Anther development, microsporogenesis and microgametogenesis in Heliconia (Heliconiaceae, Zingiberales)

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Flora 202 (2007) 148–160 www.elsevier.de/flora

Anther development, microsporogenesis and microgametogenesis in Heliconia (Heliconiaceae, Zingiberales) Daniela Guimara˜es Sima˜oa,, Vera Lucia Scatenaa, Ferry Boumanb a

Departamento de Botaˆnica, Instituto de Biocieˆncias, Universidade Estadual Paulista, Caixa Postal 199, 13506-900 Rio Claro, SP, Brazil b Hugo de Vries Laboratory, IBED, University of Amsterdam, Kruislaan 318, 1098 SM Amsterdam, The Netherlands Received 25 November 2005; accepted 16 May 2006

Abstract Anther development, microsporogenesis and microgametogenesis in several species of Heliconia were investigated as part of a complementary embryological study of the Heliconiaceae. All studied Heliconia species present bithecate and tetrasporangiate anthers with fertile pollen grains; only H. rivularis, a natural hybrid, presented sterile pollen grains of variable size and no content. The anther wall has an uniseriate epidermis and endothecium, the latter with helicoidal thickenings, although some cells of the middle layers also showed thickenings; the biseriate tapetum is of amoeboid non-syncytial type, since the tapetum cells did not fuse together forming a true plasmodium. The microsporogenesis is successive leading to isobilateral tetrads. The inaperturate pollen grains had a very reduced exine consisting of a thin, more or less continuous layer with small spines upon; the pollen grain shape is variable among the species, all of them presenting heteropolar pollen, except H. angusta with isopolar ones. Most of these characteristics were shared with other studied Zingiberales, although more studies need to be done. r 2006 Elsevier GmbH. All rights reserved. Keywords: Amoeboid non-syncytial tapetum; Heliconia; Microgametogenesis; Microsporogenesis; Pollen grain; Zingiberales

Introduction The family Heliconiaceae belongs to the order Zingiberales, a monophyletic group of tropical monocotyledons that can be divided into two informal assemblages: the banana families (Musaceae, Strelitziaceae, Lowiaceae and Heliconiaceae) and the ginger families (Zingiberaceae, Costaceae, Cannaceae and Marantaceae) (Cronquist, 1981; Dahlgren et al., 1985; Corresponding author. Present address: Faculdade de Cieˆncias Integradas do Pontal, Universidade Federal de Uberlaˆndia, Campus do Pontal, Av. Jose´ Joa˜o Dib no 2545, CEP 38302-000, Ituiutaba-MG, Brazil. Tel: +55 34 3269-2389. E-mail address: [email protected] (D.G. Sima˜o).

0367-2530/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.flora.2006.05.003

Kirchoff, 1991; Kress, 1990; Kress and Specht, 2003; Kress et al., 2001; Tomlinson, 1962). The members of Zingiberales usually present inaperturate pollen grains, with reduced exine and thickened intine, probably as an adaptation to tropical environments (Furness and Rudall, 1999a; Johri et al., 1992; Kress et al., 1978). The order is unique among other monocotyledons because it presents besides of the two main tapetum types, secretory and amoeboid, an intermediate type of the latter, named amoeboid nonsyncytial tapetum (Pacini, 1997), or just invasive tapetum (Furness and Rudall, 1998, 2001). The pollen grains of Heliconia species are similar to most Zingiberales (Davis, 1966; Johri et al., 1992), being structurally well studied (Kress and Stone, 1983; Kress

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et al., 1978; Stone et al., 1979). Characters of the pollen grains, as shape, polarity and exine sculpturing, have also been used in taxonomical studies, since they are quite variable among species (Andersson, 1985, 1992; Kress and Stone, 1983; Santos, 1978). However, other features related to the anther development have not been described in detail or are incomplete for Heliconiaceae [see compilations by Davis (1966) and Johri et al. (1992)]. The aim of this paper is to investigate the anther development, including the microsporogenesis and microgametogenesis in several species of Heliconia, as part of an embryological study of the Heliconiaceae, and to compare these data with those of other families in the Zingiberales.

Materials and methods Five species and a natural hybrid of Heliconia were examined, all of them occurring in natural areas of the Atlantic Forest, in Sa˜o Paulo State, southeastern Brazil. Voucher specimens were deposited in the Herbarium Rioclarense (HRBC): H. angusta Vell. (Sima˜o et al. 15), H. hirsuta L. f. (Sima˜o and Mantovani 69), H. rivularis Emygdio and E. Santos (Sima˜o et al. 111), H. spathocircinata Aristeg. (Sima˜o et al. 107), H. subulata subsp. gracilis (Petersen) L. Andersson (Sima˜o and Cardoso 91), and H. velloziana Emygdio (Sima˜o et al. 16). Floral buds in various developmental stages were fixed in FAA 50 and stored in 70% ethanol (Johansen, 1940). For light microscopy, the samples were dehydrated through a normal butyl alcohol series and embedded in glycol methacrylate. The samples were sectioned at 5–10 mm with a glass knife using a rotary microtome. Sections were stained either with the PAS reaction (1% periodic acid-Schiff0 s reagent) and 0.05% toluidine blue (Feder and O’Brien, 1968), or with 0.05% toluidine blue alone (O’Brien et al., 1964). For transmission electron microscopy (TEM), floral buds and anthers of H. spathocircinata were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer. The samples were washed in buffer, post fixed in 1% osmium tetroxide (2 h), stained with 2% uranyl acetate (4 h), dehydrated through an acetone series and embedded in Spurr resin (Spurr, 1969). Ultra-thin sections (60–90 nm) were stained with uranyl acetate (45 min) and lead citrate (10 min), and then examined using a Philips CM 100 transmission electron microscope. For scanning electron microscopy (SEM), pre-anthesis anthers from all species, were dehydrated through an ethanol series, critical point dried, coated with goldpalladium, and examined using a JEOL JSM-5410 SEM.

