosomes (Fig. 6g), profiles of SER and RER (Fig. 6g-h), ribosomes (Fig. 6g), mitochondria (Fig. 6f, h). The plasmalemma was irregularly formed with invaginations (Fig. 6b, f–h), sometimes with vesicles (Fig. 6f–h). TEM studies of lip basal cavity also proved the secretory epidermis with striated cell wall covered by cuticle with micro-channels and the remnants of secreted material on its surface (Fig. 7a-b). The irregular plasmalemma sometimes had cell wall protuberances (Fig. 7b).
secretory activity: the nectary in the lip base and groove and the osmophoric activity on the lip lobes and on the margins of all tepals. The micromorphological studies proved the occurrence of shallow lip cavity and the groove on the adaxial surface (Fig. 4f). The cuticle striation was more folded on the epidermal cells of groove, sometimes swelled, protruding, not appearing on each cell at the same time (Fig. 4g). On the lip lobes elongated ellipsoidal cells were observed (Fig. 4h). The lip tissue consisted of a single layer of epidermal cells, few layers of subepidermal cells, and parenchyma with collateral vascular bundles (Fig. 5a). The epidermal cells exhibit a distended cuticle (Fig. 5b-c). The Sudan Black B test (SBB) displayed highly vacuolated cells with parietal cytoplasm having several lipid droplets per cell (Fig. 5d). The test for the presence of proteins (ABB) and the test of ruthenium red showed no proteinaceous material (not shown) and no mucilage (Fig. 5e), respectively. The starch grains were observed in buds (PAS) (Fig. 5f-g), but their distribution was restricted: they were not found in the external epidermis of the sepals in bigger buds (Fig. 5f), totally absent in tissue of open flower. Such occurrence of starch grains could indicate the utilization of starch grains from the external epidermis of the sepals (Fig. 5f), then from the lip epidermis (Fig. 5g) and finally parenchyma cells in open flowers. Ultrastructural analysis of the lip groove showed epidermal cells with large nucleus (Fig. 6a) and one to few vacuoles per cell, sometimes contained flocculent material (Fig. 6a-b). The thick, striated cell wall
3.5. Gynostemium The gynostemium was short, with the column articulated with the lip (Fig. 7c). The staminodium formed a wide petaloid structure with raphides (Fig. 7c-d, also in Fig. 2g-i, 4a, d, f). The anther had two pollinia formed by two halves containing loosely connected pollen grains (Fig. 7d-e). The stigma was ventral, elliptic (Fig. 7f). The test with neutral red (see Fig. 4d-e) showed the accumulation of stain in the stigma, rostellum and viscidium, which were covered by fluid, also slightly in the anther, which was withered in open flowers (Fig. 4d). 26
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Fig. 2. Transverse sections of buds: from about 1,3 mm width (a) to the bud before anthesis (j): labellar secretory tissue in the groove (a–d, f–g, i), lip base (j) and lip lobes (a–d, f–g, i–j); substomatal cavities (asterisks) below the external epidermis of sepals (a-d, f–g, i–j), widened in bigger buds (i); secondary cell wall thickenings in endothecium - type I (d, e - detail of d, f–g, i–j, black arrowsheads); numerous raphides in idioblasts in all floral parts (a-b), also in the staminodium (g–i, h - detail of g). ds – dorsal sepal, gr - lip groove, i - idioblast with raphides, lb - lip lobes, lba - lip base, ls - lateral sepal, po - pollinia, pt – petal, s - stoma, sc, asterisk - substomatal cavity; st - stigma, vb - vascular bundle.
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Fig. 3. a. Flower – side view, sepals and petals surround the gynostemium like a helmet, sepals with abaxial keel (LM); b. abaxial (outer) surface of the lateral sepal with keel alongside the sepal (SEM); c. abaxial (outer) surface of the petal with folded margins (SEM); d. irregularly shaped cells, sometimes rectangular, with striated cuticle on the inner surface of petal (SEM); e. keel of lateral sepal with anomocytic stomata (SEM); f. abaxial (outer) surface of the dorsal sepal base with twocelled trichomes and anomocytic stomata (SEM). ds – dorsal sepal, ls - lateral sepal, pt – petal.
