(Annonaceae): Structure Indicates a Role in Pollination

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? :

1 Nectary and elaiophore work together in flowers of Xylopia aromatica (Annonaceae):

2 structure indicates a role in pollination

4 Elder Antônio Sousa Paiva1([email protected])

5 Natália Arias Galastri1,2 ([email protected])

6 Denise Maria Trombert Oliveira1 ([email protected])

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

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]

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

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).

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

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%

125 toluidine blue in acetate buffer, pH 4.7 (O’Brien et al. 1964, modified), and mounted with

126 Entellan. Histochemical tests were performed on free-hand sections of freshly collected

127 flowers and also on fixed and embedded floral samples, sectioned as indicated above. We

128 employed Sudan red B for locating lipids (Brundrett et al. 1991); lugol for detecting starch

129 (Johansen 1940); and ferric chloride plus sodium carbonate to verify the occurrence of

130 phenolic compounds (Johansen 1940). The slides were analyzed, and images obtained with an

131 Axioskop 40 microscope (Carl Zeiss, Germany) with a digital camera.

132 Samples for transmission electron microscopy were collected from regions and phases

133 defined by light microscopy study. Samples were obtained during three different periods to

134 follow structural variation during secretory activity: (1) prior to anthesis, about 12 hours

135 before the opening of petals; (2) beginning of the pistillate phase (6AM in the first day of

136 anthesis), characterized by a strong sweetDraft odor and receptive stigmas; and (3) beginning of

137 the staminate phase (6AM in the second day), when pollen is released. Samples were fixed in

138 Karnovsky solution (Karnovsky 1965) for 24 hours, post-fixed in 1% osmium tetroxide in

139 0.1M phosphate buffer, pH 7.2, and processed according to Roland (1978). The ultrafine

140 sections were contrasted with uranyl acetate and lead citrate and examined using a Tecnai G2-

141 Spirit transmission electron microscope (Philips/FEI Company, Eindhoven, Netherlands) at

142 80 KV.

143 Samples for scanning electron microscopy were fixed in 2.5% glutaraldehyde in 0.1M

144 phosphate buffer, pH 7.2, dehydrated in an ethyl alcohol series, subjected to critical-point

145 drying, glued on aluminum supports and metalized with gold (Robards 1978). The samples

146 were then examined using a Quanta 200 scanning electron microscope (FEI Company,

147 Eindhoven, Netherlands).

148

149 Results

150 Morphology and structural aspects

151 The perianth of X. aromatica has two whorls of three free petals each (Fig. 1A–C).

152 Although free, the inner petals are slightly juxtaposed at their bases. The petals are reddish in

153 the proximal region and white in the median and distal regions. A curvature of approximately

154 90° into the flower delimits the pollination chamber in the basal region of the petals. It is due

155 to the thickening of the adaxial portion of the mesophyll, which forms the roof of the

156 pollination chamber (Fig. 1B). Towards the center of the flower, above the stigmas, each

157 inner petal curves again at an angle of slightly less than 90°, projecting towards the distal

158 region of the flower and delimiting the channel that allows access to the chamber (Fig. 1B–

159 C), which is formed by the juxtaposition of the three inner petals. The pollination chamber is

160 small and partially closed, maintaining an apical opening and small lateral openings between

161 the inner petals. The chamber is inconspicuousDraft prior to anthesis (Fig. 1B) but enlarges during

162 anthesis creating a space (Fig. 1C) where pollinators can move. Alternating outer petals

163 obstruct the lateral openings between inner petals.

164 The latero-proximal region of the inside of the pollination chamber is covered by a

165 non-secretory epidermis with short non-glandular trichomes (Fig. 1D). The petals possess

166 thick mesophyll and secretory epidermis in the roof of the pollination chamber and in the

167 narrow access channel. These epidermal cells are elongate, juxtaposed, and have dense

168 cytoplasm; the surface is slightly papillose and devoid of stomata and trichomes (Fig. 1D–G).

