Protoplasma (2017) 254:353–366 DOI 10.1007/s00709-016-0956-0 ORIGINAL ARTICLE Floral ultrastructure of two Brazilian aquatic-epiphytic bladderworts: Utricularia cornigera Studnička and U. nelumbifolia Gardner (Lentibulariaceae) Bartosz J. Płachno1 & Małgorzata Stpiczyńska2 & Kevin L. Davies3 & Piotr Świątek4 & Vitor Fernandes Oliveira de Miranda5 Received: 6 January 2016 /Accepted: 22 February 2016 /Published online: 5 March 2016 # The Author(s) 2016. This article is published with open access at Springerlink.com Abstract Utricularia cornigera and Utricularia nelumbifolia was the presence of relatively large, polymorphic plastids are giant, aquatic-epiphytic species of carnivorous blad- (chromoplasts) containing many plastoglobuli. Similar derwort from southeastern Brazil that grow in the central plastids are known to occur in the fragrance-producing ‘urns’ of bromeliads. Both species have large, colourful (osmophores) and oil-producing (elaiophores) tissues of flowers. The main aim of our study is to ascertain whether several orchid species. Thus, these palate papillae may the prominent floral palate of U. cornigera and play a key role in providing the olfactory stimulus for U. nelumbifolia functions as an unguentarius—i.e. an or- the attraction of insect pollinators. Nectariferous tri- gan that bears osmophores. Floral tissues of both species chomes were observed in the floral spurs of both species, were investigated using light microscopy, scanning elec- and in U. nelumbifolia, free nectar was also recorded. The tron microscopy, transmission electron microscopy and location, micro-morphology, anatomy and ultrastructure histochemistry. Floral palates of U. cornigera and of the floral palate of the two species investigated may U. nelumbifolia provide clear visual signals for pollinat- thus indicate that the palate functions as an unguentarius. ing insects. In both species, the palate possesses diverse Furthermore, the flowers of these taxa, like those of micro-morphology, comprising unicellular, conical to vil- U. reniformis, have features consistent with bee liform papillae and multicellular, uniseriate, glandular tri- pollination. chomes that frequently display terminal branching. The most characteristic ultrastructural feature of these papillae Keywords Bladderwort . Carnivorous plant . Floral micro-morphology . Lentibulariaceae . Osmophore . Palate . Pollination . Sect. Iperua . Ultrastructure Handling Editor: Andreas Holzinger * Bartosz J. Płachno [email protected] Introduction One of the largest families of carnivorous plants is 1 Department of Plant Cytology and Embryology, Jagiellonian Lentibulariaceae, its largest genus being Utricularia L., University in Kraków, 9 Gronostajowa St., 30-387 Kraków, Poland which is well known for its bladder-type traps that capture 2 Faculty of Biology, University of Warsaw, Botanic Garden Al. prey (Juniper et al. 1989;Adamec2011). Utricularia spe- Ujazdowskie 4, 00-478 Warsaw, Poland cies possess a bilabiate corolla extending posteriorly to 3 School of Earth and Ocean Sciences, Cardiff University, Main form a floral spur. The colour of the corolla, which typi- Building, Park Place, Cardiff CF10 3AT, UK cally measures 5 mm–2cm(Taylor1989), changes to 4 Department of Animal Histology and Embryology, University of yellow and/or violet. The largest flowers for the genus Silesia, 9 Bankowa St., 40-007 Katowice, Poland occur in aquatic-epiphytic species which grow in the cen- ‘ ’ 5 Departamento de Biologia Aplicada à Agropecuária, Faculdade de tral urns of bromeliads (Utricularia cornigera Ciências Agrárias e Veterinárias, Univ Estadual Paulista—UNESP, Studnička, Utricularia nelumbifolia Gardner and Câmpus Jaboticabal, São Paulo, Brazil Utricularia humboldtii Schomb., all species of sect. 354 B.J. Płachno et al. Iperua P. Taylor), some terrestrial/lithophyte species trichomes (osmophores) densely distributed upon the (Utricularia reniformis A.St.-Hil sect. Iperua, modified floral appendages (Płachno et al. 2015). Utricularia longifolia sect. Phyllosperma P. Taylor) and According to Taylor (1989), the lower lip of the corolla of some epiphytic species (e.g. Utricularia alpina Jacq. sect. Utricularia is expanded, forming the palate. This structure is Orchidioides A.DC.