Botany Structure and function of secretory glochids and nectar composition in two Opuntioideae (Cactaceae) species Journal: Botany Manuscript ID cjb-2020-0004.R2 Manuscript Type: Article Date Submitted by the 06-Apr-2020 Author: Complete List of Authors: Silva, Stefany; Universidade Estadual Paulista Julio de Mesquita Filho, Botany Machado, Silvia; UNESP Nepi, Massimo; Università degli Studi di Siena Rodrigues,Draft Tatiane; UNESP, Botany Keyword: anatomy, cactus, nectar, secretory spines, ultrastructure Is the invited manuscript for consideration in a Special Not applicable (regular submission) Issue? : https://mc06.manuscriptcentral.com/botany-pubs Page 1 of 39 Botany Structure and function of secretory glochids and nectar composition in two Opuntioideae (Cactaceae) species Stefany Cristina de Melo Silva, Silvia Rodrigues Machado, Massimo Nepi, Tatiane Maria Rodrigues S.C.M. Silva UNESP - São Paulo State University, Institute of Biosciences, Department of Botany, 18618- 970, Botucatu city, São Paulo State, Brazil S.R. Machado UNESP - São Paulo State University,Draft Institute of Biosciences, Department of Botany, 18618- 970, Botucatu city, São Paulo State, Brazil Massimo Nepi University of Siena, Department of Life Sciences, 53100, Siena, Italy. T.M. Rodrigues UNESP - São Paulo State University, Institute of Biosciences, Department of Botany, 18618- 970, Botucatu city, São Paulo State, Brazil Corresponding author: Tatiane Maria Rodrigues (e-mail: [email protected]) 1 https://mc06.manuscriptcentral.com/botany-pubs Botany Page 2 of 39 ABSTRACT Cactaceae exhibit highly modified spines considered as extrafloral nectaries (EFNs). Despite their ecological and taxonomical relevance in this family, little is known on their structure and function. We described the anatomy, ontogenesis, and ultrastructure of the secretory glochids in two Opuntioideae species. Young cladodes of Brasiliopuntia brasiliensis and Nopalea cochenillifera were processed for light and electron microscopy studies. The secretion composition was analyzed by high performance liquid chromatography. Secretory glochids were soft, massive and barbed, as well as translucent. Hyaline droplets on the secretory glochid apex were collected by aggressive ants. Secretory glochids originated from the areolar meristem, beginning as small protuberances formed by protoderm and ground meristem. Mature secretory glochids consisted of a central multiseriate axis of ground cells covered by uniseriate epidermis with continuousDraft cuticle, and exhibited three regions: i) dilated vascularized base with parenchyma cells exhibiting features associated to nectar secretion; ii) elongated median region with juxtaposed fusiform non-lignified parenchyma cells; and iii) tapered apical portion with immature fibers loosely arranged cells. The exudate was sucrose- dominant with a similar amino acid profile in both species. Our results shed light on the secretory activity of glochids in Cactaceae and their role in cactus-ant interactions. Keywords: anatomy; Brasiliopuntia brasiliensis; cactus; nectar; Nopalea cochenillifera; secretory spines; ultrastructure 2 https://mc06.manuscriptcentral.com/botany-pubs Page 3 of 39 Botany Introduction Cladodes, succulent stems bearing areoles with spines, are a synapomorphy of Cactaceae (Nyffeler 2002; Judd et al. 2008). Two different morphological categories of spines are described in this family: larger and rigid spines consisting exclusively of lignified cells along their entire length; and smaller, hyaline and flexible spines covered by epidermis with beard-shaped cells and containing a non-lignified basal region, called glochids. The first type is widespread in Cactaceae, while glochids are restricted to Opuntioideae members (Gibson and Nobel 1986; Mauseth 2006). In several members of Cactaceae, including Opuntioideae, highly modified spines are involved in nectar secretion and have been considered as extrafloral nectaries (EFNs) (Díaz- Castelazo et al. 2005; Mauseth et al. 2016; Sandoval-Molina et al. 2018). However, detailed studies on the EFN structure in CactaceaeDraft are available for only few taxa and even species with the most obvious and highly modified glands are poorly studied (Mauseth et al. 2016). The morphology and localization of EFNs in Cactaceae have systematic value (Mauseth et al. 2016; Sandoval-Molina et al. 2018). In addition, the ecological importance of EFNs in members of the family has been emphasized in studies showing mutualistics interactions with ants and the secretion seasonality (Pickett and Clark 1979; Blom and Clark 1980; Oliveira et al. 1999; Ness 2006). Studies on the morphogenesis, structure and functioning of the EFNs in Cactaceae can present high added value, providing support and information for