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Results Floral morphology and anatomy The anthesis flowers of Heliconia present the median sepal free from the perianth (Figs. 1, 3) and only their anthers and stigma are exposed (Figs. 3, 4). Anthesis flowers last only one day, and at this time anthers with longitudinal dehiscence release the pollen grains (Figs. 4, 27). In H. rivularis, a natural hybrid, no open flowers and fruits were observed, although floral buds developed normally on the inflorescences until the anthesis stage (Fig. 2). The floral buds are subtended by bracteoles, being inserted in different parts of the inflorescence (Fig. 5). Flowers present three sepals and three petals, arranged in two whorls (Fig. 5). The two lateral petals, which are in contact with the free median sepal, present fiber bundles associated or not with vascular bundles (Fig. 16). Androecium is arranged in two whorls, with five fertile stamens and one staminode in the outer whorl (Fig. 6), and the anthers are bithecate and tetrasporangiate, with a single vascular bundle in the connective (Figs. 6, 9).

Anther wall There were no significant differences among the species of Heliconia studied here, most of them having similar patterns of anther wall and pollen grain development. The anther wall, during microsporogenesis, is composed by both uniseriate epidermis and endothecium, three middle layers, occasionally four in H. velloziana, and by two layers of tapetum cells (Figs. 10, 11). At late microgametogenesis, the anther wall is composed only by the epidermis, the endothecium and the thickened adjacent layers (Fig. 21). The thin-walled epidermal cells are longitudinally elongated (Fig. 27), becoming papillous as anther develops (Figs. 17, 19, 21). In the connective, close to the vascular bundle, the epidermal cells have a conspicuous cuticle (Figs. 19, 21), being persistent at anthesis (Fig. 22). The endothecium has initially one layer with cells of similar size as those from the epidermal and middle layers. As anther develops, these cells increase (Figs. 7, 10, 11) and their walls become thickened after microspore formation (Fig. 17). At that stage, the endothecium shows helicoidal thickenings (Fig. 17), except in the region of dehiscence (Fig. 21). The middle layers are compressed as anther develops (Figs. 10, 11), and do not persist until the pre-anthesis stage (Fig. 21). However, some cells from the layers adjacent to the endothecium, and others located in the

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Figs. 1–4. Inflorescences of Heliconia. 1. H. spathocircinata, with three anthesis flowers. 2. H. rivularis, a natural hybrid, with closed anthesis flowers. 3,4. H. angusta, anthesis flower showing: stigma in central position, and five anthers with longitudinal dehiscence.

region of the connective, show thickenings (Figs. 17, 19, 21) and do persist during pre-anthesis. The tapetum is composed of two layers of isodiametric cells with dense cytoplasm and evident nucleus (Figs. 7, 8). At early microsporogenesis, the tapetum

cells surround the sporogenous tissue, and may present more than one nucleus (Fig. 8). During the microsporocyte divisions, the cytoplasm becomes less dense and vacuoles are formed in the tapetum cells (Fig. 11). The cell walls then break down, and the cytoplasmatic content

Figs. 5–10. Microsporogenesis in Heliconia species at sporogenous cell and microsporocyte stages, transverse sections, photomicrographs. 5. H. spathocircinata, part of the cincinnus with three primordia of floral buds enclosed in bracteoles. 6. H. spathocircinata, floral bud showing the androecium, composed of five stamens and one staminode, and the style in the central position. 7. H. spathocircinata, half of one anther, showing the anther wall and the sporogenous cells. 8. H. subulata, sporogenous and tapetum cells in division. Asterisks indicate some binucleate tapetum cells. 9. H. spathocircinata, floral bud showing bithecate and tetrasporangiate anthers, at the early microsporocyte stage. 10. H. spathocircinata, microsporocytes enclosed in a callose layer and tapetum cells around them [bars: 5 ¼ 300 mm; 6, 9 ¼ 100 mm; 7, 10 ¼ 20 mm; 8 ¼ 10 mm; abbreviations (for all figures): b: bracteoles, en: endothecium, ex: exine, gc: generative cell, in: intine, m: microsporocyte, ml: median layers, r: raphides, s: staminode, sc: sporogenous cells, ta: tapetum cells, th: threads, vc: vegetative cell].