169 The epidermal cells of the roof of the chamber are involved in the synthesis and exudation of

170 nectar, as evidenced by positive tests for glucose. The viscous nectar is produced in small

171 amounts, just enough to dampen the nectary surface. The secretion of lipids is observed

172 immediately above the nectary in the portion that delimits the access channel (Fig. 1D–E).

173 Nectar secretion begins in the pistillate phase of anthesis, while lipids are produced since

174 prior to anthesis, both lasting until the end of the staminate phase. Thrips are commonly found

175 inside the pollination chamber, and they consume all the secretory epithelium in some

176 flowers.

177 While the regions of the inner petals delimiting the pollination chamber are

178 anatomically distinct (Fig. 1B, D), the portion above the chamber (elaiophore), is free and

179 more homogeneous, with an epidermis composed of cells with dense protoplast and non-

180 glandular trichomes.

181 For both secretory structures, the mesophyll of the inner petals consists of

182 parenchymatous subglandular tissue with cells of varying shapes and sizes interspersed with

183 numerous phenolic idioblasts (Fig. 1D) with amorphous or flocculated content. The

184 vasculature is formed by collateral vascular bundles, which do not branch towards the

185 secretory cells of the glandular region.

186 The epidermal cells of the nectaryDraft have a palisade arrangement, are dilated distally and

187 covered by a smooth and well-adhered cuticle; no pores or cuticular ruptures are present (Fig.

188 1F). The cuticle of the elaiophore is indistinct, and the secretion accumulates on the glandular

189 surface (Fig. 1G), which becomes viscous and sticky.

190

191 Ultrastructural organization of secretory structures

192 Floral nectary

193 In the pre-secretory phase (prior to anthesis), the subglandular parenchyma possesses

194 large vacuolated cells (Fig. 2A), in which phenolic substances predominate; there are few

195 other cytoplasmic organelles, among which plastids with starch grains (Fig. 2A–B) detaches.

196 A marked characteristic of the epidermal cells is their dense cytoplasm with numerous

197 mitochondria and plastids in which there is dense stroma and starch grains (Fig. 2C–D). The

198 starch grains of some plastids show evidence of hydrolysis, becoming heterogeneous with a

199 flocculent matrix (Fig. 2E).

200 The subglandular parenchyma presents little alteration in this stage relative to the

201 previous stage, with evidence of reduced intravacuolar phenolic content and starch hydrolysis

202 (Fig. 3A). Significant changes are observed in the epidermal cells, especially in the structure

203 of plastids, with evident starch hydrolysis (Fig. 3B–C). The fusion of plastids occurs (Fig.

204 3C), with these organelles exhibiting a pronounced reduction in stroma density and the

205 dissolution of the starch, which produces an amorphous and flocculent material (Fig. 3C).

206 Although the double membrane of the plastids can still be observed in some places (Fig. 3D),

207 the distinction between plastids and vacuoles is generally difficult at this stage (Fig. 3E). With

208 the complete disorganization of the plastidial envelope, only a single membrane remains

209 delimiting the vacuole that results from these plastids.

210 In addition to the mitochondria described in the previous phase, dictyosomes are

211 present, which possess five to seven Draftcisternae and are frequently associated with the rough

212 endoplasmic reticulum (Fig. 3F–G). Secretion products were observed in the periplasmic

213 space (Fig. 3D) and into the subcuticular space (Fig. 3G–H). This material has the same

214 appearance as that observed inside vesicles and vacuoles, which results of starch hydrolysis.

215 The secretion accumulated in the subcuticular space forms pockets (Fig. 3G) that rupture and

216 spill the secretion to the external environment (Fig. 3H).

217

218 Elaiophore

219 Although initiated prior to anthesis, the lipid secretion intensifies in the pistillate phase

220 of anthesis, which will be described here. In this phase, the epidermal cells possess dense

221 cytoplasm and a vacuole disposed in the distal portion (Fig. 4A). Among the organelles, the

222 most representative are plastids, smooth endoplasmic reticulum, and mitochondria. Plastids

223 possess dense stroma, osmiophilic inclusions, and an inconspicuous endomembrane system

224 (Fig. 4B). Significant development of the smooth endoplasmic reticulum is observed in these

225 cells, which forms a broad network dispersed throughout the extravacuolar cytoplasm (Fig.

