Androecium and gynoecium anatomy of Bromeliaceae species

Flora 263 (2020) 151538

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Androecium and gynoecium anatomy of Bromeliaceae species a,b,

a

T a

Fernanda Maria Cordeiro de Oliveira *, Ana Claudia Rodrigues , Makeli Garibotti Lusa , Gladys Flavia de Albuquerque Melo-de-Pinnab a b

Federal University of Santa Catarina, Department of Botany, Campus Reitor João David Ferreira Lima, s/n – Trindade, Florianópolis, SC, 88040-900, Brazil University of São Paulo, Department of Botany, Rua do Matão 277, Cidade Universitária, São Paulo, SP, Brazil

A R T I C LE I N FO

A B S T R A C T

Edited by: Louis Ronse De Craene

In this study, we performed a detailed anatomical analysis of the androecium and gynoecium in 16 species belonging to three out of the eight Bromeliaceae subfamilies, with a first-time description of the interlocular zone in anthers, a diagnostic character for Quesnelia and Aechmea species. Other potential taxonomic characters were observed: protuberances formed by growth in the connective region of anthers, which only occurs in species belonging to the Nidularioid complex; and an endothecium-like tissue in the anthers, near to the connective region, characterizing Dyckia species. Besides these characters, the vascularization of filaments and style are discussed from a phylogenetic perspective. In that sense, the vascularization of style is supplied by three vascular bundles that may represent a synapomorphy for Pitcairnoideae and should be further investigated in the family. In addition, the vertical zonality of carpels is described for analyzed inferior and superior ovaries and the position of the nectaries is discussed in this context. Regarding the secretory structures present on the gynoecium we provided novel data for future evolutionary studies on this group. Furthermore, characters presented in this study are discussed from an ecological point of view.

Keywords: Secretory structures Septal nectaries Pollen tube transmitting tissue Endothecium-like tissue Interlocular zone Floral vascularization

1. Introduction The Bromeliaceae are mostly distributed in the Neotropical region, with the exception of Pitcairnia feliciana (A. Chev.) Harms & Mild, which occurs in West Africa (Smith et al., 1974; Jacques-Félix, 2000). The family has 3400 species and 56 genera (Butcher and Gouda, 2020), and, along with Typhaceae and Rapateaceae, form a sister clade to the other Poales (APG IV, 2016). The family was, for a long time, subdivided into three: Bromelioideae, Pitcairnioideae s. l. and Tillandsioideae. These three subfamilies were differentiated by foliar margin, ovary position, and fruit and seed types (Smith and Downs, 1974, 1977, 1979). Nevertheless, through advances in molecular systematics, it is now known that Pitcairnioideae s. l. does not represent a monophyletic group. Thus, Bromeliaceae was reorganized into eight subfamilies: Bromelioideae, Tillandsioideae, Pitcairnioideae s. s., Navioideae, Puyoideae, Brocchinioideae, Hechtioideae and Lidmanioideae (Givnish et al., 2007, 2011). Due to the great morphological diversity within Bromeliaceae, recent phylogenetic studies have shown that a variety of genera do not represent monophyletic groups, as seen in the phylogenies proposed by Silvestro et al. (2014) and Aguirre-Santoro et al. (2016), for example. In

order to organize the family’s genera under monophyletic groups, multiple nomenclature changes have been recently proposed (Barfuss et al., 2016). Several morphological characters currently used in genera delimitation are homoplastic, such as presence of petal appendages, branched inflorescence and presence of pedicels (Schulte and Zizka, 2008; Aguirre-Santoro et al., 2016). In that sense, anatomical features have been investigated in phylogenetic context trying to identify structural synapomorphies for groups lack them, such as the Nidularioid Complex (Oliveira et al., 2018). This complex is composed by Nidularium Lem., Wittrockia Lindm., Neoregelia L.B.Sm, Canistropsis (Mez) Leme and Edmundoa Leme (Leme, 1997, 1998, 2000). Although not all of these genera are considered to be monophyletic, the Nidularioid Complex emerges as a monophyletic clade (Silvestro et al., 2014; Aguirre-Santoro et al., 2016). Based on ancestral state reconstruction analyses, Oliveira et al. (2018) proposed that the presence of elongated cells at the peltate trichome wings could represent a structural synapomorphy for the Complex. Moreover, new studies of structural diversity of flowers are desirable to try establishing structural synapomorphies that could aid in the delimitation of this complex, as already reported for other plant groups (Taylor and Robinson, 1999; Sajo et al., 2004a).

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Corresponding author. E-mail addresses: [email protected] (F.M.C.d. Oliveira), [email protected] (A.C. Rodrigues), [email protected] (M.G. Lusa), [email protected] (G.F.d.A. Melo-de-Pinna). https://doi.org/10.1016/j.flora.2020.151538 Received 15 June 2019; Received in revised form 12 December 2019; Accepted 7 January 2020 Available online 11 January 2020 0367-2530/ © 2020 Elsevier GmbH. All rights reserved.

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The flowers in Bromeliaceae are trimerous, pentacyclic, usually with a showy and colorful perianth. The petals have been widely studied, with particular interest in the presence of appendages in some of the genera, due to their taxonomic importance. These petal appendages, commonly used for generic delimitation, are the result of an outgrowth in the adaxial portion of the petals; they are not vascularized and are formed last during flower development (Brown and Terry, 1992; Sajo et al., 2004a; Oliveira et al., 2016). To date, the study of the floral anatomy in Bromeliaceae has provided diagnostic characters for taxonomic purposes. Previous works in the group have mainly described ovary and ovule anatomy and their significance for the systematic of the family (Novikoff and Odintsova, 2008; Nogueira et al., 2015; Kuhn et al., 2016). Ovule development has been described for some species, in which the chalazal and micropylar appendages would be among the most striking features of the family (Sajo et al., 2004b; Fagundes and Mariath, 2014; Mendes et al., 2014). In addition, nectar produced in the septal nectaries have been reported to attract pollinators (Smith and Downs, 1974; Sajo et al., 2004a). According to Sajo et al. (2004a), septal nectaries are related to the evolution of epigyny and represent a singular feature among Poales. The septal nectary, a nectar producing tissue that is found in the wall of a plant ovary/hypanthium, is the best known secretory structure present in the reproductive organs of Bromeliaceae (Fiordi and Palandri, 1982; Bernadello et al., 1991; Sajo et al., 2004a). This tissue has been vastly investigated with different purposes, regarding its structure and position on gynoecium (Schmid, 1985; Sajo et al., 2004a; Novikoff and Odintsova, 2008), the ultrastructure of its cells (Fiordi and Palandri, 1982), and the composition of its secretion (Galetto and Bernadello, 1992; Kromer et al., 2008; Hornung-Leoni et al., 2013). The same cannot be said about the anatomical structure of the obturator and the pollen tube transmitting tissue in the family, nor about the origin and composition of its secretions (Sajo et al., 2004a; Nogueira et al., 2015; Oliveira et al., 2016). These tissues are responsible for pollen-tube guidance and nutrition, from the style to the ovule’s micropyle on ovary locules (Tilton and Horner, 1980; Hudák et al., 1993; Singh and Walles, 1993; Herrero, 2000; Erbar, 2003; McCormick and Yang, 2005). Therefore, we performed a detailed anatomical study of the androecium and gynoecium of Bromeliaceae from developed floral buds of 16 species, including three of the eight subfamilies, in order to bring new data to further taxonomic and phylogenic studies in the family. We also aimed to describe the presence of septal nectaries and their position in the carpel, discussing the concept of the carpel zonation proposed by Leinfellner (1950). In addition, the described characters are discussed from an ecological perspective, aiming to gain a better understanding of the reproductive biology of the analyzed species.