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Figs. 11–15. Microsporogenesis in H. spathocircinata at microspore dyad and tetrad stages, transverse (TS) and longitudinal (LS) sections, photomicrographs. 11. Microspores during the first meiotic division. Note the tapetum cells with large vacuoles (TS). 12. Anthers with many developing tetrads (LS). 13. Microspores arranged in an isobilateral manner, at early tetrad stage (LS). 14. Microspore tetrad enclosed in substances produced by tapetum cells (LS). 15. Microspore tetrad (LS) [bars: 11, 14 ¼ 20; 12 ¼ 100; 13, 15 ¼ 10 mm; abbreviations see Figs. 5–10].

enters in contact with the free microspores, but without forming a plasmodium. In mature anther, just before anthesis, no tapetum cells could be observed (Fig. 21).

Microsporangium and microsporogenesis The anther primordium has a bilobed shape and is composed of meristematic tissue enveloped by an epidermal layer. The anther becomes tetralobed (Fig. 9)

after the development of the four sporangia and the cells around them (Figs. 6, 7). The archesporial cells divide periclinally to form the primary parietal cells and the primary sporogenous cells. The parietal cells then divide both anticlinal and periclinally to form the endothecium, the middle layers and the tapetum. The primary sporogenous cells undergo some divisions (Figs. 7, 8) before forming the microsporocytes (Figs. 9, 10).

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Figs. 16–22. Anther wall and pollen grain in Heliconia, transverse sections, photomicrographs. 16. H. rivularis, floral bud showing the septum rupture (arrows) between the pollen sacs. 17. H. velloziana, anther wall with helicoidal thickenings of the endothecium and of the adjacent median layers. 18. H. spathocircinata, pollen grain at the binucleate stage, in equatorial view. 19. H. spathocircinata, pollen sacs united in a single locule due to the septum rupture. 20. H. spathocircinata, pollen grains at pre-anthesis. 21. H. velloziana, endothecium and adjacent median layers with thickened walls. 22. H. spathocircinata, anther at anthesis stage [bars: 16 ¼ 300; 17, 20 ¼ 20; 18 ¼ 10; 19, 21, 22 ¼ 100; 20 ¼ 20 mm; abbreviations see Figs. 5–10].

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Cells of the sporogenous tissue divide (Figs. 7, 8), giving rise to microsporocytes with prominent nucleus, dense cytoplasm and a callose layer around their walls (Figs. 9, 10). In the first meiotic division, microsporocytes divide to form dyads (Fig. 11) and, after the second division, they form isobilateral tetrads (Figs. 12–15). Both divisions occur with the formation of wall between microspores, thus characterizing the microsporogenesis as successive. During microspore development, the two pollen sacs of each theca merge into a single locule, because the septum between them degenerates (Figs. 16, 19, 21). The region of the septum presents idioblasts containing raphides, formed at the beginning of anther wall development (Fig. 10), and later found close to the pollen grains during floral anthesis (Fig. 28). Other structures, similar to threads, are occasionally observed in the mature anthers, as in H. angusta (Figs. 27) and H. hirsuta (Fig. 28).

Microgametogenesis and pollen grain The microspore develops after tetrad release, and divides forming a two-celled microgametophyte, i.e., the pollen grain, containing both vegetative and generative cells (Fig. 18). H. rivularis presents thecae with few quantities of microspores, when compared with such other species as H. angusta (Fig. 19) and H. velloziana

(Fig. 21), and sterile pollen grains without content (Fig. 16). The sporoderm is characterized by a very reduced exine consisting of a thin, more or less continuous, layer with spines upon, which may be either larger, as in H. spathocircinata (Fig. 30) and in H. velloziana (Figs. 31, 32), or smaller, as in H. subulata (Fig. 29). The intine is thicker on the distal hemisphere (Figs. 23, 25), where it presents small channels (Figs. 24, 26). Pollen grains are mostly heteropolar (Figs. 28–30); only H. angusta presents isopolar pollen with a clear ring running the equatorial region (Fig. 27). The pollen grain shape varies among species only in the equatorial view, most are spherical in polar view. In H. angusta, pollen grains are also spherical in equatorial view, with convex proximal and distal hemispheres (Fig. 27). H. hirsuta presents oblate pollen in equatorial view, with a convex distal hemisphere, and a slightly concave proximal hemisphere (Fig. 28). In H. subulata, pollen grains are also oblate in equatorial view, with a convex distal hemisphere, and a planar proximal hemisphere (Fig. 29). In H. spathocircinata, grains are peroblate, with planar proximal and slightly concave distal hemispheres (Fig. 30). In H. velloziana, pollen grains are circular in polar view, with a planar proximal hemisphere and slightly convex distal hemisphere (Fig. 31). At pre-anthesis stage, pollen grains present a great quantity of starch grains (Figs. 20, 25, 26). The pollen

Figs. 23–26. Pollen grain of H. spathocircinata, TEM photomicrographs. 23. Pollen grain at the binucleate stage, in equatorial view. 24. Sporoderm in the thickened region of intine. 25. Pollen grain with great quantity of starch grains, in equatorial view. 26. Sporoderm with reduced exine and the intine channels [bars: 23, 25 ¼ 10; 24 ¼ 3; 26 ¼ 2 mm; abbreviations see Figs. 5–10].