226 4C–D). Oil droplets are present in the cytosol, especially in the region with the highest

227 density of endoplasmic reticulum (Fig. 4D). The lipids produced initially accumulate in the

228 periplasmic space (Fig. 4C), while oil droplets are observed on the anticlinal faces of the cell

229 wall. The lipids are deposited mainly facing the middle lamella (Fig. 4E), where the secretion

230 flows towards the surface of the elaiophore (Fig. 4F).

231 After crossing the cell wall, the secretion accumulates on the external face forming a

232 thick (sometimes surpassing 10µm) lipid layer (Figs. 1E, 1G, 4F). The thick lipid layer

233 contrasts with the thin cuticle observed in the nectariferous portion (see Figs 1D, 3G–H). The

234 accumulation of secretion on the surface of these cells makes it difficult to distinguish the

235 cuticle, which is inconspicuous, even using electron microscopy.

236 The parenchyma cells of the Draft subglandular tissue in the elaiophore are vacuolated,

237 some with phenolic content. Mitochondria and plastids with starch grains deserve special

238 mention.

239

240 Discussion

241 This article is the first report of secretory structures related to the release of nectar and

242 lipids, acting together in flowers of Annonaceae. Although nectaries have been described in

243 species of this family, the occurrence of elaiophores in the flowers of X. aromatica is an

244 unprecedented record in Annonaceae.

245 Floral nectaries have been reported for a small number of genera (Alphonsea,

246 Orophea, and Pseuduvaria) of this family (Silberbauer-Gottsberger et al. 2003; Su and

247 Saunders 2006; Okada 2014; Xue et al. 2017). Although reports of floral nectaries in

248 Annonaceae have appeared in the literature several times, some of the reports are very old

249 (see Baillon 1866), and little is known about the structure of these nectaries because there are

250 no detailed anatomical descriptions. In addition to the absence of anatomical and

251 ultrastructural studies, the occurrence of floral nectaries has never been reported for Xylopia.

252 In contrast, Andrade et al. (1996) reported the absence of nectaries for Xylopia brasiliensis

253 Spreng. when describing pollination in this species, which is mediated predominantly by

254 Coleoptera.

255 According to Simpson and Neff (1981), floral rewards are any floral product used by

256 animals that causes them to visit flowers several times to pollinate them. Pollen and nectar are

257 recognized as the main primary floral rewards (Faegri and Pijl 1979; Simpson and Neff

258 1981), but especially nectar because it is an important element for maintaining plant-

259 pollinator relationships (Faegri and Pijl 1979). Lipid secretion in flowers, in turn, may serve

260 to increase adhesion between pollen and the body of the pollinator (Gonçalves-Souza et al.

261 2018) or as another floral reward (BuchmannDraft 1987).

262 In Annonaceae, the size of the pollination chamber, the color of flowers, and the

263 reward system act by selecting insects that are attracted and can effectively enter the flowers.

264 Thus, these floral traits represent relevant factors in the evolution of pollination in the group

265 (Gottsberger 1994; Gottsberger and Silberbauer-Gottsberger 2006). While chamber size and

266 flower color have been emphasized in the floral biology of Annonaceae, the production of

267 secretions has been rarely documented. This is a significant knowledge gap, and we believe

268 that the secretory floral structures have been underreported, as demonstrated here for X.

269 aromatica flowers. Although Saunders (2010) indicated that floral glands occur in many

270 phylogenetically distant lineages of Annonaceae, suggesting multiple independent

271 evolutionary origins, the gap in detailed works searching for secretory structures can lead to

272 inaccurate character reconstruction.

273 According to Endress (1994), few plants are pollinated by thrips, usually those with

274 flowers with narrow openings toward the center of the flower, numerous closely-arranged

275 stamens, white color, and rewards such as pollen and nectar. All these features are found in

276 the flowers of X. aromatica. For this species, the white color of the petals, the presence of the

277 nectary, and the release of a strong sweet odor effectively attract thrips to the flowers.