series (50–100% EtOH). After that, samples were critical-point dried with CO2 (CPD 030, Balzer) following the method proposed by Gersterberger and Leins (1978), then mounted on stubs and sputtercoated with gold. Analysis was performed using a Zeiss DMS-940 Scanning Electron Microscope. 3. Results 3.1. Androecium All the studied species are trimerous, presenting three sepals/sepal lobes, three petals/petal lobes, six stamens divided into two whorls and a tricarpelate gynoecium (Fig. 1A–F). In species where sepals and petals are connate at the base, three lobes are observed at the basal portion of the flower, and both sepals and petals are free at the middle-upper part. In these cases, at the basal portion of the flower, a stamen-corolla tube is formed, as seen in the basal region (Fig. 1A). At the middle-upper portion, three morphological conformations may occur: 1) the stamens are free, i.e., not adnate, but the petals usually have callosities and/or appendages involving the filaments. This morphological conformation can be seen in Cryptanthus bahianus (basal part, as represented in Fig. 1A), Nidularium procerum (transition between basal and middle portions, as represented in Fig. 1B), Vriesea platynema and Quesnelia testudo (upper portion with free filaments, as represented in Fig. 1C; detail of petal appendages in SEM in Fig. 2A), and Dyckia tuberosa (the only species here without petal appendages or callosities). 2) the epipetal stamens are adnate to the petals, and the antesepalous stamens are surrounded either by longitudinal petal callosities or by petal appendages at the middle portion of the flower (Fig. 1D – note the adnation of epipetal stamens; Fig. 2B – note detail of petal appendages in SEM). At the upper portion, all stamens are entirely free. This pattern occurs in Aechmea bromeliifolia, A. gamosepala, A. cylindrata, A. nudicaulis and N. amazonicum. In these species, stamens that alternate with calyx lobes are free. 3) At the middle portion of the flower, the antesepalous stamens are adnate and are surrounded by either longitudinal petal callosities or by petal appendages (Fig. 1E), as observed in Nidularium longiscapum and Neoregelia spectabilis. In this morphological conformation, stamens that alternate with the corolla lobes are free and at the upper part of the flower, all the stamens are free. In species that do not have a petal-stamen tube, such as Encholirium horridum, Pitcairnia flammea and Vriesea inflata (Fig. 1F), the three sepals and petals are almost completely free, excepting a very short region at the base, where sepals, petals, stamens and carpels are adnate. In transverse sections of all analyzed flowers, at the portion where the filaments are free, they can be rounded, triangular or flat (Fig. 1; Fig. 2C–H). In some species, flat and rounded filaments occur simultaneously in the same flower (Table 2). The epidermis of the filaments is uniseriate and does not show cell wall thickening (Fig. 2C–I), but it does show prominent cuticle deposition in the distal portion of the filament in Neo. spectabilis, N. longiflorum, N. amazonicum, Q. testudo and V. platynema (Fig. 2I). In frontal view, the cuticle has a striate pattern (Fig. 2J–K). The filament mesophyll is filled by homogeneous parenchyma in all studied species (Fig. 2C–H), and idioblasts with raphides are present in A. gamosepala, A. cylindrata, C. bahianus, E. horridum, P. flammea and Q. testudo. Regarding filament vascularization, a single collateral bundle occupies the central part of the filaments (Figs. 2C, E, 3 A). However, in E. horridum, three vascular bundles are observed (Fig. 2F), but only two vascular bundles are seen in N. procerum, P. flammea, V. inflata and D. tuberosa (Figs. 2D; G–H; 3 B; Table 2). In these cases, bundles that vascularizes the filament arose as a single concentric bundle at the pedicel (Fig. 3B) and are united at the connective region at anthers, as represented in schematic drawing B in Fig. 3. The anthers are dithecal, tetrasporangiate and have introrse dehiscence (Fig. 4A–F). The epidermis is uniseriate, papillose, and composed

2. Material and methods A total of 16 species from nine genera representing three out of the eight subfamilies of Bromeliaceae were analyzed (see Table 1). For each species, developed flower buds in pre-anthesis were sampled. Three individuals were sampled per species, and three flower buds of each species were collected. Developed flower buds were collected and fixed in FAA 50 (formaldehyde, acetic acid and ethanol 50 – Johansen, 1940) for 48 hs and then stored in 70% ethanol. The samples were then progressively dehydrated with ethanol/tertiary butyl alcohol (50–100%) solutions, and subsequently embedded in Paraplast® (Ruzin, 1999). Serial transverse and longitudinal sections (8–12 μm) were made using a rotary microtome (Reihertt-Jung Auto Cut 2040) and either stained with Toluidine O (Sakai, 1973), or deparaffinized with xylol and stained with 1% aqueous Astra blue and 1% Safranin in a 50% ethanol solution (Bukatsch, 1972). All material was analyzed with a Leica DMLB microscope equipped with a digital camera (Leica DFC 310 FX). For scanning electron microscopy (SEM), samples were previously fixed in FAA 50 and then dehydrated in a graded ascending ethanolic 2

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Table 1 Studied species, grouped by subfamily, their respective vouchers and geographical occurrence. The acronym of Herbarium follows Thiers (2020): HUPG = State University of Ponta Grossa, UPCB = Universidade Federal do Paraná. Subfamily

Specie

Bromelioideae

Aechmea Aechmea Aechmea Aechmea

bromeliifolia (Rudge) Baker gamosepala Wittm. cylindrata Lindm. nudicaulis (L.) Griseb.

Aechmea ornata Baker Cryptanthus bahianus L.B. Sm. Nidularium amazonicum (Baker) Linden & E. Morren ex Lindm. Nidularium longiscapum B.A. Moreira & Wand. Neoregelia spectabilis (T. Moore) L.B. Sm. Nidularium procerum Lindm. Quesnelia testudo Lindm. Pitcairnoideae

Tillandsioideae

Dyckia tuberosa (Vell.) Mez Encholirium horridum L.B. Sm. Pitcairnia flammea Lindl. Vriesea inflata (Wawra) Wawra Vriesea platynema Gaudich.

Voucher

Occurence

S.N.A. Miyamoto e A.C. Azevedo 150 (HUPG) S.N.A. Miyamoto, M. Engels & V.K. Kowalski 95 (HUPG) S.N.A. Miyamoto & F.M.C. de Oliveira 61 (HUPG) S.N.A. Miyamoto, A.C. Azevedo, B.N.S. Lima, G. Migliorini & M. Santana 44 (HUPG) S.N.A. Miyamoto & V.K. Kowalski 115 (HUPG) F.M.C de Oliveira s/n (HUPG) M.E. Engels s/n (UPCB) B.N.S. Lima, V.K. Kowalski, S.N.A. Miyamoto 50 (HUPG) F.M.C. de Oliveira 54 (HUPG) F.M.C. de Oliveira 55 (HUPG) B.N.S. Lima, V.K. Kowalski, S.N.A. Miyamoto 15 (HUPG) F.M.C. de Oliveira, S.N.A. Miyamoto, M.E. Engels & V.K. Kowalski 38 (HUPG) F.M.C. de Oliveira 60 (HUPG) F.M.C. de Oliveira 61 (HUPG) M.E. Engels 310 (HUPG) M.P.M. Martínez 218 (HUPG) V.K. Kowalski & R. Kowlaski 22 (HUPG)

Ponta Grossa – PR Matinhos – PR Quatro Barras – PR Paranaguá – PR Guaratuba – PR Recife – PE Antonina – PR São Paulo – SP Ponta Grossa – PR Morretes – PR Guaraqueçaba – PR São Paulo – SP São Paulo – SP Jaguariaíva – PR Morretes – PR Balsa Nova – PR

synascidiate zone with three locules (Fig. 9A). In this zone, no ventral suture is observed, since carpels are congenitally united. Above this region, a hemisynascidiate zone is present (Fig. 9B–E), and it can be divided into two different regions: the first one is represented by the presence of one central septal nectary with a common channel (Fig. 9B–C); the second region is represented by three disconnected channels of septal nectaries and a postgenital fusion at the center (Fig. 9D–E). In analyzed species, the first region of the hemisynascidiate zone shows one of the two different morphologies: a non-labyrinthine common nectarial cavity, in which the nectary epidermis has a straight contour, as seen in N. procerum (Fig. 7A; Table 2); or a labyrinthine common nectarial cavity, in which reentrances in the nectary epidermis form a kind of labyrinthic pattern (Fig. 8A–C; Table 2). All nectaries of Bromelioideae have their opening region at the hypanthium floor (Fig. 9F). The hemisymplicate zone occurs above the hemisynascidiate zone and is characterized by visible ventral sutures (Fig. 9F). The fertile region of the carpels is located at the upper portion of the hemisynascidiate zone until the hemisymplicate zone (Fig. 7A and 9 F). Above the hemisymplicate zone occurs the asymplicate zone, which also characterizes the style (Fig. 9G–H), and the stigma (Fig. 9I). Pitcairnioideae and Tillandsioideae species have nearly-superior ovaries, with a small portion adnate to the sepals, petals and stamen. Above this region, the ovaries are free (Fig. 10A–C; Table 2) and have an uniseriate outer epidermis (Fig. 10D–F) with thin cell walls. In D. tuberosa, the epidermal cells are anticlinally elongated in transversal section, and stomata are present (Fig. 10D). The ovarian mesophyll is homogeneous, and only E. horridum has an aerenchyma close to the inner wall of the ovary (Fig. 10E). In these species, most of the nectary tissue is located below the locules at the floral base, thus being termed herein as infralocular nectaries (Fig. 11A–D) in which the nectariferous tissue has a labyrinthine form (Fig. 11B, D). In these cases, the aperture of the nectary is located at the septal region (Fig. 10C). Fig. 11C shows the detail of the nectary epithelium with elongated epidermal cells and a densely stained cytoplasm. In Fig. 12 the gynoecium zonality concept for Pitcairnoideae species is represented (excepting P. flammea). The floral base is composed of the hypocarpous zone, where the nectariferous tissue is present and no locules are observed (Fig. 12A). Above this region occurs the hemisynascidiate zone (Fig. 12D). In the transition of hypocarpous and the hemisynascidiate zone, is possible to see the nectary openings (Fig. 12B–D). Also, is possible to note in the hemisynascidiate zone, the presence of congenital union between two carpels (Fig. 12B–D, dotted ellipses). Above the hemisynascidiate zone, occurs a very short

of conical cells with thin walls. Cuticle deposition emphasizes the conical shape of the epidermal cells (Fig. 5A–D, arrows). In the anther epidermis, the cuticle can be deposited in a regular pattern (Fig. 6A; Table 2), or it can form a striated surface (Fig. 6B–F; Table 2). Near the stomium region of Aechmea and Quesnelia species, an interlocular zone can be observed, and at this stage of floral development, i.e., just before anthesis, it is characterized by the presence of elongated cells (Fig. 4A–B; 5D–E; Table 2). The endothecium consists of one to four cell layers with annular secondary thickening (Fig. 5C). The middle layer and the tapetum are not observed in advanced floral buds in pre-anthesis phase. Idioblasts containing raphides are present in the anthers of A. cylindrata, A. gamosepala, A. ornata, D. tuberosa, E. horridum, and V. inflata (Fig. 5F). The connective is vascularized by a concentric vascular bundle located between the two theca (Figs. 4A–E; 5 F–H). In D. tuberosa, we found the presence of endothecium-like tissue at the connective region near the vascular tissue (Fig. 5F–G; Table 2). Only in the anthers of N. longiscapum aerenchyma occurs at the connective region near the vascular tissue (Fig. 5H). A protuberance is present at the connective region of A. ornata, Neo. spectabilis, N. procerum, N. longiscapum and N. amazonicum, and it is characterized by a growth of this region (Fig. 4A, C; Table 2).

