Seed morphoanatomy and its systematic relevance to ... - Springer Link

Plant Syst Evol (2012) 298:1881–1895 DOI 10.1007/s00606-012-0688-3

ORIGINAL ARTICLE

Seed morphoanatomy and its systematic relevance to Tillandsioideae (Bromeliaceae) Raquel I. Magalha˜es • Jorge E. A. Mariath

Received: 15 May 2011 / Accepted: 30 July 2012 / Published online: 6 September 2012 ! Springer-Verlag 2012

Abstract The morphoanatomy of mature seeds of 14 species, representing Vriesea and Tillandsia genera, was investigated in light and scanning electron microscopy to establish structural patterns of the seed coat, endosperm and embryo, allowing the distinction between both genera and contributing to the systematic of Tillandsioideae. All species studied have basal plumose appendages in a parachute-like arrangement. In Vriesea, the embryo occupies about 1/3 of the seed, and the rest is filled by the endosperm. In Tillandsia, the area occupied by the embryo within the seed varies according to the species analyzed. The reserves stored in the endosperm and in the embryo differ considerably: both between Vriesea and Tillandsia genera and among Tillandsia species. In the median region of the hypocotyl-radicle axis of Tillandsia embryo, there is a constriction zone that separates the root portion from the rest of the embryo. This feature was not observed in Vriesea species. Our results indicate that few morphoanatomical features of the mature seed coat are good for use in phylogenetic analysis, without considering the ontogenetic study of these structures. On the other hand, characteristics of the embryo and endosperm are very informative. They are useful to separate Vriesea and Tillandsia and also to distinguish groups of species within Tillandsia genus. Keywords Vriesea

Embryo ! Poales ! Monocots ! Tillandsia !

This article is part of the first author’s Dissertation at Universidade Federal do Rio Grande do Sul, Brazil. R. I. Magalha˜es ! J. E. A. Mariath (&) LAVeg, Departamento de Botaˆnica, Instituto de Biocieˆncias, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS 91540-000, Brazil e-mail: [email protected]

Introduction Tillandsioideae has unique features, capable of distinguishing it from other traditional subfamilies of Bromeliaceae. Among these features there are leaves with entire margins, fruits of capsule septicidal type and plumose seeds adapted to wind dispersal (Smith and Downs 1977; Szidat 1922). In the division of Bromeliaceae proposed by Smith and Downs (1974, 1977, 1979), seed morphology, although little explored, had great value in the delimitation of Bromelioideae, Pitcairnioideae and Tillandsioideae. However, with the rearrangement of Bromeliaceae proposed by Givnish et al. (2007), that reorganized the family in eight subfamilies (Brocchinioideae, Bromelioideae, Hechtioideae, Lindmanioideae, Navioideae, Pitcairnioideae, Puyoideae and Tillandsioideae), morphological features previously used in taxa circumscriptions are no longer able to express the true relationship among them. This rearrangement of Bromeliaceae made it necessity for new studies to contribute to more detailed morphological features and improve circumscriptions at infrafamilial and infrageneric levels (Barfuss et al. 2005; Givnish et al. 2007; Palacı´ et al. 2004). The complexity of Tillandsioideae, whose genera delimitations are unclear and often questionable, can be seen along its taxonomic history. Frequently, there are new proposals to elevate a subgenus to genus, to create a new genus from the segregation of a major genus or to change species from one genus to another one (Barfuss et al. 2005; Espejo-Serna 2002; Grant 1993). Barfuss et al. (2005) discussed some possible phylogenetic relationships within Tillandsioideae and noticed that some Vriesea Lindl. and Tillandsia L. species, classified according to Smith and Downs (1977), appeared to be erroneously positioned.

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R. I. Magalha˜es, J. E. A. Mariath

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Vriesea has approximately 260 species, of which 145 are endemic to the Brazilian Atlantic Forest (Luther 2008; Martinelli et al. 2008; Smith and Downs 1977). According to Smith and Till (1998), some characteristics, such as bract color, petal shape and stamen position, divide the genus into two sections: Xiphion and Vriesea. Tillandsia comprises about 600 species, and it is the biggest genus of Bromeliaceae (Luther 2008). According to the classification of Smith and Till (1998), it is divided into six subgenera: Allardtia, Anoplophytum, Diaphoranthema, Phytarrhiza, Pseudalcantarea and Tillandsia. This classification is based on characteristics of petals, stamens and pistils. Currently, Tillandsia and Vriesea are distinguished only by the presence or absence of petal appendages (Brown and Terry 1992; Smith and Downs 1977); however, it is believed that these structures appeared independently. If used individually, they do not constitute a good character to separate genera, because their limits become confused, and unreliable placements are created among species (Benzing 2000; Brown and Terry 1992). Seeds provide excellent diagnostic traits because they are less susceptible to environmental changes (Gross 1988). Moreover, some embryo characteristics are usually constant within a genus and may work as important indicators of relationships among species (Von Teichman and Van Wyk 1991). If we consider the large number of species belonging to Tillandsioideae, studies involving seed morphological and anatomical descriptions are scarce in this subfamily. Since the classical work of Billings (1904) regarding the development of Tillandsia usneoides (L.) L. and of Gross (1988), who examined the seed morphology of several Bromeliaceae genera, only Cecchi-Fiordi et al. (1996, 2001) and Morra et al. (2002) described morphological and anatomical features of Tillandsia seeds. However, these descriptions include a very small number of species, do not include the addition of distinctive taxonomic features and do not make comparisons with other genera. Concerning Vriesea seeds, the situation is even worse. After Gross (1988), there were no studies involving the morphoanatomy of these structures. Palacı´ et al. (2004) analyzed the seed coat development in Catopsis Griseb. species and Tillandsia complanata Benth. In addition to contributing some comparative data on seed integument, they showed that some features have great value for the systematic of Tillandsioideae, especially at the generic level. Attempting to identify features that contribute to improving the circumscriptions of Vriesea and Tillandsia and provide additional reproductive morphological data to further phylogenies, we investigated the morphoanatomy of mature seeds of 14 species belonging to both genera, looking for structural patterns of the seed for each of them. Considering that these are large and economically