278 According to Kirk (1984), thrips frequently ingest the floral nectar and suck the contents of

279 pollen leaving only the pollen wall. Saunders (2010, page 588) concluded that perianth glands

280 in Annonaceae presumably provide “an alternative to the sweet stigmatic exudate produced

281 during the pistillate phase”.

282 As observed in the nectary and elaiophore of the inner petal of X. aromatica, dense

283 cytoplasm, a large nucleus, and numerous mitochondria are common characteristics of tissues

284 with high metabolic activity (Lüttge 1971). So, they are good indicators of secretory activity

285 (Mercadante-Simões and Paiva 2013). The cell machinery of plant secretory structures is

286 highly correlated with secretory products.Draft Indeed, changes in the cellular composition of

287 organelles, for example, can occur during the secretory process and reflect the chemical

288 nature of the products. Although unusual, the distinct cell ultrastructure of the gland of the

289 inner petals of X. aromatica can explain the dual function, as we recorded the simultaneous

290 synthesis of lipids and nectar in different regions.

291 Our data showed that there is a large amount of starch before nectar secretion in petals

292 of X. aromatica, which subsequently declines due to its hydrolysis concurrent with the release

293 of nectar, as recorded for most floral nectaries already studied (Fahn 1988; Nepi et al. 2011;

294 Płachno et al. 2018).

295 The structural changes in the plastids and their conversion into vacuoles seem to be

296 standard features of plastidial dynamics in nectaries (Paiva and Machado 2008; Guimarães et

297 al. 2016). This conversion has also been reported during other secretory processes, such as in

298 osmophores (Gonçalves-Souza et al. 2017).

299 Nectar and lipids are released in distinct ways, even considering the similarity of the

300 nectary and elaiophore surface in the flowers of X. aromatica, on which a thin cuticle and the

301 absence of stomata stand out. Subcuticular spaces and cuticle ruptures were observed in the

302 nectary, allowing free passage of secretory products, and reinforcing this common route for

303 nectar release in this type of nectary as proposed by Paiva (2017). On the other hand, there is

304 no evidence of subcuticular spaces or cuticle ruptures in the elaiophore. However, lipids can

305 pass through the cuticle due to their identical chemical nature, which is consistent with the

306 results reported here.

307 The use of oil or resins as adhesives for pollen seems to be shared in unrelated groups,

308 as Philodendron (Araceae) (Gonçalves-Souza et al. 2018 and references therein) and oil-

309 producing species of Cucurbitaceae (Possobom and Machado 2017). Various sticky

310 substances, like polysaccharides (SchnetzlerDraft et al. 2017) or oil (Steiner 1985; Pacini and

311 Hesse 2005), seem to act as adhesives for pollen. Moyano et al. (2003) reported the presence

312 of a sticky secretion acting as an adhesive for pollen in Leonurus sibiricus L. (Lamiaceae): “it

313 prevents the pollen being easily wiped off, helping it remains adhered to the insect

314 integument”. Glands specialized in the synthesis of sticky substances for pollen adhesion have

315 not been previously recorded for Annonaceae, and pollen grains mixed with lipids need to be

316 evaluated regarding their viability. Many of the pollinators described for Neotropical

317 Annonaceae have a smooth body, such as some Coleoptera and thrips, so any adherence

318 strategy seems to be relevant to pollen transport, as evidenced for other plants (Steiner 1985;

319 Moyano et al. 2003; Pacini and Hesse 2005). According to Ananthakrishnan (1982), effective

320 pollination by thrips depends on flowering time, the amount of flower rewards (pollen and

321 nectar), and easy access to flowers and rewards. The nectary of X. aromatica is near the

322 stamens in the roof of the pollination chamber. Therefore, insects entering the flower first

323 pass through the channel bounded by the elaiophore, and advance to the nectary in search of

324 food, finding the pollen grains, which they can eat or carry to another flower. Nectaries of

325 other Annonaceae, such as A. glandulosa (Xue et al. 2017) and Orophea spp. (Okada 2014),

326 were described on the adaxial face of the base of the inner petals, in a position very similar to

327 that observed in X. aromatica. Still, no mention was made of the presence of an elaiophore for

328 the simultaneous production of lipids. Momose et al. (1998) observed pollen adhered to the

329 body of 67% of the thrips collected during the staminate phase of Popowia pisocarpa Endl.