3.2. Gynoecium and nectary An inferior ovary characterizes the gynoecium in species from the Bromelioideae subfamily, with the presence of a gynoecial hypanthium (Fig. 7A–B; Table 2). The epidermis of the hypanthium is uniseriate with each cell containing a spherical silica crystal (Fig. 7C). The epidermal cells also show thickening in their anticlinal and internal periclinal walls in A. nudicaulis, A. cylindrata, and A. gamosepala (Fig. 7C). Stomata are present in A. nudicaulis and A. cylindrata, and peltate trichomes can be found in A. bromeliifolia, A. ornata, A. cylindrata and Q. testudo (Fig. 7D). The mesophyll is homogeneous (Fig. 7E–F), but in A. nudicaulis and Q. testudo the hypoderm has cells with thickened walls (Fig. 7C), and in N. procerum and N. longiscapum (Fig. 7E) an aerenchyma formed by brachiform cells is present at the hypanthium (Table 2). The hypanthium is vascularized by the presence of the sepal, petal, stamen, and dorsal and ventral carpellary bundles (Fig. 7A). The ovary of Bromelioideae species also shows a septal interlocular nectary (Fig. 7A; 8A–D), which is characterized by cells with densely stained cytoplasm and a prominent nucleus (Fig. 8E–F). In Fig. 9, the zonality concept for Bromelioideae species is represented. The inferior part of the ovary is represented by a short sterile 3

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hemisymplicate zone, where it is possible to note the presence of the nectary openings and the presence of ventral sutures, indicating postgenital fusion (Fig. 13D–E) and a larger asymplicate zone on ovary (Fig. 13F–G) and the style (Fig. 13H) and stigma (Fig. 13I). This last zone is characterized by the presence of visible ventral sutures at the center of the ovary and style, indicating postgenital fusion (Fig. 10A). Also, both hemisymplicate and asymplicate zones are fertile. All studied species have tricarpelar gynoecium with three locules. In Bromelioideae species, placentation is axile at the hemisynascidiate zone and parietal at the hemisymplicate zone (Fig. 7A–B). Tillandsioideae and Pitcairnoideae species show parietal placentation, given that the ventral suture is present (Fig. 10A–B). All analyzed ovules are anatropous, bitegmic (Fig. 14A–F), and both ovular and placental vascularization are performed by ramification of the ventral carpellary bundles (Fig. 3A–B). Ovules of D. tuberosa (Fig. 14B), E. horridum, N. amazonicum, P. flammea and V. platynema show entire chalazal appendages, which represent an acute protrusion of the chalazal region (Fig. 14C, F). Papillate obturator tissue was observed at the placenta (Fig. 14G–H), constituted by one layer of palisade secretory cells with prominent nucleus, short or elongated at the analyzed stages (Table 2). Three separated stylar channels, with uniseriate outer epidermis represent the style, and in some species this epidermis can be papillose (Fig. 15; Table 2). The inner epidermis differentiates into the pollen tube transmitting tissue (PTTT), having cells with prominent nucleus and dense stained cytoplasm (Fig. 15E). The style mesophyll is homogeneous, and in some cases, it exhibits idioblasts with raphides (Fig. 15). In all Bromelioideae and Tillandsioideae species as well as in P. flammea, the style is vascularized only by the dorsal carpellary bundles (Fig. 15A–B, E), with the exception of E. horridum (Fig. 15D) and D. tuberosa (Fig. 3B, 15C) where vascularization is performed by dorsal and lateral carpellary bundles. Stigmas constitute the spiral-conduplicate type for studied species belonging to Bromelioideae and Pitcairnoideae, representing the distal part of the carpels’ asymplicate zone (Fig. 16A–D). Stigmas constitute the simple-erect type only in V. platynema and V. inflata, in which the carpels are united, representing the distal portion of the asymplicate zone (Fig. 16E). The apical region is morphologically developed in papillae (Fig. 16F–G), which are anatomically very similar in the studied species. The abaxial epidermis has striate cuticle (Fig. 16H). The mesophyll is homogeneous in all studied species (Fig. 17). The adaxial epidermis is differentiated into a pollen tube transmitting tissue (PTTT) with secretory cells (Fig. 17G–I). The stigma is vascularized only by carpel dorsal bundles in all Bromelioideae and Tillandsioideae and P. flammea analyzed species (Fig. 17A–E, H), except for D. tuberosa and E. horridum, in which the style is vascularized by ventral and lateral carpellary bundles (Fig. 17F).

Fig. 1. Light microscopy micrographs of transverse sections showing morphoanatomical aspects of developed flower buds of (A) Cryptanthus bahianus L.B.Sm., (B) Nidularium procerum Lindm., (C) Quesnelia testudo Lindm. (D) Nidularium amazonicum (Baker) Linden & E. Morren ex Lindm., (E) Nidularium longiscapum B.A. Moreira & Wand., (F) Vriesea inflata (Wawra) Wawra. A: Presence of a true stamen-petal tube formed by the adnation of the filaments and the petals at the basal portion of flower. B: Transition zone between basal and middle portion of the flower. Presence of longitudinal callosities (arrow) towards the antesepalous rounded filaments. C: Middle portion of flower, with free dorsiventral filaments. D: Middle portion of flower, with the presence of longitudinal callosities towards the antesepalous filament, simulating a stamenpetal tube (arrow) and the epipetalous stamens adnate to the sepals. E: Middle portion of the flower, with presence of dorsiventral epipetalous filaments. Petal appendages (arrows) surrounding the antesepalous filaments (forming a stamen-petal tube). F: Superior triangular ovary and the rounded/triangular filaments. An = Anther, Fi = Filament, Fi-P = Antepetalous Filament, Fi-S = Antesepalous Filament, Ov = Ovary, Pe = Petal, Pe-Fi = Petal-Stamen Tube, Se = Sepal, St = Style.

4. Discussion 4.1. General anatomy All studied species show a stamens arrangement relative to the petals that morphologically simulates a tube at least towards the basal portion of the flower, except P. flammea, E. horridum and V. inflata. This is possible by the adnation of stamens to petals at the basal portion at the flower, forming a petal-stamen tube. At the middle portion of the flower, one whorl of stamens is adnate to the petals. In such case, the free whorl is surrounded by longitudinal petal callosities or by petal appendages, as it can be seen in N. longiscapum. With this morphological arrangement, the free stamens simulate a tube. The formation of a petal-stamen tube is probably related to pollination by hummingbirds, which is predominant in the family (Benzing, 2000; Canela and Sazima, 2003, 2005). It is worth noting that all analyzed Aechmea species, as well as N. amazonicum possess the same pattern of stamen adnation in wich the antesepalous stamens became detached at a lower level and surrounded by petal appendages while the epipetalous stamens is still

hemisymplicate zone, characterized by the presence of congenital union at the center of ovary (Fig. 12E, dotted ellipse). Above this region occurs a large asymplicate zone of the ovary, in which carpels are postgenitally united (Fig. 12F–H, dotted lines). The style (Fig. 12I–J) and stigma (Fig. 12K) also represents the distal part of asymplicate zone. In Fig. 13 the gynoecium zonality concept for Tillandsioideae and P. flammea is represented. These species present a nearly superior ovary. The lower region of ovary is united at the stamens, petals and sepals, constituting a very short hypanthium (Fig. 13A). These species are characterized by a very short hypocarpous zone (Fig. 13B) where the majority of nectariferous tissue are present and no locules can be observed. A short hemisynascidiate zone is present, above this region, where we see the presence of nectariferous tissue at the center of carpels, as well as the ovary’s locules (Fig. 13C). Finally, there is a short 4

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Fig. 2. A–B: Scanning Electron Microscopy (SEM) of petal’s adaxial basal region in (A) Quesnelia testudo Lindm. and (B) Aechmea gamosepala Wittm. C–I: Light microscopy of transversal sections from developed flower buds, highlighting the filaments in (C) Nidularium amazonicum (Baker) Linden & E. Morren ex Lindm., (D) Pitcairnia flammea Lindl., (E) Vriesea inflata (Wawra) Wawra, (F) Encholirium horridum L.B. Sm., (G) Nidularium procerum Lindm., (H) Dyckia tuberosa (Vell.) Mez, (I) Nidularium amazonicum (Baker) Linden & E. Morren ex Lindm. J–K: Scanning Electron Microscopy (SEM) of the distal portion of the filaments in (J) N. amazonicum and (K) Q. testudo. A and B: Presence of a pair of petal appendages on adaxial surface of the petals. C: Presence of longitudinal callosities towards the filaments. D: Presence of two vascular bundles (arrows) in the filament. E: Triangular filaments with one vascular bundle. F: Rounded filaments, with three vascular bundles (arrows). G: Flat filaments with two vascular bundles (arrows). H: Flat filament with two vascular bundles (arrows) I: Detail of epidermis of filament in transversal cross section. Presence of a cuticle layer that emphasizes the conical shape of these cells (arrows). J: Filament surface, showing the cuticle deposition with striate pattern. K: Filament surface, showing irregular deposition of cuticle in striate pattern. Ap = Petal appendage; Ca = Longitudinal callosities; Fi = Filament. Arrows indicate vascular bundles.

