Botany Nectary and elaiophore work together in flowers of Xylopia aromatica (Annonaceae): structure indicates a role in pollination Journal: Botany Manuscript ID cjb-2020-0090.R3 Manuscript Type: Article Date Submitted by the 21-Aug-2020 Author: Complete List of Authors: Paiva, Elder; Universidade Federal de Minas Gerais Instituto de Ciencias Biologicas, Botany Galastri, Natália; Faculdade de Tecnologia de Jahu, Coordenadoria de Meio AmbienteDraft e Recursos Hídricos Oliveira, Denise; Universidade Federal de Minas Gerais, Departamento de Botânica Keyword: Annonaceae, elaiophore, floral rewards, nectary, Xylopia Is the invited manuscript for consideration in a Special Not applicable (regular submission) Issue? : © The Author(s) or their Institution(s) Page 1 of 28 Botany 1 1 Nectary and elaiophore work together in flowers of Xylopia aromatica (Annonaceae): 2 structure indicates a role in pollination 3 4 Elder Antônio Sousa Paiva1([email protected]) 5 Natália Arias Galastri1,2 ([email protected]) 6 Denise Maria Trombert Oliveira1 ([email protected]) 7 8 1 Departamento de Botânica, Instituto de Ciências Biológicas, Universidade Federal de Minas 9 Gerais, Belo Horizonte, Minas Gerais, Brazil 10 2 Coordenadoria de Meio Ambiente e Recursos Hídricos, Faculdade de Tecnologia de Jahu, 11 Jaú, São Paulo, Brazil 12 Draft 13 Short title: Nectary and elaiophore in Xylopia aromatica 14 15 Correspondence 16 E. A. S. Paiva, Departamento de Botânica, Universidade Federal de Minas Gerais, Av. 17 Antonio Carlos 6627, 31270-901, Belo Horizonte, Minas Gerais, Brazil. 18 Phone: +55 31 34092683 19 Fax: +55 31 34092671 20 E-mail: [email protected] 21 22 23 24 25 © The Author(s) or their Institution(s) Botany Page 2 of 28 2 26 Abstract: Secretory structures that produce floral rewards have been rarely reported for 27 Annonaceae. We identified a glandular region in Xylopia aromatica, which consisted of a 28 nectary and an elaiophore. This study aimed to describe the structure and secretory process of 29 these glandular structures, highly correlated to the reproductive biology of this species. 30 Anatomical and ultrastructural studies were performed prior to and during anthesis, focusing 31 on the channel and pollination chamber. The floral nectary is placed in the roof of the 32 chamber. It has a secretory epidermis and subglandular parenchyma and is immediately 33 contiguous with the elaiophore, a portion that delimits the pollination channel and produces 34 lipids. The release of nectar begins in the pistillate phase, while the elaiophore starts secreting 35 prior to anthesis, both of which finishing during the staminate phase. Lipids form a sticky 36 layer covering the channel surface, which provides access to the chamber. The cell machinery 37 of the epidermis for both, nectary andDraft elaiophore, is highly correlated with the exudates, 38 despite their high structural similarity. Nectar attracts pollinators to the pollination chamber, 39 while lipids seem to act in pollen adhesion to the body of pollinators. Both of exudates appear 40 to act in complementary ways during pollination. 41 Key words: Annonaceae, elaiophore, floral rewards, nectary, secretion, Xylopia 42 43 44 45 46 47 48 49 50 © The Author(s) or their Institution(s) Page 3 of 28 Botany 3 51 Introduction 52 Xylopia L. is the only genus of Annonaceae with a pantropical distribution and is the 53 second largest genus in the family with 160–180 species worldwide (Johnson and Murray 54 2018). Xylopia aromatica (Lam.) Mart. occurs in several vegetation types of the Brazilian 55 cerrado (Lorenzi 1992; Durigan et al. 2004). The flowers are monocline, erect and whitish, 56 and present a small pollination chamber formed by closing of the three inner petals. The 57 flowers emit a strong sweet odor during anthesis, which is produced in an osmophore placed 58 at the distal portion of outer and inner petals (Paiva, EAS. unpublished data). The species is 59 the only member of Annonaceae of the cerrado for which thrips (Thysanoptera) are the main 60 pollinators, while beetles of the genus Cillaeus Laporte (Nitidulidae), and eventually certain 61 Staphylinidae and Chrysomelidae, are secondary pollinators (Gottsberger 1970; Silberbauer- 62 Gottsberger et al. 2003; Gottsberger andDraft Silberbauer-Gottsberger 2006). Annonaceae stands 63 out for its intricate floral biology, which is strongly focused on plant-insect interaction. 64 Structure, metabolism, and floral rewards, like heating, odor, shelter (Gottsberger 1999), 65 nectar (Okada 2014; Xue et al. 2017), nutritious tissue (Gottsberger and Webber 2018), or 66 according to Lau et al. (2017), stigmatic secretions can be involved in pollination depending 67 on the species. 