330 (Annonaceae). Although these authors did not verify the occurrence of adhesive substances,

331 their existence seems to be consistent with adhesion for pollen grains since insect samples

332 prepared for scanning electron microscopy still had pollen grains adhered to, which appears to

333 be improbable without the action of something sticky. In addition to the stigmatic exudate,

334 and the possibility of other sticky secretions such as polysaccharides and lipids, the sticky

335 character of the pollen of some speciesDraft of Annonaceae should also be considered. This was

336 indeed suggested by Ratnayake et al. (2007, page 1259) for pollen of Xylopia championii

337 Hook.f. and Thomson: “the freshly dehisced pollen tetrads were loosely connected to each

338 other, presumably due to the presence of a sticky pollenkitt”.

339 Pollen adherence to the insect body seems to depend on insect morphology, as can be

340 inferred from Gottsberger et al. (2011). During a study of pollination in some African

341 Annonaceae, these authors stated (page 504) that “pollen stuck very well on the hairy

342 scarabaeid beetles, with about 1,300 pollen tetrads counted on individuals (as compared to

343 only three to six pollen tetrads encountered on the curculionid beetles)”. On the other hand,

344 we must consider that sticky stigmatic exudates, commonly found in Annonaceae flowers

345 (Gottsberger 2012), can act as a pollen adhesive. Indeed, according to Gottsberger et al.

346 (2011), the pollen of most Annonaceae is sticky and adheres well even to smooth, hairless

347 beetles. Therefore, even considering some adhesive properties (not tested) of the pollen of X.

348 aromatica, the presence of the sticky secretion on the entrance of the pollination chamber

349 seems to constitute a way to assure better pollen adherence to the smooth body of the thrips.

350 The adhesion of pollen of X. brasiliensis to pollinator bodies was reported by Andrade et al.

351 (1996, page 318), who stated that “Brachypnoca spp. (Chrysomelidae) were often seen

352 leaving the flowers at the end of afternoon carrying pollen and petal fragments adhered to

353 their bodies”. Different species of Xylopia have very similar flowers, so it is possible that

354 elaiophores like the observed in X. aromatica may be acting in X. brasiliensis flowers,

355 allowing the adhesion not only of the pollen but also of the petals fragments.

356 The complex secretory system that we observe in the flowers of X. aromatica interacts

357 in a highly specialized way with the process of pollination. Despite this, we observed the

358 maintenance of structural characters in this species that are commonly associated with

359 cantharophily, such as thick petals and protogynous dichogamy. Saunders (2012), studying

360 evolutionary shifts in the pollination systemDraft within the Annonaceae, already stated that many

361 floral adaptations are similar in the family (e.g., protogyny and partially enclosed floral

362 chambers), even for different pollinator guilds. Simultaneously, Gottsberger (2012, page 245)

363 highlighted, “As cantharophily is plesiomorphic (...) in Annonaceae (...) Even non-

364 cantharophilous species retain one or more characteristic features of beetle-pollinated

365 species”. The analysis of glands in other species and phylogenetic groups of Annonaceae,

366 searching for nectaries and elaiophores, could shed light on the evolution of these traits in this

367 basal family of angiosperms, which has a very conservative pollination syndrome.

368 Finally, nectaries are important structures for successful pollination, and detailed floral

369 studies should be performed in other species of Annonaceae to verify the existence of

370 nectaries. It is probable that the presence of stigmatic exudation, sometimes abundant, has

371 confused researchers about the presence of floral nectaries in Annonaceae, making them

372 difficult to find and leading to underestimating their occurrence. This is especially important

373 because, according to Erbar (2014), epithelial nectaries are less conspicuous, and they are the

374 most common type of floral nectaries among basal angiosperms.