In the studied species, the epidermis in the distal portion of filaments, styles and anthers is papillose and conical. Nevertheless, this shape, which is observed in both transversal and longitudinal sections, is caused by cuticle deposition and not by the cell wall thickening or cell shape. These traits have already been reported in petals of Aechmea distichantha Lem. and Canistropsis billbergioides (Schult. & Schult. f.) Leme by Oliveira et al. (2016), as well as in stigmas of species of Bromelioideae and Tillandsioideae by Souza et al. (2016), and in anthers of

adnate to the petals. Even though Aechmea does not represent a monophyletic genus (Sass and Specht, 2000), this character should be subject of additional investigation to be used in ancestral character reconstructions. In N. longiscapum and Neo. spectabilis, antepetalous stamens are free, while antesepalous stamens are adnate to the petal lobes, also forming a petal-stamen tube. In this case, free filaments are also surrounded by longitudinal petal callosities, simulating a floral tube. 5

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Table 2 Anatomical characters of the androecium and gynoecium of the studied species of Bromeliaceae. Character 1: Presence of ornamented cuticle in the anthers (+), absence (–). Character 2: Number of vascular bundles in the filaments. Character 3: Presence of raphides in the filaments (+), absence (–). Character 4: Flattened filaments (+), round or triangular filaments (–). Character 5: Presence of interlocular zone in anther (+), absence (–). Character 6: Anther with protuberance in the connective region (+), absence of the protuberance (–). Character 7: Presence of aerenchyma in anther (+), absence (–). Character 8: Presence of mechanical tissue near the connective region (+), absence (–). Character 9: Presence of idioblast with raphides in the anther (+), absence (–). Character 10: Superior ovary, or almost (+), inferior ovary (–). Character 11: Ovary/ hypanthium outer epidermal cells with wall thickening in anticlinal and internal periclinal walls (+), ovary/ hypanthium outer epidermal cells without wall thickening in anticlinal and internal periclinal walls (–). Character 12: Ovary outer epidermal cells anticlinally elongated (+), not elongated (–). Character 13: Presence of aerenchyma in the ovary/hypanthium walls (+), absence (–). Character 14: Presence of ornamented cuticle in the style (+), absence (–). Character 15: Presence of stomata in the walls of the ovary/hypanthium (+), absence (–). Character 16: Presence of peltate trichomes in the hypanthium walls (+), absence (–). Character 17: Hypodermal cells with thickened walls in hypanthium (+), hypodermal cells not thickened (–). Character 18: Obturator with short cells (+); obturator with long cells (–). Character 19: Lateral and dorsal carpellary bundles in the style and stigma (+), absence (–). Character 20: Ovules with chalazal appendages (+), ovules without chalazal appendages (–). Character 21: Type of septal nectaries in cross section. 1:Nonlabyrinthine common nectarial cavity (inferior ovary); 2: Labyrinthine common nectarial cavity (inferior ovary); 3: Infralocular nectaries with labyrinthine common nectarial cavity. Species/ characters Bromelioideae Aechmea bromeliifolia A. cylindrata A. gamosepala A. nudicaulis A. ornata Cryptanthus bahianus Neoregelia spectabilis Nidularium amazonicum N. longiscapum N. procerum Quesnelia testudo Pitcairnoideae Dyckia tuberosa Encholirium horridum Pitcairnia flammea Tillandsioideae Vriesia inflata V. platynema

1

2

3

4

5

6

7

8

9

10

– – + + + – – + – + +

1 1 1 1 1 1 1 1 1 2 1

– + + – – + – – – – +

– + + – – + – + – + +

+ + + + + – – – – – +

– – – – + – + + + + –

– – – – – – – – + – –

– – – – – – – – – – –

– + + – + – – – – – –

– – – – – – – – – – –

– + +

2 3 2

– + +

+ + +

– – –

– – –

– – –

+ – –

+ + –

– +

2 1

– –

– +

– –

– –

– –

– –

+ –

11

12

13

14

15

16

17

18

19

20

21

– + + + – – – – – – –

– – – – – – – – – – –

– – – – – – – – + + –

– + – – + – – + + – +

– + – + – – – – – – –

+ + – – + – – – – – +

– – – + – – – – – – +

– + + – + + + + + + +

– – – – – – – – – – –

– – – – – – – + – – –

2 1 1 1 1 1 2 2 2 1 2

+ + +

– – –

+ – –

– + –

+ – –

+ – –

– – –

– – –

+ + +

+ + –

+ + +

3 3 3

+ +

– –

– –

– –

– –

– –

– –

– –

+ –

– –

– +

3 3

Fig. 3. Comparative diagrams of inner stamens whorl and gynoecium vascularization. The center represents the pedicel; the margins represent the upper portion of stamens and gynoecium. A: Aechmea gamosepala Wittm. The filaments are vascularized by a single vascular bundle (Fi) since the pedicel (center of the diagram). The carpels are vascularized by carpellary bundle that arose as a single bundle in the pedicel (center of diagram), but divides into two ventral carpellary bundles (Vc) and a central dorsal carpellary bundle (Dc). The ramifications of ventral carpellary bundles (Vc’) vascularizes the septal nectaries and the placentae as well as the ovules. B: Dyckia tuberosa (Vell.) Mez. The filaments are vascularized by two vascular bundles at its entire length (Fi). However, at the pedicel (center of the diagram), the stamens complexes arose as a single bundle. At the connective region, in anthers, the two bundles of filaments are united. The carpels are vascularized by carpellary bundle that arose as a single bundle in the pedicel (center of diagram), but divides into two ventral carpellary bundles (Vc) and a central dorsal carpellary bundle (Dc). At its base, the ventral carpellary bundle divides into two: one of the ramifications of ventral carpellary bundle (Vc’) vascularizes the plancentae as well as ovules. The other ramification, the lateral carpellary bundle (Lc), vascularizes the style and stigma. Dc = Dorsal carpellary bundle; Fi = Filament bundle; Lc = Lateral carpellary bundle; Vc = Ventral carpellary bundle; Vc’ = Ramification of ventral carpellary bundle. 6

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Fig. 4. Light microscopy of transversal sections of developed flower buds, highlighting the anthers in (A) Aechmea ornata Baker, (B) Quesnelia testudo Lindm., (C) Nidularium procerum Lindm., (D) Nidularium longiscapum B.A. Moreira & Wand., (E) Encholirium horridum L.B. Sm. and (F) Cryptanthus bahianus L.B. Sm. A and B: Presence of interlocular zone (arrows). A: Presence of a protuberance in connective region (arrowhead). C–F: Absence of interlocular zone. D: Presence of an aerenchyma in the connective region. Ar = Aerenchyma; Co = Connective; En = Endothecium; Fi = Filament; Po = Pollen sac. Arrows indicate the interlocular zone. Arrowheads indicate the presence of a protuberance in the connective region.

pollinators. In the studied species, idioblasts with raphides are present in both androecium and gynoecium. The presence of idioblasts containing raphides in vegetative organs of Bromeliaceae is well documented (Krauss, 1949; Tomlinson, 1969). In flowers, they have been documented in anthers by Sajo et al. (2005), and in all whorls by Oliveira et al. (2016). The presence of raphides is usually associated with a need to neutralize large amounts of oxalic acid produced (Brighina et al., 1984), in addition to making the plant less palatable to herbivores (Mauseth, 1988). Coté (2009) studied different types of crystals in vegetative and reproductive organs of Diefferenbachia seguine (Araceae). He argues that idioblasts with raphides might not act to deter herbivory, at least in the staminodia, since this structure is a reward for pollinators, usually beetles, and consumed by them when they are trapped within the spathe. Gebura and Winiarczyk (2016) point that calcium is essential for the growth of the male gametophyte. Accordingly, the presence of raphides, or needle-shaped crystals of calcium oxalate, is an important source of calcium in the anthers of Commelinaceae. Also regarding anthers, D’Arcy et al. (1996) investigated the role of calcium oxalate in Ericaceae. In the investigated species, raphides were shown to occur as an oxalate package located near the stomium, and it was suggested to act as a reward for some pollinators. In Araceae, Barabé et al. (2004) describe the presence of extracellular calcium oxalate crystals in the inflorescence, mainly acting to

species of the genus Dyckia by Carvalho et al. (2016). Within Poales, an epidermis with conical aspect was observed in the anthers of Rapateaceae (Venturelli and Boumanm, 1988; Oriani and Scatena, 2013; Ferrari and Oriani, 2016) and Mayacaceae (Carvalho et al., 2009). Several authors have discussed the ecological implications of conical epidermis with cuticle deposition in the petals. According to Koch et al. (2008) and Whitney et al. (2011), this feature could enhance the grip of insect pollinators to petals during flower visitation, while also avoiding the accumulation water on the surface of petals. Oriani and Scatena (2013) have also observed ornamented cuticle in the epidermis of the petals, anthers, and styles in Rapateaceae. The authors report that the presence of a thick cuticle could be an adaptation against excess light by increasing solar radiation reflection. The same authors infer that cuticle ornamentation could be related to pollinator attraction. Choi et al. (2011) noted the same pattern of striation in cuticle deposition in the tepal epidermal cells of Allium (Amarilidaceae). The authors suggest that light could be reflected in a different way, depending on the pattern of cuticle striation, attracting and guiding pollinators to the flowers. As pointed out by Choi et al. (2011), we believe that the presence of a thick, ornamented cuticle in the filaments, anthers, styles, and stigmas in Bromeliaceae is also related to pollinator attraction. We reasoned that when the cuticle is present in filaments and styles, occurring in their upper position, the anthers and stigmas are exposed during anthesis, promoting light reflectance that attracts 7