4. Discussion
Cymbidium lowianum Rchb.f. and C. devonianum Paxton (Davies et al., 2006). Additionally, the stomata present on the keels on the abaxial surface of sepals and large substomatal cavities, as described in Epipactis palustris (Kowalkowska et al., 2015a) and Bulbophyllum levanae (Wiśniewska et al., 2018) may be connected with fragrance emission, as in Acianthera (De Melo et al., 2010). They could function as osmophores, releasing fragrance from the bud stage, which could signalize the presence of developing flower for pollinators. The flowers are totally green, so we hypothesized that at first fragrance from sepals of buds could attract pollinators. The secretory function was not observed on the stem trichomes. They are only providing a good grip for pollinators (Claessens and Kleynen, 2011). The groove of N. ovata is formed by a single-layered epidermis, few layers of subepidermal cells and underlying parenchyma, described previously in species with superficial nectaries, such as Bulbophyllum wendlandianum (Kowalkowska et al., 2015b), in hypochile of E. palustris
The inconspicuous flowers of Neottia ovata secrete nectar onto the labellum surface and have a distinct and somewhat sweet scent that attracts many insect species (Brys et al., 2008). The survey of anatomical features of N. ovata confirmed that the lip base and the groove function as a nectary (the secretory tissue was present in buds about 1,3 mm), noted in previous ecological observations (Dressler, 1990; Claessens and Kleynen, 2011). The sweet scent comes from the aromatic constituents of the nectar and also scent is released by the lip lobes and margins of all tepals, which were stained in the test of neutral red. Such test was used to identify osmophores (Vogel, 1990; Kowalkowska et al., 2017). Because of the cytotoxicity of secretory material, the fragrance is produced and released periodically in osmophores (Stern et al., 1987; Vogel, 1990). The striation of cuticle may play a role as visual cues for pollinators reflecting light, noted in 28
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Fig. 4. a. Flower – frontal view, the outer whorl built by dorsal sepal and two lateral sepals, the inner whorl formed by two lateral petals and lip divided into two lobes, with groove and shallow lip base, gynostemium built by staminodium, anther, stigma (LM); b. longitudinal, median groove - slightly raised, especially at the end, older flower (LM); c. the nectar visible in a basal, shallow cavity in the lip base and the groove alongside the lip (LM); d. flower after staining with neutral red; note accumulation of stain in the tissue of the lip base and the groove (nectary); and on anther, rostellum, viscidium, stigma; and the lib lobes and the margins of all tepals (osmophores) (LM); e. detail of d (LM); f. flower with the shallow lip cavity and the groove on the adaxial surface (SEM); g. folded cuticle striation on the epidermal cells of groove, sometimes swelled, protruding (white arrowheads) (SEM); h. elongated, ellipsoidal cells on lip lobes (SEM); an - anther, co - column, ds – dorsal sepal, g – gynostemium, gr - lip groove, lb - lip lobes, lba - lip base, ls – lateral sepal, po - pollinnia, pt – petal, ro – rostellum, sm - staminodium, st – stigma, vi viscidium.