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Figs. 27–32. Anther and pollen grain of Heliconia, SEM photomicrographs. 27. H. angusta, anther with pollen grains. Observe threads among the pollen grains. 28. H. hirsuta, pollen grains, in equatorial view. Note raphides and threads close to the pollen grains. 29. H. subulata, pollen grain, in equatorial view. 30. H. spathocircinata, pollen grain, in equatorial view. 31. H. velloziana, pollen grain, in distal polar view. 32. H. velloziana, exine ornamentation [bars: 27 ¼ 50; 28–31 ¼ 10; 32 ¼ 5 mm; abbreviations see Figs. 5–10].

grains do not have any aperture region, i.e., they are inaperturate, and are thus shed in monads (Figs. 25, 27, 31). Divisions of the generative cell leading to 3-celled pollen grains were observed in some pollen grains of H. angusta and H. spathocircinata. In the latter, there are some occasional vegetative cells with two nucleoli. The pollen tube entered the ovule through the micropyle and was already visible on the nucellar epidermis in oneday anthesis flowers (Sima˜o et al., 2006).

Discussion Although there are not enough data on pollen and anther development for making a comparative study among the eight families of Zingiberales, since few species have been studied so far (Andersson, 1998b;

Davis, 1966; Johri et al., 1992; Larsen, 1998), we have listed some important characters in Table 1. The number of fertile stamens in the banana group are five (six in Ensete and Ravenala), while the ginger group presents a single fertile stamen, so that in Cannaceae and Marantaceae only half of the anther is fertile (Dahlgren et al., 1985; Kirchoff, 1991; Kress, 1990). The reduction in the number of the fertile stamens in the order is interpreted by many authors as evolutionary stages of modifications of stamens into staminodes, initiated with one staminode in Heliconiaceae (Kirchoff, 1991; Kress, 1990; Tomlinson, 1962). In relation to the anther wall development, Dahlgren et al. (1985) reported the Monocotyledonous type for all Zingiberales. Our results showed however that in Heliconia the anther wall did not conform to the typical Monocotyledonous type reported by Davis (1966).

Monocot3

? Secretory

? ? Successive

?

Inaperturate

Reduced or developed Isopolar20

Anther wall Anther wall development type

Middle layers (number of layers) Tapetum type

Number of tapetal layers Number of nuclei in tapetal cells Microsporogenesis

Form of tetrads

Pollen grains Number of apertures

Exine Polarity

Reduced Isopolar21

Inaperturate13

?

Many ? Successive

? Secretory

Monocot3

5–62 4

Str

Reduced ?

Inaperturate

? Possibly secretory ? ? Possibly successive ?

Monocot3

5 4

Low

1 4

Zin

1 4

Cos

Reduced Mostly heteropolar

Inaperturate

Isobilateral; decussate, linear and T-shaped

Inaperturate14 or sulcate15 Spiraperturate16, sulcate17 or foraminate18 Reduced or developed Developed ? Heteropolar

Isobilateral, decussate, linear and T-shaped12

Monocot3

1 2

Can

Reduced Isopolar

Inaperturate

2–4 Amoeboid and am. non-syncytial 4–5 2–4 Successive or simultaneous Isobilateral, and decussate Isobilateral

Monocot3 or no Monocot3 Monocot3 regular pattern 1–44 ? 4–55 Amoeboid or am. non- Amoeboid6, and am. non- Secretory8 and am. nonsyncytial7 syncytial9 syncytial 1–411 2–3 1–5 1–2 ? 2 Successive Successive Successive

5 4

Hel

Reduced ?

Inaperturate19

?

? Am. nonsyncytial10 Many ? Successive

Monocot3

1 2

Mar

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?, no available information. References: Davis (1966), Stone et al. (1979), Stone et al. (1981), Dahlgren and Clifford (1982), Dahlgren et al. (1985), Kronestedt and Walles (1986), Tiwari and Gunning (1986), Kress (1990), Johri et al. (1992), Kirchoff and Kunze (1995), Andersson (1998b), Larsen (1998), Furness and Rudall (1998, 1999a, b, 2001), Prakash et al. (2000), Kress et al. (2001), Sima˜o, Scatena and Bouman (this work). 1 In Ensete. 2 In Ravenala. 3 Monocotyledonous type according Davis (1966). 4 2 layers (this work). 5 7–9 layers in Tapeinochilos. 6 In Amomum, Elettaria and Etlingera. 7 In Globba. 8 In Tapeinochilos. 9 In Costus. 10 In Calathea and Marantochloa. 11 2 layers (this work) 12 In Alpinia. 13 In Strelitzia. 14 In 18 genera. 15 In Alpinia, Amomum, Curcuma, Globba and Zingiber. 16 In Tapeinochilos. 17 In Dimerocostus. 18 In Costus. 19 In Calathea, Maranta, Marantachloa and Phrynium. 20 In Musa. 21 In Phenakospermum and Strelitzia.

5–61 4

Mus

Families

Anther and pollen grain development features in Zingiberales families

Androecium Number of fertile stamens Number of microsporangia

Characters

Table 1.