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Fig. 5. Light microscopy of transversal sections in developed flower buds showing details of the anthers in (A, F–G) Dyckia tuberosa (Vell.) Mez, (B) Encholirium horridum L.B. Sm., (C, H) Nidularium longiscapum B.A. Moreira & Wand., (D) Quesnelia testudo Lindm. and (E) Aechmea ornata Baker. A: Presence of conical epidermis with thin cuticle deposition. B–D: Presence of conical epidermis with cuticle deposition (arrows). D–E: Presence of an interlocular zone (thick arrows). F–G: Presence of endotheciumlike tissue in the connective region (arrowheads). H: Detail of anther, showing the aerenchyma. Ar = aerenchyma; Co = Connective; En = Endothecium; Fi = Filament. Asteriscs indicate idioblasts with raphids.

thickening patterns in the endothecium layer, the author concluded that endothecium thickening might provide useful characters to distinguish different groups among angiosperms. In this context, the author provides a detailed description of six main patterns: Bankzellen (baseplate present), Griffzellen (baseplate absent, but thickening bars on inner periclinal wall branching), Netzfazern (reticulate thickening), Ringfasern (annular thickening), Spiralfasern (helical thickening) and U-Klammern (U-shaped thickening). In the studied species, our data show that the thickening of endothecium cells is U-shaped with basal anastomosis. This corroborates data shown by Manning (1996), who classified the endothecium of all Bromeliaceae species as following this pattern. For Typhaceae and Rapateaceae, the closest families to Bromeliaceae, the author cited the presence of a helical thickening pattern.

attract pollinators by their reflective capacity. In contrast, our observations indicate that raphides occur inside idioblasts in the gynoecium and androecium. Even though the location of the raphides observed by us is not superficial, when viewed under stereomicroscopy, Bromeliaceae flowers have a characteristic brightness (personal observation). In Bromeliaceae, nectar produced by septal nectaries, not floral parts, is offered as a reward to pollinators, such as hummingbirds. In this case, the presence of idioblasts with raphides could attract pollinators due to the brightness they confer to floral parts, as suggested by Barabé et al. (2004). The endothecium promotes stomium rupture by differential shrinkage upon anther drying (Schmid, 1976; Esau, 1977; Endress, 1994). According to Manning (1996), after a long revision about 8

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Fig. 6. Scanning Electron Microscopy (SEM) of the anthers’s surface in (A) Dyckia tuberosa (Vell.) Mez, (B) Enchlirium horridum L.B. Sm., (C) Neoregelia spectabilis (T. Moore) L.B. Sm., (D) Nidularium amazonicum (Baker) Linden & E. Morren ex Lindm., (E) Aechmea nudicaulis (L.) Griseb. and (F) Vriesea platynema Gaudich. A: Presence of regular deposition of cuticle. B: Presence of a slight cuticle deposition in striate pattern. C–F: Striated pattern of cuticle deposition.

the Hamamelidae subclass (Hufford and Edress, 1989), and it seems to play a role in anther dehiscence. In Bromeliaceae, this character can be seen only in Aechmea and Quesnelia species, reflecting its phylogenetic relation potential use to distinguish these genera from other Bromelioideae. Another useful character to distinguish Bromelioideae species is the presence of a protuberance in the connective region. First reported here, this character also requires further study regarding its value to phylogenetic studies, given it only occurs in species belonging to the Nidularioid Complex (N. procerum, N. amazonicum, N. longiflorum and Neo. spectabilis). This complex comprises the genera Neoregelia, Nidularium, Canistropsis, Edmundoa and Wittrockia, which are characterized by inflorescences with developed superior and primary scape bracts that can accumulate water and resemble a nest over the foliar rosette (Leme, 1997). These genera have a difficult delimitation, and recent phylogenetic studies show that none of them represents a monophyletic group (Silvestro et al., 2014; Evans et al., 2015). However, the Nidularioid complex seems to represent a monophyletic group, emerging as a Nidularioid clade in most recent phylogenies (Silvestro et al., 2014; Evans et al., 2015; Heller et al., 2015). In this context, the presence of a protuberance in the connective region may represent a structural synapomorphy for this group. Studying flowers of species belonging to the Nidularioid complex (Canistropsis billbergioides, Canistrum auratiacum, Edmundoa lindenii,

Apart from endothecium cells, this thickening can occur in some anther tissue with endothecium-like cellular characteristics located near the connective regions, thus named endothecium-like tissue. Similar to the endothecium, this tissue also has an annular or helicoidal thickening composed of cellulose and lignin. This tissue functions in a manner similar to that of the endothecium, i.e., promoting anther dehiscence (Bhandari, 1984). In the studied species, this tissue only occurs in Dyckia tuberosa. Carvalho et al. (2016), when analyzing three species of Dyckia, have also described the presence of this tissue in anthers of D. ibicuiesis and D. racinae. Considering this character was described only for Dyckia species, we suggest that it may represent a synapomorphy for the genus. Elongated cells near the stomium form an interlocular zone, but only in Aechmea and Quesnelia species. This is the first record of this zone being present in Bromeliaceae, and it could represent a potential character for taxonomical delimitation distinguishing Aechmea and Quesnelia from other Bromelioideae species. In images provided by Oliveira et al. (2016), this character can also be seen in Aechmea distichantha, corroborating the proximity between Aechmea and Quesnelia, as demonstrated through phylogenetic studies (Faria et al., 2004; Almeida et al., 2009; Schulte et al., 2009). According to Hufford and Edress (1989), the interlocular zone is a region between the pollen sacs of a theca that becomes disrupted prior to dehiscence. This zone is found in anthers of several eudicot family species, such as members of 9

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Fig. 7. Light microscopy of transversal sections in developed flower buds in the hypanthium region of the studied species from Bromelioideae subfamily (A and E) Nidularium procerum Lindm., (B) Nidularium amazonicum (Baker) Linden & E. Morren ex Lindm., (C) Aechmea nudicaulis (L.) Griseb., (D) Aechmea ornata Baker., (F) Neoregelia spectabilis (T. Moore) L.B. Sm. A: Presence of septal nectary at same level as the placentae insertion region. B: Presence of septal nectary cavity in placentae insertion region. C: Detail of outer wall of hypanthium, showing the thickening of inner periclinal wall of epidermal cells. D: Detail of outer wall of hypanthium, showing a peltate trichome. E: Detail of hypanthium wall, showing aerenchyma next to the inner wall. F: Detail of hypanthium wall, showing the absence of aerenchyma next to the inner wall. Ar = Aerenchyma; DCb = Dorsal Carpellary bundle; Hi = Hypanthium; Lo = Ovary locule; Op = Nectary opening. P-St = Petal stamen complex blundle; Pl = Placenta; Sb = Sepal bundle; Sn = Septal nectary. Thin arrows indicate epidermal cells with silica crystal. Thick arrows indicate epidermal cells with thickened anticlinal and internal periclinal walls. Asteriscs indicates the hypoderm.

accumulate water, we do not believe that the presence of aerenchyma are related to hypoxia. In the studied species, chalazal appendages are present only in N. amazonicum for Bromelioideae species, in V. platynema for Tillandsoideae species, and in all Pitcairnoideae species analyzed. It is known that this structure is formed by a growth in the epidermal and subepidermal layers of the chalazal region, and that the final product of this growth is an entire, or fimbriated, protuberance (Palací et al., 2004; Sajo et al., 2004b; Fagundes and Mariath, 2014; Mendes et al., 2014; Kuhn et al., 2016). These appendages are not exclusive to Bromeliaceae ovules, since they are present in the ovules of several other monocotyledon families, such as Tofieldiaceae and Nartheaceae (Remizowa et al., 2006). In Poales, specifically, the Bromeliaceae, Rapateaceae, and Juncaceae all have chalazal appendages in their ovules (Venturelli and Boumanm, 1988; Sajo et al., 2004b; Oriani et al., 2012). According to Smith and Downs (1977) and Palací et al. (2004), the presence of chalazal appendages is related to the development of feathery seeds in Tillandsioideae. In Catopsis, for instance, Palací et al. (2004) observed that the chalazal appendages of the ovules are fimbriated and grow along with the development of seeds, generating their feathery appendages that assist in wind dispersal. For species in the genus Tillandsia, the appendages are entire. The authors discuss that the

Nidularium inocentii, Neoregelia johanis and Wittrockia superba), Nogueira et al. (2015) also reported the presence of aerenchyma in the ovary walls. The authors associated the presence of this character with a hypoxic condition of ovaries in direct contact with water. Such contact arises from the submersion of inflorescences in water that accumulates in the central tank formed by the overlapping of leaf sheaths, or due to water accumulation by primary and floral bracts. The authors also highlight that the presence of aerenchyma in the ovary walls could be considered a synapomorphy for the Nidularioid clade. Four species belonging to the Nidularioid complex were also analyzed in the present study (N. amazonicum, N. longiflorum, N. procerum and Neo. spectabilis). Neo. spectabilis is the only one whose inflorescences are submerged in the central tank. The other three species show an elongated floral peduncle and well-developed primary bracts, permitting the accumulation of rainwater. Since the species with submerged inflorescences does not show such feature, this trait might not necessarily be related to hypoxia. However, it could represent a synapomorphy for the Nidularioid complex, thus corroborating the hypothesis of Nogueira et al. (2015), but with reversions in the Nidularioid complex clade. In the remaining studied species, aerenchyma is present only in the ovary walls of E. horridum and since this species inflorescences are not submerged and its floral bracts do not 10