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Fig. 5. Histochemical tests: a. lip groove consisted of a single layer of epidermal cells, few layers of subepidermal cells, and parenchyma with collateral vascular bundles (TBO); b. detail of a, cells of the groove with distended cuticle (black arrowheads), secretory cells with large nucleus (TBO); c. distended cuticle in the groove (white arrowheads) (Auramine O); d. highly vacuolated cells with parietal cytoplasm having several lipid droplets per cell (SBB); e. lack of mucilage in the groove (Ruthenium Red); f. starch grains present in the parenchyma (black arrows), a few or not visible in the external epidermis of the sepals in bigger buds (PAS); g. starch grains (black arrows) noted in parenchyma of lip base, not present in the epidermis of the lip and sepals (PAS). ab - abaxial (outer) surface, ad - adaxial (inner) surface, ds - dorsal sepal, ee - external (outer) epidermis, gr - lip groove, i - idioblast with raphides, ie - inner (adaxial) epidermis, l - lipid droplet, lb - lip lobes, lba - lip base, ls - lateral sepal, n - nucleus, pa – parenchyma, pt - petal, s - stoma, sc - substomatal cavity, se - secretory epidermis, sm - staminodium, t - trichome, va - vacuole, vb - vascular bundle.
Kowalkowska et al., 2018; Naczk et al., 2018). Nectar can be accumulated under the cuticle and released by its rupture under the growing pressure outside the cell (Durkee, 1983; Curry et al., 1991), which was observed in Epipactis (Kowalkowska et al., 2015a, 2018). The presence of irregular plasmalemma, dictyosomes, and profiles of ER could also indicate the granulocrine mode of secretion, proposed by Fahn (1979). The pre-nectar (substances transported into nectary tissue to be transformed into nectar by the nectary parenchyma or epidermal cells; definition after Nepi, 2007) is transported through the symplast of the secretory parenchyma, then loaded onto ER or dictyosomes. The vesicles originated from ER or dictyosomes are fused with irregular plasmalemma and the nectar is released outside the cells. Such a process has frequently been reported in orchid secretory tissues (i.e. Kowalkowska et al., 2012, 2015b, 2018). The irregular plasmalemma with invaginations (observed in groove) and cell wall protuberances (observed in lip base) along the outer periclinal wall may function highly specialized cells - transfer cells, where an intensive transport of solutes is high through the plasmalemma (Gunning and Pate, 1974; Schnepf and Christ, 1980). The wall ingrowths, with the presence of cuticular micro-channels or pores, play a role in the nectar resorption.
(Kowalkowska et al., 2015a) and E. helleborine (Kowalkowska et al., 2018). In nectaries of about 12.6% of angiosperms (Frei, 1955), the presence of collateral vascular bundles are frequently noted, e.g. in E. palustris (Kowalkowska et al., 2015a), in B. wendlandianum (Kowalkowska et al., 2015b) and in Epipogium aphyllum (Święczkowska and Kowalkowska, 2015). At first, the carbohydrates of nectar are deposited in cells as starch and then is hydrolyzed as a source of energy during production and secretion of nectar and scent (Vogel, 1990; Pacini and Nepi, 2007; Nepi, 2007), which is a common feature of nectaries and osmophores (Stern et al., 1987; Pacini and Nepi, 2007; De Melo et al., 2010). The abundant starch grains present in buds, absent in sepals and lip epidermis in larger buds and almost absent in open flowers (visible in plastids in TEM) of N. ovata suggests gradual reduction during floral development and nectar secretion, which was also noted in E. helleborine (Kowalkowska et al., 2018). The nectar was gathered under the cuticle, which was visible in the groove and lip base, as in previously described Epipactis (Kowalkowska et al., 2015a, 2018). The main mode of nectar release is by diffusion through micro-channels, which is often observed in orchids (i.a. 30
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Fig. 6. Ultrastructural analysis of lip groove (TEM): a. epidermal cells with large nucleus, one to few vacuoles per cell, thick striated cell wall with cuticle, swollen on some cells (black arrowheads); b. vacuole with flocculent material, irregular plasmalemma; c. micro-channels in the cuticle and the secretory material on its surface (black arrowheads), irregular plasmalemma; d. details of c, micro-channels (white arrowheads), a few secretory material (black arrowheads); e. detail of a, micro-channels (white arrowheads), a material in the cell wall (black arrows); note that it is the same material as in cuticle; f–h. the dense parietal cytoplasm contained chloroplasts with starch grains, lipid droplets, mitochondria, irregular plasmalemma with vesicles, dictyosomes, profiles of SER and RER, ribosomes, mitochondria. c - cuticle, cw - cell wall, d - dictyosome, fl flocculent material, l - lipid droplet, m mitochondrion, n - nucleus, pl - plasmalemma, RER - rough endoplasmic reticulum, ri – ribosomes, SER - smooth endoplasmic reticulum, sg - starch grains, va - vacuole, ve - vesicles.