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It resembles the Basic type, with additional divisions of both median layer and tapetum. So, we believe, this feature needs to be examined more carefully in other Zingiberales, since it is a well established taxonomic character, although there are few exceptions (Davis, 1966). Some authors reported more recently no regular pattern of anther wall development in some Ericaceae (Hermann and Palser, 2000) and in Acorus species (Duvall, 2001). The outer middle layer of the species studied here showed thickenings similar to those observed in the endothecium, and persisted until anthesis. On the other hand, the inner layers did not show any thickenings, and were not present during late pollen grain development. This was observed in the Heliconia studied by Prakash et al. (2000), and in other Zingiberales, as some Zingiberaceae, whose middle layers usually present thickenings (Bhandari, 1984; Johri et al., 1992). According to Prakash et al. (2000), the thickenings of the middle layer may be related to the anther size and to the pollination syndrome. Our data agree with these authors, since the studied Heliconia have relatively large anthers and are pollinated by hummingbirds (Berry and Kress, 1991; Buzato et al., 2000; Stiles, 1975). In addition to the thickenings of the middle layers found in Heliconia anthers, fiber bundles were also present in the two lateral petals, close to the free sepal, as an adaptation of the Heliconia flowers to the pollinating agent. According to Andersson (1998a) and Kress et al. (1999), in the Heliconia species, the perianth is united and only the median sepal frees from the tube at anthesis, allowing pollinators to access the nectar at the perianth base. It is believed that these fiber bundles in the petals give better support to this region, avoiding damages to flowers and, consequently to the fruit development. The dehiscence region of the Heliconia anthers did not show any endothecium or middle layer thickenings, but presented idioblasts containing raphides. According to Mauseth (1988), the dehiscence region of the anther may present cells with calcium oxalate, which would work as a defense against insects that feed on pollen. In addition to those idioblasts found in the septum, raphides were also found in other organs of Heliconia: in their rhizomes and leaves (Sima˜o and Scatena, 2001; Tomlinson, 1959, 1962), in the inflorescence bracts (Sima˜o and Scatena, 2004) and in the ovary and pericarp, especially in the mesocarp (Sima˜o et al., 2006). There is no information available for the anthers of the remaining Zingiberales; only some raphides were found not in the anther wall, as in Heliconia, but in the sporogenous tissue of Elletaria species (Johri et al., 1992). Long threads were also observed in pre-anthesis and mature anthers, among the pollen grains, like the ones found in the Heliconia species studied by Rose and Barthlott (1995). According to them, these structures,

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named pollen-connecting threads, probably helps in the pollination of Heliconia, since they can adhere to the pollen grains, forming large groups which will be transported by the pollinators (Rose and Barthlott, 1995). These threads are also found in Strelitzia species, which belongs to Strelitziaceae, although they show important differences in their morphology, origin and function (Rose and Barthlott, 1995). The tapetum of the studied species is composed of two cell layers. Prakash et al. (2000) found from one until four layers of tapetum cells in the Heliconia studied by them, indicating that the number of tapetal layers probably is variable among Heliconia species. In other studied Zingiberales, as in some species of Strelitziaceae (Davis, 1966), Costaceae (Furness and Rudall, 1998; Stone et al., 1981), Cannaceae (Tiwari and Gunning, 1986), Zingiberaceae and Marantaceae (Furness and Rudall, 1998), the tapetum is also composed of more than one layer. According to Bhojwani and Bhatnagar (1974) and Bhandari (1984), despite the presence of tapetum formed by two or more layers of cells, the presence of a single layer is more common among angiosperms. Further studies should be done to obtain complete information within Zingiberales, and to confirm, or not, the multi-seriate tapetum as a characteristic pattern of the order. In relation to the number of nuclei in the tapetum cells, Prakash et al. (2000) also reported binucleate cells in the Heliconia at the early stages and during the microsporocyte meiosis. On the other hand, Stone et al. (1979), who studied pollen ontogeny in four Heliconia species, did not mention any binucleate cells in the tapetum. For Zingiberales, there are reports of binucleate tapetum cells in Costus (Johri et al., 1992) and binucleate to tetranucleate cells in Canna (Tiwari and Gunning, 1986). Since this character is evident in all the species here studied and, according to some authors (Bhojwani and Bhatnagar, 1974; D’Amato, 1984; Maheshwari, 1950; Oksala and Therman, 1977) the multinucleate condition is a common character in the tapetum cells, these authors probably may have not observed the early stages of anthers development, when most binucleate cells appear. The results found here suggest that Heliconia present an intermediate type of amoeboid tapetum, called amoeboid non-syncytial, or just invasive, similar to the one found in Canna (Tiwari and Gunning, 1986). In this type of tapetum, cells lose their walls in the late stages of tetrad formation, and protoplasts enclose the microspores, but do not form a multinucleate plasmodium, as in an amoeboid tapetum (Tiwari and Gunning, 1986). As more detailed studies on anther development are done, more intermediate types of tapetum have been found, showing the complexity of this tissue (Hesse and Hess, 1993). For instance, in anthers of Nymphaea colorata, there is a reorganization of the tapetal cells