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Fig. 8. Light microscopy of transversal sections in developed flower buds, at the region of hypanthium, highlighting the septal nectaries in (A) Quesnelia testudo Lindm., (B and E) Neoregelia spectabilis (T. Moore) L.B. Sm., (C) Nidularium amazonicum (Baker) Linden & E. Morren ex Lindm., (D and F) Aechmea gamosepala Wittm., A–C: Hemisynascidiate zone, with a common nectary cavity. D: Basal portion of nectaries. Presence of three disconnected channels, corresponding to the gynoecium’s hemisynascidiate zone with nectary closed in the center postgenitally. E: Detail of septal nectary showing the great amount of vascular bundles. F: Detail of septal nectary showing secretory cells with strongly stained cytoplasm. Hi = hypanthium; Lo = ovary locule; Sn = septal nectary.

winged seeds in some Pitcarnioideae (Varadarajan and Gilmartin, 1988). In Dyckia, the entire asymmetric-shaped chalazal appendages are related to the asymmetrical growth that characterizes the wings of seeds, as observed in D. racinae (Dorneles et al., 2014). In Dyckia tuberosa, we observed an entire chalazal appendage with a pronounced asymmetric growth toward the concave side of the ovule. This morphology observed on the ovules resembles that of the future seed. Thus, in this case, it can be concluded that the chalazal appendage is responsible for the formation of seed wings in Dyckia species.

feathery appendages of Tillandsia seeds originate from the growth of both inner and outer integuments of the ovule. Hence, the authors propose that the feathery appendages of the seeds of Vriesea and Tillandsia are not homologous, despite having the same purpose of aiding seed dispersal through wind. In the studied species of Vriesea, only V. platynema shows a welldeveloped, entire chalazal appendage. Both inner and outer integuments showed a more pronounced growth when compared to the remaining studied species. The pronounced growth of the ovule integuments was also observed in the 25 species of Vriesea analyzed by Kuhn et al. (2016). Thus, feathery appendages of the seeds of Vriesea species are most likely derived from the growth of integuments, as reported by Palací et al. (2004) for species of Tillandsia. However, to confirm such hypothesis, ontogenetic studies of the seeds of Vriesea species are needed. Chalazal appendages also seem to be related to the development of

4.2. Vascularization Filaments are usually vascularized by one vascular bundle in Bromeliaceae. Filaments can be vascularized by more than one vascular bundle, such as the cases seen in Pitcarnioideae (Dyckia tuberosa, D. racinae, Encholirium subsecundum, E. horridum, and P. flammea), in 11

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Fig. 9. Light microscopy of transversal sections of Aechmea gamosepala Wittm. developed flower bud. A: Hypanthium, synascidiate zone. B–C: Hypanthium, hemisynascidiate zone with the presence of one central septal nectary cavity. D–E: Hypanthium, hemisynascidiate zone with the presence of three disconnected channels of septal nectaries and a postgenital fusion at the center (dotted lines). F: Hypanthium, hemisymplicate zone with postgenital fusion at the center (dotted lines) and the presence of nectary apertures. G: Hypanthium, asymplicate zone, base of style. H: Style, asymplicate zone I: Stigma, asymplicate zone. Arrows shows postgenical fusion. Hi = hypanthium; Lo = ovary locule; Op = Nectary opening; Sn = septal nectary; Sc = stylar channel; St = stigma. Arrows indicate the occurrence of postgenical fusion. Dotted lines indicate postgenical fusion.

on data from both the literature and the studied species, we can say that the presence of more than one vascular bundle at the pedicels was originated several times over in Bromeliaceae, and it occurs in species of Bromelioideae, Tillandsioideae and Pitcairnioideae. Nevertheless, filaments with more than one vascular bundle seem not to be related with filament size or shape. This is evidenced on Table 2, character 2 (number of vascular bundles on filaments) and character 4 (flattened filament represented by +). Flattened filament are bigger than rounded or triangular filaments, and still can have only one bundle, as seen in C. bahianus, N. amazonicum, Q. testudo and V. platynema (see Table 2). On the other hand, filaments with more than one vascular bundle can present rounded or triangular shape, as seen in V. inflata (Table 2). In the ovary region of the gynoecium of studied species, both dorsal and ventral carpellary bundles are responsible for vascularization.

Tillandsioideae (Vriesea oligantha, V. inflata) and in Bromelioideae (Nidularium procerum) (Arrais, 1989; Carvalho et al., 2016). Data about the remaining subfamilies are not available in the literature. Stamen vascularization is generally formed by a single vascular bundle that can be concentric, or not, in most Angiosperms (Eames, 1931; Puri, 1951; Schmid, 1976). However, filaments vascularized by more than one bundle (generally three bundles) occur in stamens of Magnoliaceae, Lauraceae and Musaceae (Eames, 1931; Wilson, 1942; Puri, 1951). Among the studied species, E. horridum has both antesepalous and antepetalous filaments vascularized by three bundles. In Dyckia tuberosa, P. flammea, V. inflata and Nidularium procerum, both whorls of filaments are vascularized by two bundles. In all cited species, the bundles that vascularize the filaments arose as one in the pedicel. Based 12

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Fig. 10. Light microscopy of transversal sections in developed flower buds in the ovary region of species from the Tillandsioideae – (A) Vriesea inflata (Wawra) Wawra and Pitcairnioideae – (B and D) Dyckia tuberosa (Vell.) Mez, (C) Pitcairnia flammea Lindl., (E–F) Encholirium horridum L.B. Sm. A: Presence of a superior ovary and a stamen-petal tube. B: Detail of superior ovary with anatropous ovules. C: Detail of a single carpel and a filament with two vascular bundles. Presence of septal nectary at the same level as that of the carpel in the center of ovary. D: Detail of epidermis, showing stomata. E: Presence of aerenchyma near the inner wall of ovary. F: Presence of uniseriated epidermis. Ar = Aerenchyma; Ep = Epidermis; Fi = Filament. Lo = Locule. Ov = Ovary. Pe = Petal. Se = Sepal. Arrows indicate vascular bundles of the filaments. Arrowheads indicates the nectary openings. Asteriscs indicate idioblasts with raphids.

production of nectar is related to pollinators, the loss of septal nectary through evolution is likely related to wind pollination throughout the Poales (Rudall, 2002). Thus, pollination by animals that are attracted by the nectar produced in the flowers is likely a plesiomorphic condition in Poales (Givnish et al., 2010). Several authors discussed the interpretation of carpel fusion and the presence of septal nectaries. Van-Heel (1988), after analyzing the development of septal nectaries in different species of monocotyledons, states that the septal nectaries development is related free or partially free carpels primordia that are united posteriorly by postgenital fusion. Rudall (2002) and Remizowa et al. (2008) reports the septal nectary to be a feature of species that have postgenitally fused carpels of syncarpous ovaries. Schmid (1985) highlight that his secretory structure is located in a chamber formed in the septum of the ovary with postgenital fusion. Remizowa et al. (2010) state that carpel primordia are initiated individually in a syncarpic gynoecium with septal nectary. The ventral portion of adjacent primordia is joined in a relatively late stage of development. The nectaries develop in the outer wall of the lateral and ventral parts of these primordia before the fusion of the carpels. Thus, after fusion, the nectaries are internalized in the septal region or in the basal region of the carpels below the locules. In flowers with inferior ovary, intercalary growth occurs from the base of the primordium, including the region of nectariferous tissue proliferation. In this case, nectaries are found in the region of the ovary septa. In flowers with superior ovary, the nectariferous tissue starts to proliferate in the basal portion of the primordia, and the region of the carpel with intercalary growth is located above the proliferating nectariferous tissue. Thus, the nectaries formed by the folding of the carpel and by the intercalary growth are located below the locules of the ovary. In the

When the ovary is inferior, as in species belonging to the Bromelioideae, ventral carpellary bundles are responsible for the vascularization of the placenta and the septal nectaries. On the other hand, in flowers of Pitcairnioideae and Tillandsioideae spp., with a nearlysuperior ovary, ventral carpellary bundles vascularize only the placenta, since the nectary is located below the locules of the ovary. In species of Pitcairnioideae, the dorsal and lateral (ramification on ventral carpellary bundle) carpellary bundles vascularize the upper region of the gynoecium, represented by the style and the stigma. Vascularization of style and stigma by three vascular bundles is not a very common character among Angiosperms (Eames, 1931; Wilson, 1942; Puri, 1951). For that matter, the condition of a single bundle could have been originated by the fusion of two lateral bundles with the median trace, or even by the loss of the lateral traces (Wilson, 1942). In Bromeliaceae, styles and stigmas vascularized by the dorsal and lateral carpellary bundles are only present in Pitcairnioideae (E. horridum and D. tuberosa) and might represent a synapomorphy for this subfamily. 4.3. Secretory structures and gynoecium zonality One of the most striking features of flowers in Bromeliaceae is the presence of septal nectaries (Sajo et al., 2004a). These nectaries are complex structures found only in flowers of Monocotyledons and were lost several times throughout the evolutionary history of the group (Fahn, 1952; Rao, 1975; Endress, 1994; Rudall, 2002; Bernadello, 2007). In the order Poales, these structures were only registered in Bromeliaceae and Rapateaceae (Sajo et al., 2004a) and may represent an ancestral condition in this order (Linder and Rudall, 2005). In the remaining lineages of Poales, this structure was lost. Because the 13