(Schnepf and Pross, 1976) and Strelitzia (Kronestedt and Robards, 1987), and in the lip nectary of Bulbophyllum cumingii (Kowalkowska et al., 2017). Lipids are frequently recorded in the nectary cells of orchids
Resorbed nectar can be translocated along the symplast and/or apoplast, and then is transported across vascular bundles to the reservoir of assimilates (Stpiczyńska et al., 2012). Cell wall ingrowths were found in septal nectaries of Tillandsia (Fiordi and Palandri, 1982), Gasteria, Aloe 31
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Fig. 7. TEM micrographs of lip basal cavity with the secretory epidermis: a. striated cell wall covered by cuticle, with the remnants of secreted material (black arrowheads); b. micro-channels in cuticle (white arrowheads) and some remnants of secreted material (black arrowheads), irregular plasmalemma with cell wall protuberances (black arrow); c. short gynostemium with column, articulated with the lip (white arrows), staminodium formed a wide petaloid structure with raphides (white arrowheads), anther without pollinia, pollinium attached to the stigma; d. gynostemium, anther with pollinia; e. two pollinia of two halves; f. ventral, elliptic stigma with rostellum (SEM). an - anther, co - column, po - pollinnia, ro – rostellum, sm - staminodium, st – stigma.
and in the "neottioid" genera, including some groups in the Spiranthoideae, Orchidoideae (Diurideae, Tropidia, Sarcoglottis, Disperis) and in the Epidendroideae (Vanilleae, Triphoreae) (Freudenstein, 1991). The endothecium secondary thickenings play a role for providing the mechanical forces for anther dehiscence and opening and release of pollen grains (Keijzer, 1987; Bonner and Dickinson, 1989; Scott et al., 2004, after Wilson et al., 2011). Althogether, these results of floral anatomy and ultrastructure will give more insight into pollination biology of N. ovata and will be useful for future projects of plant conservation planning.
(Figueiredo and Pais, 1992; Paiva, 2009). Several lipid droplets found in the plastid neighbourhood, as in previously examined orchids (Kowalkowska et al., 2017, 2018), suggest their participation in the production and exudation of substances and their subsequent transport by ER profiles or vesicles. They are likely constituents of nectar, as the nectar could also contain lipids, amino or organic acids, enzymes, antioxidants, vitamins, mineral ions, and secondary metabolites, next to the main components: sugars (glucose, fructose, sucrose) (Baker and Baker, 1975; Galetto et al., 1998; Lüttge and Schnepf, 1976). Lipids are also regarded as physical equivalents of fragrances (Swanson et al., 1980; Pridgeon and Stern, 1983; Curry et al., 1988; Kowalkowska et al., 2012). Numerous idioblasts were visible in all tepals, which could cause the light reflection and directing the pollinators attention to the centre of flowers (van der Cingel, 2001; Franceschi, 2001). However, their quantity in part of the staminodium was astonishing. Such idioblasts, with raphides of calcium oxalate crystals, are often described in orchid tissues (i.e. Kowalkowska and Magońska, 2009; Kowalkowska et al., 2015a; 2015b, 2018; Naczk et al., 2018; Wiśniewska et al., 2018), frequently accompanied by the secretory cells (nectaries, resin glands, elaiophores) (Davies and Stpiczyńska, 2012). They possibly help to prevent herbivory (Prychid and Rudall, 1999) or might be related to the exclusion of additional calcium from the cytosol (Paiva and Machado, 2008). In the anther, the endothecial cell thickenings were composed of numerous complete rings or helical segments in a closely spaced parallel arrangement. In Neottia the bars are occasionally half-open. This thickening variety was described as type I in Apostasia and Neuwiedia,