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during the anther development, leading them to different cycles of activity (Rowley, 1993; Rowley et al., 1992). These tapetal cells change from noninvasive to invasive type during their development, this tapetum being named as ‘‘cyclic-invasive’’ (Rowley, 1993; Rowley et al., 1992). The data about the tapetum, in the present work, agree with Pacini (1997) who, in his revision of the tapetum types in angiosperms, attributed an amoeboid non-syncytial tapetum to Heliconia. Stone et al. (1979) and Prakash et al. (2000), who studied pollen and anther ontogeny in Heliconia, respectively, considered the Heliconia tapetum as amoeboid, although the latter (Prakash et al., 2000) reported a similarity between Heliconia and Canna tapetum, studied by Tiwari and Gunning (1986), in the early stages of microsporogenesis. Furness and Rudall (1998, 2001) reported that most Zingiberales present an amoeboid or amoeboid nonsyncytial tapetum. For them, the amoeboid nonsyncytial type, which was probably not observed in previous works, could represent a synapomorphy for Cannaceae, Costaceae and Marantaceae. According to these authors, Musaceae, Strelitziaceae and, possibly, Lowiaceae present a secretory tapetum. In their revisions (Furness and Rudall, 1998, 2001), Heliconiaceae appears having an amoeboid tapetum, but the results presented here show that the Heliconia species also share this apomorphic character with Cannaceae, Costaceae and Marantaceae. The two main tapetum types, i.e., the secretory and amoeboid, are widely distributed in monocotyledons, probably evolved many times in the group, and are considered as an important taxonomic character (Furness and Rudall, 1998). Among monocotyledons, the order Commelinales, which is close to Zingiberales within the commelinid clade (APG II, 2003), is the only one to present both main types of tapetum, since all others present either a secretory or amoeboid tapetum, with very few exceptions (Furness and Rudall, 1998). According to Pacini (1997), the different types of tapetum have the same functions, despite of the variation in the structure. For this author, such variations may result from different types of adaptation of the pollen grains to the most varied habitats and pollinator agents. In the Heliconia species, the microsporogenesis is successive, since in the first meiotic division of the microsporocytes, a callose wall is formed, separating the two cells of the dyad. Among the Zingiberales studied so far, all of them also present the successive type, which is a uniform feature within the order (Furness and Rudall, 1999b), although there are reports of simultaneous microsporogenesis in Canna indica (Davis, 1966; Furness and Rudall, 2001; Johri et al., 1992). The pollen grains of Heliconia species have no apertures, as we have presented here and as mentioned

in previous works (Kress and Stone, 1983; Kress et al., 1978; Stone et al., 1979), although functionally the pollen is considered to be monoaperturate (Kress and Stone, 1983; Kress et al., 1978; Stone et al., 1979). Inaperturate pollen grains are also found in most of Zingiberales (Davis, 1966; Furness and Rudall, 1999a; 2001; Johri et al., 1992; Knox, 1984), and could be regarded as a synapomorphy for the order (Kress, 1990; Kress et al., 2001), since only Costaceae and few members of Zingiberaceae have spiraperturate or sulcate pollen grains (Furness and Rudall, 1999a; Stone et al., 1981). Many authors (Furness and Rudall, 2001; Knox, 1984; Kress et al., 1978; Pacini, 1997) suggested that the absence of apertures in the Zingiberales pollen grains is related to the reduced exine found in these species. As the exine is the outer layer of the sporoderm and one of its functions is to protect the microgametophyte (Bhojwani and Bhatnagar, 1974), this layer is much reduced in highly moist environments (Pacini, 1997). So, the reduced exine of most Zingiberales could be an adaptation of the pollen grain to the moist tropical environments (Knox, 1984), and it is also considered as an apomorphic character shared by the Zingiberales families (Kress, 1990; Kress et al., 2001). Only some Zingiberaceae and Costaceae have a thickened exine, probably as a secondarily derived feature (Kress, 1990; Stone et al., 1981). Pollen grains of the studied species were mostly heteropolar, since only H. angusta presented isopolar ones. The pollen grains of H. angusta were also different from the others in having an evident ‘‘ring’’ around the equatorial region. This character can also be observed in pollen grains of H. talamancana and H. maculata, in the later species being less evident (Kress and Stone, 1983), and could be a special sporoderm feature of the Heliconia pollen grains. Among the Heliconia studied in this work, H. rivularis was the only species with no open flowers in anthesis during our field visits. This observation corroborates Andersson (1992), according to whom H. rivularis could be a natural hybrid since it presents characters from two different species: the spiral bracts of H. spathocircinata and the greenish-yellow flowers of H. velloziana. We observed these two species occurring together with H. rivularis, which reinforces the suggestion by Andersson (1992). The presence of anthers with rare pollen grains, of variable size and no content, gives more evidence that H. rivularis is a hybrid, since studies on natural hybrids (Kress and Stone, 1983) and cultivars of Heliconia (Lee et al., 1994; Prakash et al., 2000) brought similar results. The results presented here for the Heliconia species are common to most features found in the Zingiberales families studied so far, as the multi-seriate tapetum with bi- or multi-nucleate cells, multi-layered middle layers

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with thickenings similar to the endothecial cells, successive microsporogenesis, and inaperturate pollen grains with reduced exine. Hence, we believe that with more complete studies, as mentioned above, features related to the anther and pollen grain development could be used to provide additional evidence on the relationships between those Zingiberales families.