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Fig. 11. Light microscopy of a longitudinal section of developed flower buds of (A) Vriesea platynema Gaudich. Light microscopy of transversal (B–D) sections of developed flower buds of Pitcairnoideae – (B–C) Dyckia tuberosa (Vell.) Mez, and Tillandsioideae – (D) Vriesea inflata. (Wawra) Wawra. A: Presence of a nectary below the ovary’s locule. B–D: Presence of the labyrinthine form of the infralocular nectaries. C: Detail of nectary cells. D: Lo = ovary locule; N = infralocular nectary; Ov = ovary.

zone; (2) the syncarpous type, as represented by fused carpels with synascidiate, symplicate, hemisymplicate and asymplicate zones; and (3) the hemisyncarpous type, in which carpels are partially fused with the hemisynascidiate, hemisymplicate and asymplicate zones. For the hemisyncarpous gynoecium, which occurs in Bromelioideae species, the author established the following four zones: In the (syn)ascidiate zone, carpels are united centrally by septa, and no suture can be observed between the axis and the carpel tissues. This zone is congenitally fused. In the (sym)plicate zone, which is located above the (syn)ascidiate zone, the carpels are partially, postgenitally united. Areas of fusion are highlighted by the presence of visible sutures. The septum is present in this region. The hemisymplicate zone is located above the symplicate zone where the septum is no longer present. In this case, carpels are still united postgenitally at the margins. This area usually forms the upper part of ovary and the style. In the asymplicate zone, the carpels are free. This zone corresponds to the stigma. Using the zonation concept described above after Leinfellner (1950); Odintsova et al. (2013) have described the gynoecium of three species of Asparagaceae (Asparagales), which also possess septal nectaries. The authors distinguish three main zones regarding the position of septal nectaries in the zones proposed by Leinfellner (1950). First, in the zone where the nectary tissue is distinct, three disconnected

analyzed species of Bromelioideae, it is possible to see the presence of a ventral suture at the upper portion of the hemisynascidiate zone of the carpels, mainly at the placentation and upper portion of the nectary, indicating that the fusion occurred postgenitally. In Pitcairnoideae and Tillandsioideae species, in which ovaries are nearly-superior, it is possible to see that all the carpels, except for a very short zone at the base that represents the hypocarpous zone with the presence of most of the nectariferous tissue, are postgenitally united (represented by hemysimplicade and asymplicate zones), with conspicuous ventral sutures. Schmid (1985) classified septal nectaries into five main types, according to their anatomy and morphology: (1) Non-labyrinthine distinct septal nectaries, (2) Labyrinthine distinct septal nectaries, (3) Nonlabyrinthine common nectarial cavity, (4) Labyrinthine common nectarial cavity, and (5) Labyrinthine common nectarial cavity with convoluted proliferations of the carpellary walls. In cross sections, we notice that at the base nectaries of inferior ovary species represent the type 1. When analyzing the middle region of these nectaries, we found differences in anatomy, providing more characters with which to distinguish Bromeliaceae species, as shown in Table 2. Leinfellner (1950) recognized three types of gynoecia and established the vertical zonality of the gynoecium: (1) the apocarpous type, as represented by non-fused carpels and having only an asymplicate 14

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Fig. 12. Light microscopy of transversal sections of Dyckia tuberosa (Vell.) Mez developed flower bud. A: Hypocarpous zone with the presence of infralocular nectary. B – D: Sections between hypocarpous and hemisynascidiate zones, with the presence of the nectary openings. Congenital union are evidenced at the center of dotted ellipses, and dotted lines and arrows indicate postgenital fusions. E: Ovary, hemisymplicate zone, presence of congenital union at the center of dotted ellipse. F – H: Ovary, asymplicate zone. I: Basal portion of style, asymplicate zone. J: Upper portion of style, asymplicate zone. K: Stigma, asymplicate zone. Lo = Ovary locule. Op = Nectary opening. Ov = Ovary. Pl = Placentae. Sc = Stylar channel. Se = Sepals. N = Nectary. Dotted lines represent postgenital fusion. Arrows indicate the region of postgenital fusion. Dotted ellipses indicate the presence of congenital union.

In the studied Bromelioideae species, which have flowers with an inferior ovary, nectaries are truly septal. They occur in the septa of the ovary, mostly at the hemisynascidiate zone of the carpel, with their openings at the basal part of the hemisymplicate zone (at the hypanthium floor). In these species, we can observe that zone 1 (zone of distinct nectary) and 2 (zone of common nectary), as described by Odintsova et al. (2013), occur at the hemisynascidiate zone of

nectariferous cavities are present. This region is located beneath the locules and the synascidiate zone. Second, in the zone of common nectary tissue, the three nectary cavities are united in the ovary center by a common epidermal surface. This zone is located at hemisymplicate zone. Third, the zone of external nectary tissue comprises the upper part of the previous zone up to the base of asymplicate zone. In this region, each septal nectary cavity unites distally with the septal groove. 15

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Fig. 13. Light microscopy of longitudinal (A) and transversal (B–I) sections of Vriesea platynema Gaudich. developed flower bud. A: Longitudinal view of the inferior portion of ovary showing the small region (between lines A and B) where sepals, petals, stamens and carpels are united constituting a hypanthium. B: Hypocarpous zone with the presence of nectariferous tissue. C: Hypanthium, hemisynascidiate zone, with the presence of three channels of nectary and the presence of postgenital fusion (dotted lines). D–E: Hypanthium, hemisimplicate zone, with the presence of three channels of nectariferous tissue and nectary openings. The septal region present postgenital fusion where the nectaries are not present (dotted lines). Sections C–E represent the hypanthium portion between lines A and B of section A. F–G: Ovary, asymplicate zone, presence of upper part of nectary opening between septal postgenital fusion (dotted lines). H: Style, asymplicate zone. I: Stigma, asymplicate zone. Ap = Petal appendage. Fi = Filament. Hi = Hypanthium. Lo = Ovary locule. Op = Nectary opening. Ov = Ovary. Pe = Petals. Pl = Placentae. Sc = Stylar channel. Se = Sepals. N = Nectary. Dotted lines represent postgenital fusion. Arrows indicate the region of postgenital fusion.

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Fig. 14. A–D: Light microscopy of transversal sections of the ovaries, highlighting the anatropous ovules in (A) Aechmea gamosepala Wittm., (B) Dyckia tuberosa (Vell.) Mez, (C) Pitcairnia flammea Lindl. and (D) Nidularium procerum Lindm. E–F: SEM of the ovules in (E) Nidularium amazonicum (Baker) Linden & E. Morren ex Lindm. and (F) Vriesea platynema Gaudich. G–H: Transversal sections of the ovaries, highlighting the obturator tissue in (G) V. platynema and (H) Aechmea ornata Baker. A: Arrowheads indicate obturator tissue with short cells. B: Presence of an entire chalazal appendage (arrow). C: Presence of an entire chalazal appendage (arrow). D–E: Absence of chalazal appendage. F: Presence of an entire chalazal appendage. G: Obturator tissue with elongated cells. H: Obturator tissue with short cells. Ov = Ovary; Pl = Placentae; Lo = ovary locule. Arrows indicate chalazal appendages and arrowheads indicate obturator tissue.

occupy the hemisynascidiate zone of carpels, as we found in the species analyzed here. After describing a new structural zone for gynoecium with septal nectaries, named hypocarpous zone, in which septal nectaries occur below the ovary’s locules in hemisyncarpous gynoecium, the authors theoretically proposed 12 types of carpel by combining possible consecutive structural zones. The authors point out that not all types described by them can be found in Bromeliaceae gynoecium, since it is a computer-based model. Within this classification, the studied Bromelioideae species belong to type A, having a short

Bromelioideae carpels and that the classification provided by Schmid (1985) is applicable only at the zone of common nectary, as shown in Table 2. The zone of distinct nectary is always classified as type 1 (nonlabyrinthine distinct septal nectaries), as described by Schmid (1985). Novikoff and Odintsova (2008) applied the concept of vertical zonality to three bromeliad species belonging to the Bromelioidae (Aechmea fulgens, Pseudoananas sagenarius and Billbergia vittata). For these species, the authors described synascidiate, hemisynascidiate, hemisymplicate and asymplicate zones. Septal nectaries in these species 17

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Fig. 15. Light microscopy of transversal sections of developed flower buds, highlighting the styles in (A and F) Nidularium longiscapum B.A. Moreira & Wand., (B and E) Quesnelia testudo Lindm., (C) Dyckia tuberosa (Vell.) Mez and (D) Encholirium horridum L.B. Sm. A–B: Presence of a dorsal vascular bundle (arrow). C–D: Presence of a dorsal (arrow) and lateral (arrowheads) carpellary vascular bundles. F: Detail of the epidermis of the style with cuticle deposition (arrows). Sc = Stylar channel. * = Idioblast with raphids. In A–D arrows indicate dorsal carpellary vascular bundle and arrowheads indicate lateral carpellary vascular bundles. In E the arrow indicates the pollen tube transmiting tissue (PTTT).