Acknowledgment This work was supported by University of Gdańsk [DS/530-L160D243-17]. References Baker, H.G., Baker, I., 1975. Studies of nectar-constituents and pollinator-plant coevolution. In: Gilbert, L.E., Raven, P.H. (Eds.), Coevolution of Plants and Animals. University Press of Texas, Austin. Bonner, L., Dickinson, H., 1989. Anther dehiscence in Lycopersicon esculentum Mill. New Phytol. 113, 97–115. Bronner, R., 1975. Simultaneous demonstration of lipids and starch in plant tissues. Stain Technol. 50 (1), 1–4. https://doi.org/10.3109/10520297509117023. Brys, R., Jacquemyn, H., Hermy, M., 2008. Pollination efficiency and reproductive patterns in relation to local plant density, population size and floral display in the rewarding Listera ovata (Orchidaceae). Bot. J. Linn. Soc. 157, 713–721. https://doi.org/ 10.1111/j.1095-8339.2008.00830.x. J. Claessens, J. Kleynen. The Flower of the European Orchid. Form and Function, 2011, Schrijen-Lippertz, Druk Voerendaal.
32
Flora 255 (2019) 24–33
A.K. Kowalkowska and A.T. Krawczyńska
mycoheterotrophic orchid Epipogium aphyllum, Orchidaceae (ghost orchid). Ann. Bot. 118 (1), 159–172. https://doi.org/10.1093/aob/mcw084. Kronestedt, E.C., Robards, A.W., 1987. Sugar secretion from the nectary of Strelitzia. Protoplasma 137, 168–182. https://doi.org/10.1007/BF01281152. Lüttge, U., Schnepf, E., 1976. Organic substances. In: Lüttge, U., Pitman, M.G. (Eds.), Transport in Plants. II B. Tissues and Organs. Springer-Verlag, Berlin, Heidelberg, New York, pp. 244–277. Naczk, A.M., Kowalkowska, A.K., Wiśniewska, N., Haliński, Ł.P., Kapusta, M., Czerwicka, M., 2018. Floral anatomy, ultrastructure and chemical analysis in Dactylorhiza incarnata/maculata complex (Orchidaceae). Bot. J. Linn. Soc. 187 (3), 512–536. https://doi.org/10.1093/botlinnean/boy027. Nepi, M., 2007. Nectary structure and ultrastructure. In: Nicolson, S.W., Nepi, M., Pacini, E. (Eds.), Nectaries and Nectar. Springer, Netherlands, pp. 129–166. Nepi, M., 2017. New perspectives in nectar evolution and ecology: simple alimentary reward or a complex multiorganism interaction? Acta Agrobot. 70 (1), 1704. https:// doi.org/10.5586/aa.1704. Nilsson, L.A., 1981. The pollination ecology of Listera ovata (Orchidaceae). Nord. J. Bot. 1, 461–480. Pacini, E., Nepi, M., 2007. Nectar production and presentation. In: Nicolson, S.W., Nepi, M., Pacini, E. (Eds.), Nectaries and Nectar. Springer, Rotterdam, pp. 167–214. Paiva, E.A.S., 2009. Ultrastructure and post-floral secretion of the pericarpial nectaries of Erythrina speciosa. Ann. Bot. 104, 937–944. https://doi.org/10.1093/aob/mcp175. Paiva, E.A.S., Machado, S.R., 2008. The floral nectary of Hymenaea stigonocarpa (Fabaceae, Caesalpinioideae): structural aspects during floral development. Ann. Bot. 101, 125–133. Pridgeon, A.M., Stern, W.L., 1983. Ultrastructure of osmophores in Restrepia (Orchidaceae). Am. J. Bot. 70 (8), 1233–1243. https://doi.org/10.2307/2443293. Prychid, C.J., Rudall, P.J., 1999. Calcium oxalate crystals in monocotyledons: a review of their structure and systematics. Ann. Bot. 84, 725–739. https://doi.org/10.1006/ anbo.1999.0975. Reynolds, E.S., 1963. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17 (1), 208–212. https://www.ncbi.nlm.nih.gov/ pmc/articles/PMC2106263/pdf/208.pdf. Ruzin, S.E., 1999. Plant Microtechnique and Microscopy. Oxford University Press, New York. Schnepf, E., Christ, P., 1980. Unusual transfer cells in the epithelium of the nectaries of Asclepias curassavica L. Protoplasma 105, 135–148. Schnepf, E., Pross, E., 1976. Differentiation and redifferentiation of a transfer cell: development of septal nectaries of Aloe and Gasteria. Protoplasma 89, 105–115. https:// doi.org/10.1007/BF01279332. Scott, R.J., Spielman, M., Dickinson, H.G., 2004. Stamen structure and function. Plant Cell 16 (Suppl. 1), S46–S60. Sprengel, C.K., 1793. Das entdeckte Geheimnis der Natur im Bau und in der Befruchtung der Blumen. Friedrich Vieweg dem aeltern, Berlin. https://doi.org/10.5962/bhl.title. 50179. Spurr, A.R., 1969. A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26, 31–43. Stern, W.L., Curry, K.J., Pridgeon, A.M., 1987. Osmophores of Stanhopea (Orchidaceae). Am. J. Bot. 74, 1323–1331. https://doi.org/10.2307/2444310. Stpiczyńska, M., Nepi, M., Zych, M., 2012. Secretion and composition of nectar and the structure of perigonal nectaries in Fritillaria meleagris. Plant Syst. Evol. 298, 997–1013. https://doi.org/10.1007/s00606012-0609-5. Swanson, E.S., Cunningham, W.P., Holman, R.T., 1980. Ultrastructure of glandular ovarian trichomes of Cypripedium caleolus and C. reginae (Orchidaceae). Am. J. Bot. 67, 784–789. https://doi.org/10.1007/s00606-012-0611-y. Szlachetko, D.L., Rutkowski, P., 2000. Gynostemia Orchidalium. Vol. 1. Apostasiaceae, Cypripediaceae, Orchidaceae (Thelymitroideae to Vanilloideae). Acta Bot. Fenn. 169, 1–380. Święczkowska, E., Kowalkowska, A.K., 2015. Floral nectary anatomy and ultrastructure in mycoheterotrophic plant, Epipogium aphyllum Sw. (Orchidaceae). Sci. World J. https://doi.org/10.1155/2015/201702. https://www.hindawi.com/journals/tswj/ 2015/201702/. Tałałaj, I., Ostrowiecka, B., Włostowska, E., Rutkowska, A., Brzosko, E., 2017. The ability of spontaneous autogamy in four orchid species: Cephalanthera rubra, Neottia ovata, Gymnadenia conopsea, and Platanthera bifolia. Acta Biol. Cracov. Bot. 59 (2), 51–61. https://doi.org/10.1515/abcsb-2017-0006. van der Cingel, N.A., 2001. An Atlas of Orchid Pollination: European Orchids. CRC Press, Brookfield. van der Pijl, L., Dodson, C.H., 1969. Orchid Flowers: Their Pollination and Evolution. University of Miami Press, Coral Gables, Florida. Vogel, S., 1990. The Role of Scent Glands in Pollination: On the Structure and Function of Osmophores. Amerind, New Delhi. Wilson, Z.A., Song, J., Taylor, B., Yang, C., 2011. The final split: the regulation of anther dehiscence. J. Exp. Bot. 62 (5), 1633–1649. https://doi.org/10.1093/jxb/err014. Wiśniewska, N., Kowalkowska, A.K., Kozieradzka-Kiszkurno, M., Krawczyńska, A.T., Bohdanowicz, J., 2018. Floral features of two species of Bulbophyllum section Lepidorhiza Schltr.: B. levanae Ames and B. nymphopolitanum Kraenzl. (Bulbophyllinae Schltr., Orchidaceae). Protoplasma 255 (2), 485–499. https://doi.org/10.1007/ s00709-017-1156-2.