Acknowledgements We thank Instituto Florestal, Fundac¸a˜o Florestal, Jardim Botaˆnico Municipal de Bauru and Mr. Djalma Zabeu for providing collecting permits. We also thank J.M.A. Braga for identifying the Heliconia species. This paper was improved by the comments and suggestions of two anonymous referees. This work was supported by a PhD grant to D.G. Sima˜o from FAPESP (00/02345-5), and by a Research grant to V.L. Scatena from CNPq (301404/2004-6).

References Andersson, L., 1985. Revision of Heliconia subgen. Stenochlamys (Musaceae—Heliconioideae). Opera Bot. 82, 1–124. Andersson, L., 1992. Revision of Heliconia subgen. Taeniostrobus and subgen. Heliconia (Musaceae—Heliconioideae). Opera Bot. 111, 1–98. Andersson, L., 1998a. Heliconiaceae. In: Kubitzki, K. (Ed.), The Families and Genera of Vascular Plants. IV. Alismatanae and Commelinanae (except Gramineae). Springer, Berlin, pp. 226–230. Andersson, L., 1998b. Marantaceae. In: Kubitzki, K. (Ed.), The Families and Genera of Vascular Plants. IV. Flowering Plants. Monocotyledons. Alismatanae and Commelinanae (except Gramineae). Springer, Berlin, pp. 278–293. APG II, 2003. An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG II. Bot. J. Linnean Soc. 141, 399–436. Berry, F., Kress, J., 1991. Heliconia—An Identification Guide. Smithsonian Institution Press, Washington, DC. Bhandari, N.N., 1984. The microsporangium. In: Johri, B.M. (Ed.), The Embryology of Angiosperms. Springer, Berlin, pp. 51–121. Bhojwani, S.S., Bhatnagar, S.P., 1974. The Embryology of Angiosperms. Vikas Publishing House, New Delhi. Buzato, S., Sazima, M., Sazima, I., 2000. Hummingbirdpollinated floras at three Atlantic Forest sites. Biotropica 32, 824–841. Cronquist, A., 1981. An Integrated System of Classification of Flowering Plants. Columbia University Press, New York, pp. 1157–1172. D’Amato, F., 1984. Role of polyploidy in reproductive organs and tissues. In: Johri, B.M. (Ed.), The Embryology of Angiosperms. Springer, Berlin, pp. 519–566. Dahlgren, R.M.T., Clifford, H.T., 1982. The Monocotyledons: a Comparative Study. Academic Press, London.

159

Dahlgren, R.M.T., Clifford, H.T., Yeo, P.F., 1985. The Families of the Monocotyledons. Springer, Berlin, pp. 350–358. Davis, G.L., 1966. Systematic Embryology of the Angiosperms. Wiley, New York. Duvall, M.R., 2001. An anatomical study of anther development in Acorus L.: Phylogenetic implications. Plant Syst. Evol. 228, 143–152. Feder, N., O’Brien, T.P., 1968. Plant microtechnique: some principles and new methods. Am. J. Bot. 55, 123–142. Furness, C.A., Rudall, P.J., 1998. The tapetum and systematics in monocotyledons. Bot. Rev. 64, 201–239. Furness, C.A., Rudall, P.J., 1999a. Inaperturate pollen in monocotyledons. Int. J. Plant Sci. 160, 395–414. Furness, C.A., Rudall, P.J., 1999b. Microsporogenesis in Monocotyledons. Ann. Bot. 84, 475–499. Furness, C.A., Rudall, P.J., 2001. Pollen and anther characters in monocot systematics. Grana 40, 17–25. Hermann, P.M., Palser, B.F., 2000. Stamen development in the Ericaceae. I. Anther wall, microsporogenesis, inversion, and appendages. Am. J. Bot. 87, 934–957. Hesse, M., Hess, M.W., 1993. Recent trends in tapetum research. A cytological and methodological review. Pl. Syst. Evol. 7 (Suppl.), 127–145. Johansen, D.A., 1940. Plant Microtechnique. McGraw-Hill Book Company, New York. Johri, B.M., Ambegaokar, K.B., Srivastava, P.S., 1992. Comparative Embryology of Angiosperms. Springer, Berlin. Kirchoff, B.K., 1991. Homeosis in the flowers of the Zingiberales. Am. J. Bot. 78, 833–837. Kirchoff, B.K., Kunze, H., 1995. Inflorescence and floral development in Orchidantha maxillarioides (Lowiaceae). Int. J. Plant Sci. 156, 159–171. Knox, R.B., 1984. The pollen grain. In: Johri, B.M. (Ed.), The Embryology of Angiosperms. Springer, Berlin, pp. 197–271. Kress, W.J., 1990. The phylogeny and classification of the Zingiberales. Ann. Mo. Bot. Gard. 77, 698–721. Kress, W.J., Stone, D.E., 1983. Morphology and phylogenetic significance of exine-less pollen of Heliconia (Heliconiaceae). Syst. Bot. 8, 149–167. Kress, W.J., Specht, C.D., 2003. Tropical Zingiberales: time, place and pattern of diversification. In: Third International Conference on the Comparative Biology of the Monocotyledons (Abstracts). Claremont, California, p. 47. Kress, W.J., Stone, D.E., Sellers, S.C., 1978. Ultrastructure of exine-less pollen: Heliconia (Heliconiaceae). Am. J. Bot. 65, 1064–1076. Kress, W.J., Betancur, J., Echeverry, B., 1999. Heliconias: Llamaradas de la Selva Colombiana. Cristina Uribe Editores, Santafe´ de Bogota´. Kress, W.J., Prince, L.M., Hahn, W.J., Zimmer, E.A., 2001. Unraveling the evolutionary radiation of the families of the Zingiberales using morphological and molecular evidence. Syst. Biol. 50, 926–944. Kronestedt, E., Walles, B., 1986. Anatomy of Strelitzia reginae flower (Strelitziaceae). Nord. J. Bot. 6, 307–320. Larsen, K., 1998. Lowiaceae. In: Kubitzki, K. (Ed.), The Families and Genera of Vascular Plants. IV. Flowering