68 As is typical in Annonaceae, X. aromatica has protogynous flowers with diurnal 69 anthesis that lasts two days. The pistillate phase takes place early in the morning of the first 70 day when pollinators, especially thrips and small beetles, can access the pollination chamber 71 (Gottsberger 1994; Silberbauer-Gottsberger et al. 2003). The staminate phase starts in the 72 morning of the second day, during which the stamens detach (Silberbauer-Gottsberger et al. 73 2003). The petals detach at the end of the second day, and insects impregnated with pollen fly 74 to other flowers where pollination occurs again (Gottsberger 1994). © The Author(s) or their Institution(s) Botany Page 4 of 28 4 75 The efficiency of pollination by thrips depends on several factors, mainly on the 76 physical interaction between the smooth insect body and the surface of the pollen grains. 77 Therefore, the availability of pollen and nectar as rewards, pollen-stigma interaction, pollen- 78 wall architecture, and mechanisms of pollen-insect adhesion are important factors for 79 successful thrips-flower interaction (Ananthakrishnan 1993). 80 Few anatomical descriptions have been published on species of Xylopia, and no 81 structural or ultrastructural data can be found in the literature on secretory structures of 82 flowers of this genus. The occurrence of glandular or specialized food reward tissues was 83 reported for several species of Annonaceae by Xue et al. (2017). Still, these authors stated that 84 “most observations of glandular or specialized tissues were based on the examination of 85 herbarium specimens”. In these conditions, the samples are dried and the exudate absent, so 86 nectar release has been confirmed for Draftjust a few taxa: Orophea Blume (Kessler 1988; Okada 87 2014), Alphonsea glandulosa Y.H.Tan & B.Xue (Xue et al. 2017), and Pseuduvaria Miq. 88 (Silberbauer-Gottsberger et al. 2003; Su and Saunders 2006; Pang et al. 2013). There is also a 89 possibility of floral nectaries occurring in Asimina Adans. because, according to Norman and 90 Clayton (1986, page 21), “The food tissue found on the surface of the inner petals of A. 91 obovata is high in carbohydrates with lower concentrations of proteins and lipids. This tissue, 92 on which small droplets of exudate are occasionally seen, is eaten by beetles”. Although 93 Norman and Clayton (1986) did not describe the presence of floral nectaries, the evidence is 94 quite strong in this regard. Besides, Goodrich et al. (2006) illustrated the inner petals of 95 Asimina, which release “glistening” liquid secretions. Indeed, many Annonaceae species have 96 perianth glands, usually disposed at the base or margins of the adaxial surface of the inner 97 petals (Saunders 2010). In most of them, the secretory activity is understudied, but nectar 98 seems to be the most probable exudate, as suggested by data presented by Saunders (2010). 99 The author mentions Xylopia as showing glandular structures in the perianth, similar to some © The Author(s) or their Institution(s) Page 5 of 28 Botany 5 100 species from tropical Africa studied by Johnson and Murray (2018). Therefore, there is a 101 knowledge gap about perianth glands in Annonaceae and the significance of their secretory 102 activity in the pollination biology of the family. 103 Considering that our preliminary observations indicated the occurrence of secretion on 104 the inner petals of X. aromatica, this work described the structure of the glandular portion and 105 the biology of the secretory process. It also aimed to determine the chemical nature of the 106 secretory products to provide information to better understand the complex floral biology and 107 interactions with pollinators of this singular species of the Brazilian cerrado. 108 109 Materials and methods 110 Samples of floral buds, and of flowers at pistillate and staminate phases of anthesis 111 were collected from five individuals ofDraft Xylopia aromatica (Lam.) Mart. occurring in cerrado 112 areas nearby Botucatu, São Paulo, Brazil (22° 43'5,1"S, 48°20'58,1"W). Fertile branches were 113 included in the Herbarium BHCB of the Universidade Federal de Minas Gerais, Belo 114 Horizonte, Minas Gerais, Brazil (Galastri N.A. 7, BHCB 154418). 115 Due to daytime anthesis, field observations were performed from 6AM to 6PM 116 (UTC−3, BRT) in both the first and second days of anthesis. To verify the occurrence of the 117 nectary, strips for glucose detection (Alamar Tecno Científica, São Paulo, Brazil) were used 118 to test for the presence of this sugar in secretion samples. 119 Anatomical studies were performed on material fixed in Karnovsky solution 120 (Karnovsky 1965) for 24 hours, then partially dehydrated and preserved in 70% ethanol 121 (Jensen 1962). After dehydration in an ethanol series and freezer embedding in (2- 122 hydroxyethyl)-methacrylate (Leica, Heidelberg, Germany), according to Paiva et al. (2011), 123 the material was sectioned into 8μm-thick transversal and longitudinal sections with a Hyrax 124 M40 rotary microtome (Zeiss, Walldorf, Germany). Sections were stained with 0.05% © The Author(s) or their Institution(s) Botany Page 6 of 28 6 125 toluidine blue in acetate buffer, pH 4.7 (O’Brien et al. 1964, modified), and mounted with 126 Entellan.