Botany New perspectives on secretory structures in Clusia (Clusiaceae - Clusiod clade): production of latex or resins? Journal: Botany Manuscript ID cjb-2019-0103.R2 Manuscript Type: Article Date Submitted by the 02-Dec-2019 Author: Complete List of Authors: Alencar, Ana Claudia; State University of Campinas, Biologia Vegetal Tölke, Elisabeth; State University of Campinas, Biologia Vegetal Mayer, Juliana; State University of Campinas, Biologia Vegetal Keyword: Cavities, glands,Draft Malpighiales, secretory structures, histochemistry Is the invited manuscript for consideration in a Special Not applicable (regular submission) Issue? : https://mc06.manuscriptcentral.com/botany-pubs Page 1 of 33 Botany New perspectives on secretory structures in Clusia (Clusiaceae - Clusiod clade): production of latex or resins? Ana Cláudia Alencar*¹; Elisabeth Dantas Tölke²; Juliana Lischka Sampaio Mayer*¹ ¹Universidade Estadual de Campinas, Instituto de Biologia, Departamento de Biologia Vegetal, rua Monteiro Lobato, 255, Campinas, SP. 2Universidade de São Paulo, Instituto de Biociência, Departamento de Botânica, rua do Matão, 277, Cidade Universitária, São Paulo, SP. [email protected] *Corresponding author: Draft [email protected] Tel. +55 (19) 9 8195-6490 [email protected] Tel. +55 (19) 9 8823-6779 1 https://mc06.manuscriptcentral.com/botany-pubs Botany Page 2 of 33 Abstract The Clusioid clade is quite diverse, encompassing the five families Clusiaceae, Bonnetiaceae, Calophyllaceae, Hypericaceae, and Podostemaceae. However, its members have important synapomorphies, such as the presence of xanthones, polyisoprenylated benzophenones, and quinones, as well as schizogenous secretory ducts and cavities, and tenuinucellate ovules. Due to the milky appearance of the exudate present in ducts of these families (except Bonnetiaceae), many authors have referred to this secretion as latex, while others have preferred to use the term resin. Faced with this confusion about the description of the exudate present in Clusiaceae, the aim of this study was to identify which classes of substances are being produced by those structures in the genus Clusia, as compared to other members of Clusiaceae and the Clusioid clade in general. Furthermore, we describe theDraft structure and distribution of the secretory ducts in species of Clusia, as well as a detailed histochemical study. These secretory ducts secrete an exudate of variable composition, but predominantly terpenic. We conclude that Clusia, as well as some representatives of the Clusioid clade, do not have latex but resiniferous secretory ducts. Keywords: Cavities, glands, Malpighiales, secretory structures, histochemistry. 2 https://mc06.manuscriptcentral.com/botany-pubs Page 3 of 33 Botany Introduction Clusiaceae (Malpighiales) are a pantropical family consisting of about 15 genera and 800 species (Stevens 2001). Clusiaceae with the four families Bonnetiaceae, Calophyllaceae, Hypericaceae, and Podostemaceae together form an assemblage referred to as the Clusioid clade. Despite being composed of families with very heterogeneous characteristics, this clade presents important synapomorphies, such as the production of shared chemical compounds (e.g., xanthones, polyisoprenylated benzophenones and quinones), schizogenous secretory ducts and cavities, and a tenuinucellate ovule (Stevens 2001; Ruhfel et al. 2011). Clusia is the largest genus of Clusiaceae and consists of approximately 300-400 species, which are distributed in the neotropical region from Central America to Southern Brazil (Stevens 2001; Gustafsson etDraft al. 2007). Clusia and all other genera of Clusiaceae are often recognized by the presence of an exudate, commonly milky or yellowish in color, which is secreted by all vegetative and reproductive organs when the plant suffers any kind of mechanical injury (Maguire 1966, 1979; Pipoly and Graff 1995a, b; Bittrich and Amaral 1997; Pipoly et al. 1998; Camara et al. 2018). In addition to this exudate, most Clusia species secrete resin in their flowers, namely in the stamens and/or staminodes (Armbruster 1984; Bittrich and Amaral 1996, 1997; Bittrich et al. 2006; Hochwallner and Weber 2006; Gustafsson et al. 2007; Amaral et al. 2017). The exudate is often described in revisions and floras of Clusia since it appears readily when the plant is cut or injured. Due to its variation in color, the exudate can be used for identification of species and even genera. Moreover, the distribution of secretory structures containing these exudates varies in the plant body, and ducts may or not be visible on the lamina. The details of the morphology and/or density of these secretory structures vary between species (Bittrich and Stevens 1998; Pipoly et al. 1998; Alencar 3 