375

376 Acknowledgements

377 We are grateful to Dr. Wilma de Grava Kempinas for permission to use her laboratory

378 at São Paulo State University (Unesp). To the Center of Microscopy at the Universidade

379 Federal de Minas Gerais (http://www.microscopia.ufmg.br) and Unesp (IB Botucatu) for

380 providing the equipment and technical support for electron microscopy. This study was

381 financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil

382 (CAPES, Finance Code 001). It was also supported by the Fundação de Amparo à Pesquisa

383 do Estado de Minas Gerais (FAPEMIG, Brazil, process CRA-PPM-00272-11). D.M.T.O. and

384 E.A.S.P. thank the Conselho NacionalDraft de Desenvolvimento Científico e Tecnológico (CNPq,

385 Brazil, processes 305686/2018-6 and 305638/2018-1 respectively) for research grants. We

386 also thank Dr. Richard Saunders and Dr. Lars Chatrou for their accurate revision of the

387 manuscript.

388

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529

530

Draft

531 FIGURE LEGENDS

532 Fig. 1. General aspects of the flower of Xylopia aromatica and its secretory structures. (A) A

533 fertile branch, showing two flowers in which the corolla has three outer and three inner petals.

534 (B–C) Longitudinal sections of a flower prior to anthesis seen with a light microscope and of

535 a flower in anthesis seen with a stereomicroscope, respectively; in the inner petals notice the

536 nectary and elaiophore delimitating the pollination chamber. (D) Longitudinal section of a

537 secretory region seen with a light microscope; arrow indicates the non-glandular portion. (E)

538 Detail of the elaiophore in the previous figure; the secreted lipids are stained with Sudan Red

539 B. (F–G) Scanning electron micrographs of the nectary and elaiophore; an abundant secretion

540 can be seen in the latter. an, anther; el, elaiophore; ip, inner petal; ne, nectary; op, outer petal;

541 ov, ovary; pc, pollination chamber; sg, stigma.

542 Draft

543 Fig. 2. Ultrastructure of the floral nectary of Xylopia aromatica during the pre-secretory

544 phase (12h prior to anthesis). (A–B) Subglandular portion with parenchyma cells with

545 phenolics in vacuoles; in (B) plastids with starch grains can be seen. (C–E) Epidermal cells.

546 (C) Secretory cell with a conspicuous nucleus and organelle-rich cytoplasm. (D)

547 Cytoplasmatic portion showing mitochondria, plastids, and small vacuoles. (E) Detail of a

548 starch grain within a plastid; note the evidence of starch hydrolysis in the form of its

549 flocculent aspect compared to (B). mi, mitochondria; pl, plastid; st, starch; va, vacuole.

550

551 Fig. 3. Ultrastructure of the floral nectary of Xylopia aromatica during the pistillate phase.

552 (A) Parenchyma cells of the subglandular region. (B–H) Secretory epidermis. (B) General

553 view of the epidermis showing cells with dense cytoplasm and vacuoles towards the nectary

554 surface. (C–E) Different stages of plastidial change and starch hydrolysis. (C–D) Plastids still

555 delimited by the double membrane and showing starch undergoing hydrolysis; in (D), long

556 arrows indicate plastidial membrane, and short arrows indicate secretory products in the

557 periplasmic space. (E) Plastids and vacuole become indistinct. (F) Cytoplasm portion showing

558 active dictyosomes; flocculent substances are seen within vacuoles. (G–H) Secretion release;

559 note the similarity between substances inside the vacuole and in the subcuticular space; in

560 (H), arrows indicate cuticle rupture where nectar is released. di, dictyosome; er, endoplasmic

561 reticulum; mi, mitochondria; pl, plastid; ss, subcuticular space; va, vacuole.