ARTICLE IN PRESS 160

D.G. Sima˜o et al. / Flora 202 (2007) 148–160

Plants. Monocotyledons. Alismatanae and Commelinanae (except Gramineae). Springer, Berlin, pp. 275–277. Lee, Y.H., Ng, N.Y., Gob, C.J., 1994. Pollen formation and fruit-set in some cultivars of Heliconia psittacorum. Sci. Hort. 60, 167–172. Maheshwari, P., 1950. An Introduction to the Embryology of Angiosperms. McGraw-Hill Book Company, New York. Mauseth, J.D., 1988. Plant Anatomy. The Benjamin/Cummings Publishing Company, Menlo Park. O’Brien, T.P., Feder, N., McCully, M.E., 1964. Polychromatic staining of plant cell walls by toluidine blue O. Protoplasma 59, 368–373. Oksala, T., Therman, E., 1977. Endomitosis in tapetal cells of Eremurus (Liliaceae). Am. J. Bot. 64, 866–872. Pacini, E., 1997. Tapetum character states: analytical keys for tapetum types and activities. Can. J. Bot. 75, 1448–1459. Prakash, N., Lee, Y.H., Goh, C.J., 2000. Anther and pollen development in Heliconia. Harvard Papers Bot. 5, 193–200. Rose, M.J., Barthlott, W., 1995. Pollen-connecting threads in Heliconia (Heliconiaceae). Pl. Syst. Evol. 195, 61–65. Rowley, J.R., 1993. Cycles of hyperactivity in tapetal cells. Pl. Syst. Evol. 7 (Suppl.), 23–37. Rowley, J.R., Gabarayeva, N.I., Walles, B., 1992. Cyclic invasion of tapetal cells into loculi during microspore development in Nymphaea colorata (Nymphaceae). Am. J. Bot. 79, 801–808. Santos, E., 1978. Revisa˜o das espe´cies do geˆnero Heliconia L. (Musaceae s.l.) espontaˆneas na regia˜o fluminense. Rodrigue´sia 30, 99–201. Sima˜o, D.G., Scatena, V.L., 2001. Morphology and anatomy in Heliconia angusta Vell. and H. velloziana L. Emygd.

(Zingiberales: Heliconiaceae) from the Atlantic forest of southeastern Brazil. Revta. Brasil. Bot. 24, 415–424. Sima˜o, D.G., Scatena, V.L., 2004. Morfoanatomia das bra´cteas em Heliconia (Heliconiaceae) ocorrentes no Estado de Sa˜o Paulo, Brasil. Acta bot. Bras. 18, 261–270. Sima˜o, D.G., Scatena, V.L., Bouman, F., 2006. Developmental anatomy and morphology of the ovule and seed of Heliconia (Heliconiaceae, Zingiberales). Plant Biol. 8, 143–154. Spurr, A.R., 1969. A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26, 31–43. Stiles, F.G., 1975. Ecology, flowering phenology, and hummingbird pollination of some Costa Rican Heliconia species. Ecology 56, 285–301. Stone, D.E., Sellers, S.C., Kress, W.J., 1979. Ontogeny of exineless pollen in Heliconia, a banana relative. An. Mo. Bot. Gard. 66, 701–730. Stone, D.E., Sellers, S.C., Kress, W.J., 1981. Ontogenetic and evolutionary implications of a neotenous exine in Tapeinochilos (Zingiberales: Costaceae) pollen. Am. J. Bot. 68, 49–63. Tiwari, S.C., Gunning, B.E.S., 1986. Development of tapetum and microspores in Canna L—An example of an invasive but non-syncytial tapetum. Ann. Bot. 57, 557–563. Tomlinson, P.B., 1959. An anatomical approach to the classification of the Musaceae. J. Linn. Soc. (Bot.) 55, 779–809. Tomlinson, P.B., 1962. Phylogeny of the Scitamineae— morphological and anatomical considerations. Evolution 16, 192–213.