https://mc06.manuscriptcentral.com/botany-pubs Botany Page 4 of 33 and Marinho 2017; Nascimento-Jr et al. 2017). There are inconsistencies about how the exudate is described in the literature. Some authors describe a "resinous juice" (Choisy 1824; Cambessedes 1828; Lindley 1836; Planchon and Triana 1860), and other authors describe the exudate as latex (Pipoly and Graff 1995a, b; Vieira 1996; Bittrich and Amaral 1996, 1997; Pipoly et al. 1998; Silva et al. 2019). Although the exudate is frequently mentioned in taxonomic studies, the anatomical and histochemical knowledge of the secretory structures in Clusiaceae is fragmentary. Vesque (1892) conducted one of the pioneering works observing and describing these secretory structures in leaves of Clusia and other genera of Clusiaceae (some currently placed in Calophyllaceae). He described them as “glandulas canaliformes oleo-resiniferas” (Vesque 1892, p. 3). More recent anatomical studies of Clusiaceae have confirmed the presence of secretoryDraft ducts or cavities, but unlike Vesque, they called the secretion latex or, more vaguely, an exudate (Fernandes 2007; Rêgo and Kikuchi 2015; Silva et al. 2017, 2019). Faced with this confusion about the description of the exudate present in Clusiaceae, the aim of this study was to identify which classes of substances they yield in the genus Clusia, with comments on the family and the Clusioid clade. Furthermore, we describe the structure, distribution, and histochemistry of the secretory ducts in species of Clusia. Material and methods Material Leaves of Clusia obdeltifolia Bittrich, Clusia criuva ssp. parviflora (Engl.) Vesque and Clusia melchiorii Gleason and staminate anthetic flowers of 19 especies of Clusia (Table 1) were collected. Fresh leaves of Clusia criuva ssp. parviflora were used only for 4 https://mc06.manuscriptcentral.com/botany-pubs Page 5 of 33 Botany histochemical tests. All other leaves and flowers were fixed in Karnovsky's solution for 24 h (Karnovsky 1965) and prepared for both histochemical tests and light microscopy. The fixed material was dehydrated using an alcohol dilution series up to 70% ethanol and embedded in plastic resin (Leica Historesin). Histochemistry The main classes of compounds present in the secretions were investigated using the following histochemical tests: 1% nile blue sulfate for neutral and acid lipids (Cain 1947), sudan III for total lipids (Jensen 1962), NADI reagent for essential oils and resins (David and Carde 1964), periodic acid–Schiff reagent (PAS) for polysaccharides (Feder and O'Brien 1968), aniline blue black for proteins (Fisher 1968), 3% ferric chloride for phenolic compounds (Johansen 1940), oil red O for rubber (Jayabalan and Shah 1986) and lugol to confirm the presence ofDraft starch (Berlyn and Miksche 1976). These tests were applied to transverse and longitudinal sections of leaves and flowers that were obtained following the same protocol described for light microscopy. Some free hand sections were made in fresh leaves of Clusia criuva ssp. parviflora to compare with the results obtained with fixed material sections and to confirm that all substances identified are the same and that none could have been lost during the fixation process. Light microscopy Tranverse and longitudinal sections 5–7 μm thick were obtained using a rotary microtome (Leica Microsystems Richmond, Inc., Wetzlar, Germany). The sections were stained with 0.05% toluidine blue (Sakai 1973) in phosphate buffer and pH 4.5 citrate and permanently mounted in Entellan® synthetic resin (Merck KGaA). To document the results, images were captured using an Olympus DP71 digital camera coupled to an Olympus BX 51 microscope. 5 https://mc06.manuscriptcentral.com/botany-pubs Botany Page 6 of 33 Results Characterization of the exudate and histochemistry The exudate in leaves of Clusia is milky (Fig. 1a) but turns to orange or brown due to oxidization when it comes in contact with air (Fig. 1b). This secretion is initially fairly fluid and becomes thicker and more viscous during the oxidative process. In contrast, the flowers produce a small amount of exudate. When the organs are cut, it has the same characteristics as the exudate of the vegetative parts. However, in stamens of C. diamantina, the exudate is amber, orange, or red, translucent, and has a denser texture and is more viscous than the exudate of the vegetative parts and other floral organs. The histochemical tests performed on the leaves and flowers, fixed or fresh, demonstrated that the secretion produced by the secretory ducts consists of resins, essential oils, lipids, phenolic compounds,Draft and proteins. As far as the content of the exudate is concerned, there is no difference between the secretion produced by the ducts of the stamens and those of other