562

563 Fig. 4. Elaiophore in the pollination channel of Xylopia aromatica during the pistillate phase.

564 (A) Typical epidermal cells showing an organelle-rich cytoplasm. (B–D) Detail of cytoplasm

565 showing predominant organelles. (B) Plastids with dense stroma and osmiophilic globules

566 inside. (C) A dense cytoplasmic portion with well-developed endoplasmic reticulum,

567 mitochondria, and evidence of exocytosisDraft in periplasmic space; inside the circle, an oil droplet

568 can be seen. (D) Oil droplets in a portion where endoplasmic reticulum is predominating. (E)

569 Anticlinal face of two contiguous cells; arrows indicate oil droplets facing the middle lamella.

570 (F) Released lipids that accumulate in the external face, sometimes surpassing 10µm thick.

571 cw, cell wall; di, dictyosome; er, endoplasmic reticulum; mi, mitochondria; od, oil droplet; pl,

572 plastid; ps, periplasmic space; se, secretion; va, vacuole.

Draft

Fig. 1. General aspects of the flower of Xylopia aromatica and its secretory structures. (A) A fertile branch, showing two flowers in which the corolla has three outer and three inner petals. (B–C) Longitudinal sections of a flower prior to anthesis seen with a light microscope and of a flower in anthesis seen with a stereomicroscope, respectively; in the inner petals notice the nectary and elaiophore delimitating the pollination chamber. (D) Longitudinal section of a secretory region seen with a light microscope; arrow indicates the non-glandular portion. (E) Detail of the elaiophore in the previous figure; the secreted lipids are stained with Sudan Red B. (F–G) Scanning electron micrographs of the nectary and elaiophore; an abundant secretion can be seen in the latter. an, anther; el, elaiophore; ip, inner petal; ne, nectary; op, outer petal; ov, ovary; pc, pollination chamber; sg, stigma.

Draft

Fig. 2. Ultrastructure of the floral nectary of Xylopia aromatica during the pre-secretory phase (12h prior to anthesis). (A–B) Subglandular portion with parenchyma cells with phenolics in vacuoles; in (B) plastids with starch grains can be seen. (C–E) Epidermal cells. (C) Secretory cell with a conspicuous nucleus and organelle-rich cytoplasm. (D) Cytoplasmatic portion showing mitochondria, plastids, and small vacuoles. (E) Detail of a starch grain within a plastid; note the evidence of starch hydrolysis in the form of its flocculent aspect compared to (B). mi, mitochondria; pl, plastid; st, starch; va, vacuole.

Draft

Fig. 3. Ultrastructure of the floral nectary of Xylopia aromatica during the pistillate phase. (A) Parenchyma cells of the subglandular region. (B–H) Secretory epidermis. (B) General view of the epidermis showing cells with dense cytoplasm and vacuoles towards the nectary surface. (C–E) Different stages of plastidial change and starch hydrolysis. (C–D) Plastids still delimited by the double membrane and showing starch undergoing hydrolysis; in (D), long arrows indicate plastidial membrane, and short arrows indicate secretory products in the periplasmic space. (E) Plastids and vacuole become indistinct. (F) Cytoplasm portion showing active dictyosomes; flocculent substances are seen within vacuoles. (G–H) Secretion release; note the similarity between substances inside the vacuole and in the subcuticular space; in (H), arrows indicate cuticle rupture where nectar is released. di, dictyosome; er, endoplasmic reticulum; mi, mitochondria; pl, plastid; ss, subcuticular space; va, vacuole.

Draft

Fig. 4. Elaiophore in the pollination channel of Xylopia aromatica during the pistillate phase. (A) Typical epidermal cells showing an organelle-rich cytoplasm. (B–D) Detail of cytoplasm showing predominant organelles. (B) Plastids with dense stroma and osmiophilic globules inside. (C) A dense cytoplasmic portion with well-developed endoplasmic reticulum, mitochondria, and evidence of exocytosis in periplasmic space; inside the circle, an oil droplet can be seen. (D) Oil droplets in a portion where endoplasmic reticulum is predominating. (E) Anticlinal face of two contiguous cells; arrows indicate oil droplets facing the middle lamella. (F) Released lipids that accumulate in the external face, sometimes surpassing 10µm thick. cw, cell wall; di, dictyosome; er, endoplasmic reticulum; mi, mitochondria; od, oil droplet; pl, plastid; ps, periplasmic space; se, secretion; va, vacuole.