taxonomy (Mauseth et al. 2016) and ecological studies (Díaz-Castelazo et al. 2005; Sandoval-Molina et al. 2018). The structure of EFNs and nectar composition affects the attractiveness of plants to nectar-foraging insect visitors (Diaz-Castelazo et al. 2005, 2017). In general, the morphological and anatomical complexity of the EFNs is linked to their vascularization degree, being that the proportion of phloem and xylem in the EFNs reflects in the nectar concentration, i.e., the more developed the phloem, the more concentrated the nectar is (Fahn 3 https://mc06.manuscriptcentral.com/botany-pubs Botany Page 4 of 39 1979; Nepi 2007). Vascularized EFNs in Opuntioideae were reported only in Opuntia stricta (Oliveira et al. 1999), while non-vascularized EFNs seem to be predominant among this subfamily (see Sandoval-Molina et al. 2018 and cited references). The nectar from EFNs in Opuntioideae species is composed of sugars such as glucose, fructose and sucrose commonly found in the exudate of many other plant species; however, the amino acid concentration seems to be higher than what has been reported in the nectar from other plant groups (Pickett and Clark 1979). In order to understand the function of the secretory glochids, we conducted histological, cytological and chemical analyses in Brasiliopuntia brasiliensis (Willd.) A. Berger and Nopalea cochenillifera (L.) Salm-Dyck, two members of Opuntioideae co- occurring in the Atlantic Forest. Draft Materials and Methods Plant species Brasiliopuntia brasiliensis and Nopalea cochenillifera are shrubby species occurring in the Caatinga (an ecological region of northeastern Brazil occupied by tropical dry forest and scrub vegetation) and Atlantic Forest (Zappi and Taylor, in prep). We sampled adult individuals of B. brasiliensis (n = 4) and N. cochenillifera (n = 5) cultivated in the experimental area of the Department of Botany of the Institute of Biosciences of Botucatu (IBB), São Paulo State University (UNESP), Botucatu city (22°53’08” S, 48°26’42” W), São Paulo State, Brazil. The ants observed collecting nectar from EFNs were collected, stored in 70% alcohol and identified by Dr. Roberto da Silva Camargo of the Laboratory of Social Insects and Pests, Faculty of Agronomic Sciences (FCA), UNESP, Botucatu. 4 https://mc06.manuscriptcentral.com/botany-pubs Page 5 of 39 Botany Vouchers were deposited in the Herbarium Irina Delanova Gemtchúnicov (BOTU), Department of Botany, IBB, UNESP, under the register numbers 33069 and 33070 for B. brasiliensis and 32966, 32967 and 33071 for N. cochenillifera. The plant identification was confirmed by Dr. Lidyanne Yuriko Saleme Aona of the Universidade Federal do Recôncavo da Bahia. Light Microscopy EFNs in the secretory stage (swollen and containing exudate drops on their apex) were observed using a Leica M205C stereomicroscope. Images were recorded using a Leica DFC 425 digital camera coupled to the stereomicroscope. The length of the secretory spines was measured using a digital caliper. For histological and ontogeneticDraft studies, EFNs from cladodes at different developmental stages (from 3 to 10 cm of length) were collected, fixed in FAA 50 (formaldehyde 37%, acetic acid glacial and ethanol 50%) (Johansen 1940), dehydrated in an ethanol series and embedded in Leica methacrylate resin. Transverse and longitudinal sections (5μm thickness) were obtained serially using a Leica RM2245 semiautomatic rotating microtome and stained with 0.05% Toluidine Blue pH 4.7 (O’Brien et al. 1964). Permanent slides were mounted using synthetic resin Permount and analyzed using a Leica DMR light microscope under bright field and under polarized light. The results were documented using a Leica DFC425 digital camera. For identification of the main classes of compounds present in the cells of the EFNs, areolar samples (n = 4) of fresh material were sectioned by hand using a razor blade. The sections were treated with Fehling reagent for the identification of reducing sugars (Sass 1951); Ruthenium red 0.02% for acid polysaccharides and mucilage (Jensen 1962); periodic acid / Schiff reagent (PAS) for polysaccharides (Taboga and Vilamaior 2001); Lugol reagent for starch grains (Johansen 1940); Sudan IV for total lipids (Johansen 1940); bromophenol 5 https://mc06.manuscriptcentral.com/botany-pubs Botany Page 6 of 39 mercuric blue for proteins (Mazia et al. 1953); ferric chloride 10% for phenolic compounds (Johansen 1940) and hydrochloric acid 10% for calcium oxalate crystals (Chamberlain 1932). The control tests were performed according to the descriptor of each technique. The material was analyzed using a Leica DMR light microscope and documented with a Leica DFC425 digital camera. For detection of acidic polysaccharides, samples treated with Acridin orange