Curry, K.J., Stern, W.L., McDowell, L.M., 1988. Osmophore development inStanhopea anfracta and S. pulla (Orchidaceae). Lindleyana 3, 212–220. Curry, K.J., McDowell, L.M., Judd, W.S., Stern, W.L., 1991. Osmophores, floral features, and systematics of Stanhopea (Orchidaceae). Am. J. Bot. 78, 610–623. Davies, K.L., Stpiczyńska, M., 2012. Comparative labellar anatomy of resin-secreting and putative resin-mimic species ofMaxillaria s.l. (Orchidaceae: Maxillariinae). Bot. J. Linn. Soc. 170, 405–435. https://doi.org/10.1111/j.1095-8339.2012.01278.x. Davies, K.L., Stpiczyńska, M., Turner, M.P., 2006. A rudimentary labellar speculum inCymbidium lowianum (Rchb.f.) Rchb.f. and Cymbidium devonianum Paxton (Orchidaceae). Ann. Bot. 97, 975–984. https://www.ncbi.nlm.nih.gov/pmc/articles/ PMC2803401/. De Melo, M.C., Borba, E.L., Paiva, E.A.S., 2010. Morphological and histological characterization of the osmophores and nectaries of four species ofAcianthera (Orchidaceae: Pleurothallidinae). Plant Syst. Evol. 286, 141–151. https://doi.org/10. 1007/s00606-010-0294-1. Delforge, P., 2006. Orchids of Europe, North Africa and the Middle East. A. & C. Black, London. Dressler, R.L., 1990. The Orchids - Natural History and Classification. Harvard University Press, London. Durkee, L.T., 1983. The ultrastructure of floral and extrafloral nectaries. In: Bentley, B., Elias, T. (Eds.), The Biology of Nectaries. Columbia University Press, pp. 1–29. Fahn, A., 1979. Ultrastructure of nectaries in relation to nectar secretion. Am. J. Bot. 66 (8), 977–985. https://doi.org/10.2307/2442240. Feder, N., O’Brien, T.P., 1968. Plant microtechnique: some principles and new methods. Am. J. Bot. 55, 123–142. https://doi.org/10.2307/2440500. Figueiredo, A.C.S., Pais, M.S., 1992. Ultrastructural aspects of the nectary spur of Limodorum abortivum (L.) Sw. Orchidaceae. Ann. Bot. 70, 325–331. https://hal. archives-ouvertes.fr/hal-00891238. Fiordi, A.C., Palandri, M.R., 1982. Anatomic and ultrastructural study of the septal nectary in some Tillandsia (Bromeliaceae) species. Caryologia 35, 477–489. Franceschi, V.R., 2001. Calcium oxalate in plants. Trends Plant Sci. 6 331-331. Frei, E., 1955. Die Innervierung der floralen Nektarien dikotyler Pflanzenfamilien. Ber Schweiz Bot Ges 65, 60–114. Freudenstein, J.V., 1991. A systematic study of endothecial thickenings in the Orchidaceae. Am. J. Bot. 78 (6), 766–781. https://www.jstor.org/stable/2445067. Gahan, P.B., 1984. Plant Histochemistry and Cytochemistry. Academic Press, London. Galetto, L., Bernardello, G., Sosa, C.A., 1998. The relationship between floral nectar composition and visitors in Lycium (Solanaceae) from Argentina and Chile: what does it reflect? Flora 193, 303–314. https://doi.org/10.1016/S0367-2530(17)30851-4. Gunning, B.E.S., Pate, J.S., 1974. Transfer cells. In: Robards, A.W. (Ed.), Dynamic Aspects of Plant Ultrastructure. McGraw-Hill, London, pp. 441–480. Heslop-Harrison, Y., 1977. The pollen-stigma interaction: pollen-tube penetration in Crocus. Ann. Bot. 41, 913–922. https://www.jstor.org/stable/42756464. Jensen, D.A., 1962. Botanical Histochemistry: Principles and Practice. Freeman, San Francisco. Johansen, D.A., 1940. Plant Microtechnique. McGraw-Hill Book Company, New York. Keijzer, C., 1987. The processes of anther dehiscence and pollen dispersal. I. The opening mechanism of longitudinally dehiscing anthers. New Phytol. 105, 487–489. Kotilínek, M., Těšitelová, T., Jersáková, J., 2015. Biological flora of the British Isles: Neottia ovata. J. Ecol. 103, 1354–1366. Kowalkowska, A.K., Margońska, H.B., 2009. Diversity of labellar micromorphological structures in selected species of Malaxidinae (Orchidales). Acta Soc. Bot. Pol. 78, 141–150. Kowalkowska, A.K., Margońska, H.B., Kozieradzka-Kiszkurno, M., 2010. Comparative anatomy of the lip spur and additional lateral sepal spurs in a three-spurred form (f. fumeauxiana) of Anacamptis pyramidalis. Acta Biol. Cracov. Bot. 52 (1), 13–18. http:// www2.ib.uj.edu.pl/abc/pdf/52_1/02_kowalkowska.pdf. Kowalkowska, A.K., Margońska, H.B., Kozieradzka-Kiszkurno, M., Bohdanowicz, J., 2012. Studies on the ultrastructure of a three-spurred fumeauxiana form of Anacamptis pyramidalis. Plant Syst. Evol. 298, 1025–1035. https://doi.org/10.1007/s00606-0120611-y. Kowalkowska, A.K., Kostelecka, J., Bohdanowicz, J., Kapusta, M., Rojek, J., 2015a. Studies on floral nectary, tepals’ structure, and gynostemium morphology of Epipactis palustris (L.) Crantz (Orchidaceae). Protoplasma 252, 321–333. https://doi.org/10. 1007/s00709-014-0668-2. Kowalkowska, A.K., Kozieradzka-Kiszkurno, M., Turzyński, S., 2015b. Morphological, histological and ultrastructural features of osmophores and nectary ofBulbophyllum wendlandianum (Kraenzl.) Dammer (B. section Cirrhopetalum Lindl., Bulbophyllinae Schltr., Orchidaceae). Plant Syst. Evol. 301 (2), 609–622. https://doi.org/10.1007/ s00606-014-1100-2. Kowalkowska, A.K., Turzyński, S., Kozieradzka-Kiszkurno, M., Wiśniewska, N., 2017. Floral structure of two species of Bulbophyllum section Cirrhopetalum Lindl.:B. weberi Ames and B. cumingii (Lindl.) Rchb. f. (Bulbophyllinae Schltr., Orchidaceae). Protoplasma 254, 1431–1449. https://doi.org/10.1007/s00709-016-1034-3. Kowalkowska, A.K., Pawłowicz, M., Guzanek, P., Krawczyńska, A.T., 2018. Floral nectary and osmophore of Epipactis helleborine (L.) Crantz (Orchidaceae). Protoplasma 255 (6), 1811–1825. https://doi.org/10.1007/s00709-018-1274-5. Krawczyk, E., Rojek, J., Kowalkowska, A.K., Kapusta, M., Znaniecka, J., Minasiewicz, J., 2016. Evidence for mixed sexual and asexual reproduction in the rare European
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