results, it appears that septal nectaries of Bromelioideae of studied species are classified as type A, as described by Novikoff and Odintsova (2008), unless nectaries are present below the locules. In the Pitcairnioideae and Tillandsioideae, we have decided to classify the nectaries as infralocular, considering the majority of nectariferous tissue occurs below the locules and only the nectary aperture occurs between the locules in the septal region. In the analyzed species representing these subfamilies, zone 1, as described by Odintsova et al. (2013), does not occur, since these species possess infralocular nectaries. In these species, we observe a ‘hypocarpous’ structural zone, as proposed by Novikoff and Odintsova (2008) for Billbergia vittata. In this case, the septal nectary cavity occurs below the locules, as it does for the Tillandsioideae and Pitcairnoideae analyzed here. According to the

synascidiate zone, where locules are visible and congenitally united; a hemisynascidiate zone, where septal nectaries are present; a hemisymplicate zone where nectary openings are visible; and an asymplicate zone. The studied species of Tillandsioideae and Pitcairnoideae belong to type K, having an hypocarpous zone, where the majority of nectariferous tissue is present; a hemisynascidiate zone, where the minority of nectariferous tissue is present at the center of carpel; a hemisymplicate zone, where the sutures are visible at the center of carpels and is possible to see the nectary openings; and a large asymplicate zone, where carpels are only postgenitally united. The species analyzed by Novikoff and Odintsova (2008) with nectaries only at the septum (A. fulgens and P. sagenarius) also belong to type A, and B. vitatta belongs to type K due to the presence of nectary tissue below the locules. With the present 18

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Fig. 16. A–H: SEM of the stigma in (A) Aechmea gamosepala Wittm., (B and F) Dyckia tuberosa (Vell.) Mez, (C) Nidularium procerum Lindm., (D) Quesnelia testudo Lindm., (E and G) Vriesea platynema Gaudich., (H) Pitcairnia flammea Lindl. A–D: Spiral-conduplicate type. E: Simple-erect type of stigma. F: Detail of stigma surface showing papillae on stigmatic surface. G: V. platynema. Detail of stigma surface showing elongated papillae on stigmatic surface. H: Detail of abaxial surface of stigma with striate pattern of cuticle deposition.

could increase the amount of nectar produced. Since the flowers we analyzed are pollinated by either hummingbirds or bats, a voluminous production of nectar is justified, as nectar is offered as a reward to pollinators. According to Remizowa et al. (2010), septal nectaries have evolved towards their internalization, which means that infralocular nectaries would be considered plesiomorphic in relation to the septal interlocular nectaries. This hypothesis suggests that through the internalization of nectaries, their secretory surface would be expanded, producing more nectar when compared to infralocular nectaries. However, when

authors, this zone is interpreted as a lower part of the gynoecium, but it can be formed by an invaginated floral apex. Only a small part of the nectariferous tissue and its opening occur in the hemisymplicade zone. Most of the nectariferous tissue do not occur at the septal region, but below the locules; for this reason, the authors suggest classifying them as gynopleural. Another characteristic of these nectaries is the labyrinth that delimits the cavity. According to Vogel (1969), the differentiation of a labyrinth formed in the inner surface of infralocular nectaries increases the area for nectar secretion. Thus, in the presence of an infralocular nectary, the author suggests that the formation of labyrinths 19

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Fig. 17. Light microscopy of transversal sections of developed flower buds, highlighting the stigmas in (A and G) Quesnelia testudo Lindm, (B) Nidularium procerum Lindm., (C and E) Nidularium longiscapum B.A. Moreira & Wand., (D) Aechmea ornata Baker, (F) Dyckia tuberosa (Vell.) Mez and (H–I) Aechmea cylindrata Wittm. A–E: Presence of dorsal carpellary bundle (arrow). F: Presence of dorsal (arrow) and lateral carpellary bundles (arrowheads). G: Presence of cuticle deposition on abaxial epidermis (arrow) and the presence of pollen tube transmitting tissue on adaxial epidermis (arrowheads). H–I: Presence of secretory cells in the adaxial epidermis, with dense stained cytoplasmic content, characterizing the pollen tube transmitting tissue (PTTT, arrowhead).

portion of the nectaries is present in the locular region, as in P. flammea. In flowers with inferior ovaries, nectaries can be active in the region of ovule insertion, or even below this region. In the style region, the internal epidermis has cells with prominent nuclei and dense cytoplasmic content that highlights its secretory nature. This tissue, known as transmitting tissue or pollen tube transmitting tissue (PTTT), stimulates through its secretion the growth and direction of the pollen tube (Endress, 1994; McCormick and Yang, 2005). Among the two types of transmitting tissue, one is formed by secretory epidermal cells in which the pollen tube grows through the stylar canal and the style is hollow; in the second type, the transmitting tissue has several cells layers underneath the epidermis. In the later, the pollen tube passes through the cells until it reaches the ovary locule where the style is solid (Went and Willemse, 1984; Erbar, 2003). In monocotyledons, the hollow style prevails. Here, cells from the stylar canal generally have dense cytoplasm and are multiple organelles

analyzing species from five of the eight Bromeliaceae subfamilies, Sajo et al. (2004a) propose that the condition of epigynous flowers, and, consequently, those with septal interlocular nectaries, would be plesiomorphic with several reversions to the hypogynous condition, or that epigynous flowers would have originated more than once in the group. When analyzing flower buds of Bromelioideae, Pitcairnioideae and Tillandsioideae species, we have verified that all species, except those in Bromelioideae, have infralocular nectaries, corroborating the results of Sajo et al. (2004b). Regarding their classification, nectaries are structured, i.e., morphologically recognizable, and show a specialized anatomical structure. They have vascular bundles responsible for supplying water and nutrients, parenchyma where nectar is stored, and epidermal cells through which the nectar is secreted (Fahn, 1979). The region where nectaries are active varies among the studied species. In species with superior ovaries, nectaries are active below the locules, even when only a small 20

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Acknowledgements

(Went and Willemse, 1984; Sajo and Rudall, 2012). According to this classification, Bromeliaceae species are classified as type 1, hollow, sometimes with secretion in the stylar canal, as observed in the present study. In Poales, hollow styles with secretory epidermis are likely plesiomorphic in the order, and have been recorded in Xyridaceae (Remizowa et al., 2012), Rapateaceae (Oriani and Scatena, 2013), Mayacaceae and Juncaceae (Oriani et al., 2012). In the inferior portion of carpels, the PTTT is usually papillate and secretory, constituting the obturator tissue (Endress, 1994). The obturator tissue has been thoroughly studied, especially for its importance to reproduction, being responsible for chemically and/or physically directing the pollen tube towards the micropyle, also nurturing the pollen tube (Tilton and Horner, 1980; Herrero, 2000; Vardar et al., 2012). When the pollen tube is chemically directed, the obturator is considered to be secretory. Its cells generally show prominent, heavily stained nuclei, which indicate secretory activity. Sometimes, the presence of secretion can be observed in the locules of the ovaries. The chemical composition of the exudate varies, containing polysaccharides, proteins and lipids (Singh and Walles, 1992; Hudák et al., 1993; Herrero, 2000; Oliveira et al., 2016). When the obturator directs the pollen tube mechanically, this tissue resembles trichomes (Tilton and Horner, 1980; Herrero, 2000). Its origin can be placental, as seen in the studied species, or even funicular. In some instances, it may have a mixed origin (Tilton and Horner, 1980). In Poales, this tissue is quite diverse in both origin and shape. In Cyperaceae, it is formed by trichomes with funicular origin (Reynders et al., 2012), and in Juncaceae and Rapateaceae, it is formed by trichomes with placental origin (Oriani and Scatena, 2012, 2013). In Mayacaceae, the obturator is secretory and originated from the placenta (Oriani et al., 2012). In Bromeliaceae, the obturator tissue has a palisade shape with evident and heavily stained nuclei, as well as secretory activity. It can be present in single or multiple cell layers (Fagundes and Mariath, 2010; Oliveira et al., 2016). In the studied species, the obturator tissue has a single layer of cells in palisade, with evident nuclei. In all species, the tissue is placental in its origin, similar to what was observed for the remaining species of the family by Fagundes and Mariath (2010) and Oliveira et al. (2016).

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5. Conclusions Results obtained in this work reveal 21 anatomical characters of both androecium and gynoecium, which could be useful for further systematic studies of the family, especially those involving vascularization at subfamily level. In particular, gynoecium vascularization deserves intensive study, and in this case, we suggest that the presence of three vascular bundles on Pitcairnoideae species may represent a synapomorphy for this group. Morphologically, the adnation of the stamens to the petals is an interesting character to use in phylogenetic studies, given that out of all studied species, only those belonging to Aechmea, plus N. amazonicum possess epipetal stamens adnate to the petals with antesepalous stamens free at the middle portion of the flower. Additionally, adnation of the stamens to the petals, simulating a floral tube, is probably related to pollination by hummingbirds. Another character that seems to be involved in attracting pollinators is the presence of conical epidermis in the distal part of filaments and style, and in the anthers and stigma, as well as the presence of idioblasts with raphides. Other characters that show phylogenetic potential involve the anatomical structure of septal nectaries at the hemisynascidiate zone in Bromelioideae species; the presence of endothecium-like tissue in species of Dyckia; the presence of a protuberance in the connective region in species belonging to the Nidularioid complex; and the presence of an interlocular zone in the anthers of Quesnelia and Aechmea species. Further studies of Bromeliaceae floral anatomy should be undertaken, analyzing other subfamilies, in order to gain a better understanding of floral evolution of this group. 21

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