—Taylor 1989; Guisande et al. 2007; often ornamented, is pubescent or glandular, and often has Studnička 2009, 2011). These all have showy flowers and diagnostic value in taxonomical studies. Moreover, the palate are often cultivated as ornamental carnivorous plants. can easily be distinguished from the rest of the corolla owing Furthermore, some have formed the subject of embryolog- to its distinctly different colour. ical (Płachno and Świątek 2012), seed and seedling struc- This study aims to identify the site of floral scent pro- ture (Studnička 2009;Płachno and Świątek 2010; duction and secretion in Utricularia sect. Iperua.Inpar- Menezes et al. 2014), ecological (Studnička 2011)and ticular, it aims to ascertain whether the prominent palate genetic studies (Clivati et al. 2012).Withthesoleexcep- functions as an unguentarius—i.e. an organ that bears tion of U. reniformis (Clivati et al. 2014), detailed obser- scent glands or osmophores (Płachno et al. 2015). The vations of plant–pollinator interactions are lacking for micro-morphology of the floral spurs of both species these species. Pollination of the small, terrestrial species was also investigated. Utricularia albocaerulea Dalz., Utricularia graminifolia Vahl. (=Utricularia purpurascens Graham) and Utricularia reticulata Sm. (sect. Oligocista A.DC.) was reported in detail by Hobbhahn et al. (2006), who showed Material and methods that they were pollinated by numerous insect pollinators, such as bees, butterflies, moths and dipterans. To date, Species used in this study include U. cornigera Studnička only two species of pollinators (Xylocopa sp. and clone U9B (which was used as the holotype, Studnička Bombus sp.) have been recorded for U. reniformis 2009) and clone U9 obtained from Botanická zahrada (Clivati et al. 2014). Recently, however, it was proposed Liberec, Czech Republic (Fig. 1a–c)andU. nelumbifolia that in the Australian species Utricularia dunlopii,where Gardner obtained from the living collections of Jagiellonian the nectary spur is reduced, pollinators are attracted large- University Botanical Garden in Kraków. Some additional ma- ly by the insectiform configuration of the flower and vol- terial was provided by Botanická zahrada Liberec, atilization of fragrance putatively produced by glandular Czech Republic (Fig. 8a, b). Fig. 1 Gross and floral morphology of Utricularia cornigera. a Cultivated clone U9B at Botanic Garden Liberec. b Floral morphology of Utricularia cornigera clone U9B: palate (arrows) with distinct nectar guides. c Floral morphology of Utricularia cornigera clone U9: palate (arrows). d Micro- morphology of palate; bar =300μm. e Conical papillae (dorsal and lateral views) of palate coinciding with position of nectary guides; pollen grain (arrow); bar =100μm Palate of aquatic-epiphytic bladderworts 355 Fig. 2 Micro-morphology of Utricularia cornigera palate. a Papillae and glandular trichomes; bar =300μm. b Glandular trichomes with acorn-shaped head; bar =50μm. c Glandular trichome with bicellular head; head cells are of similar size; bar =30μm. d Glandular trichome with bicellular head; head cells differ in size; bar =20μm Floral structure and histochemistry Biology and Imaging, Institute of Zoology, Jagiellonian University in Kraków. The distribution of secretory glandular trichomes and unicel- Semi-thin sections (0.9–1.0 μm thick) were prepared lular papillae was determined by examining entire flowers for light microscopy (LM) and stained for general histol- using a Nikon SZ100 stereoscopic microscope. We interpret ogy using aqueous methylene blue/azure II (MB/AII) for the term ‘palate’ in Utricularia as the inflated base of the 1–2 min (Humphrey and Pittman 1974) and examined lower lip of the corolla, which differs both morphologically with an Olympus BX60 light microscope. The periodic and in terms of colour from the remaining part of the perianth. acid-Schiff (PAS) reaction was also used to reveal the Floral parts bearing papillae and glandular trichomes, presence of insoluble polysaccharides, and Sudan Black namely the palate and spur, were examined using light micros- B was used to detect the presence of lipids (Jensen copy (LM), scanning electron microscopy (SEM) and trans- 1962). Staining for total proteins was achieved using mission electron microscopy (TEM), as follows: Firstly, the Coomassie brilliant blue R250 or Ponceau 2R (Fisher epidermis of the floral palate was examined during anthesis, 1968; Ruzin 1999). Material was also tested for lipids, and pieces of floral tissue were excised and fixed in 2.5 % (v/v) starch and mucilage using a saturated ethanolic solution glutaraldehyde 2.5 % (v/v) formaldehyde in 0.05 M sodium of Sudan III, aqueous IKI (iodine-potassium iodide) solu- cacodylate buffer (pH 7.0) for 2 h at 4 °C, washed three times tion and ruthenium red solution, respectively (Ruzin in 0.05 sodium cacodylate buffer pH 7 and post-fixed in 1 % 1999). (w/v) osmium tetroxide solution for 1.5 h at 0 °C. Dehydration A Nikon Eclipse E200 camera and an Olympus BX60 using a graded ethanol series and infiltration and embedding microscope were used for general photography and using an epoxy embedding medium kit (Fluka) followed. micrometry/photomicrography, respectively. Following polymerization at 60 °C, sections