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important genera, this work will also provide some important features that might be helpful to horticultural breeders and to researchers interested in tropical biodiversity and evolution of flowering plants.

Materials and methods Material collection and biometric characterization Mature seeds of Vriesea (V. carinata, V. corcovadensis, V. flammea, V. gigantea, V. incurvata, V. platynema, V. psittacina and V. rodigasiana) and Tillandsia (T. aeranthos, T. geminiflora, T. recurvata, T. stricta, T. tenuifolia and T. usneoides) were collected at the Bromeliaceae Collection in the Botanical Garden of Porto Alegre (under greenhouse conditions) and in counties of Rio Grande do Sul, Brazil (Table 1). To ensure the use of mature seeds of both genera and make the results of comparisons more reliable, seeds were removed only from fresh fruits that had opened by themselves and from at least three individuals of each species. To obtain values of seed length and relative embryo size, we measured ten seeds of each species with a Wild M7A stereomicroscope. The seed portion occupied by the embryo is an average of the value obtained by dividing the embryo length by the seed length (without appendages and endostome). Material preparation for light microscopy The botanical material was fixed with 1 % glutaraldehyde and 4 % formaldehyde (McDowell and Trump 1976). After fixing, it was washed with phosphate buffer 0.1 M, pH 7.2 (Gabriel 1982), followed by dehydration in increasing ethanol series (10–100 %) and inclusion in historesin hydroxyethylmetacrilate (Gerrits and Smid 1983). After processing and inclusion, 6-lm-thick sections were prepared using a rotation microtome Microm HM 340 E. To visualize the structures in light microscopy, the material was stained with toluidine blue 0.05 %, pH 4.4 (O’Brien and McCully 1981). To verify the nature of the substances accumulated in the seeds, some histochemical tests were performed, such as Coomassie blue for proteins (Southworth 1973), Lugol’s reagent for starch (Johansen 1940) and Sudan black B for lipids (Jensen 1962). The analysis and recording of images were performed with a Leica DMR HC microscope, with a Leica DFC 500 digital camera, using Leica Application Suite-LAS software. Material preparation for scanning electron microscopy For scanning electron microscopy, the material was washed in sodium phosphate buffer, dehydrated in ascending

Seed morphoanatomy and its systematic relevance to Tillandsioideae Table 1 List of analyzed species Taxon

Vriesea corcovadensis Mez Vriesea flammea L.B.Sm.

Vriesea incurvata Gaudich. Vriesea rodigasiana E.Morren

Vriesea psittacina Lindl.

Registration number

Dom Pedro de Alcaˆntara, RS

ICN 144794

Terra de Areia, RS Oso´rio, RS

BROM00534 BROM00549

Porto Alegre, RS Cambara´ do Sul, RS

BROM00620

Not informed

BROM00814


ICN 144795


BROM00624

Results


BROM00619

Shape, size and seed coat

Maquine´, RS Maquine´, RS Saõ Lourenço do Sul, RS Maquine´, RS

BROM00090

Itati, RS Viamaõ, RS Viamaõ, RS

BROM00852

ICN 140786

BROM00800 BROM00812 BROM00813 BROM00063

Porto Alegre, RS

BROM00103 BROM00351

Porto Alegre, RS

ICN 183677

Terra de Areia, RS

BROM00748

Piratuba, SC

BROM00718

Canela, RS Barracaõ, RS

BROM00804

Vriesea Sect. Xiphion Vriesea gigantea Gaudich. Vriesea platynema Gaudich.

BROM00786

Tillandsia L. Subg. Anoplophytum Tillandsia aeranthos (Loisel.) L.B.Sm.

Porto Alegre, RS

ICN 188807

Tillandsia geminiflora Brogn.

Camaqua˜, RS

ICN 183675

Porto Alegre, RS Caçapava do Sul, RS

ICN 183676 BROM00632

Machadinho, RS Saõ Pedro do Sul, RS

BROM00714

Tillandsia stricta Sol. ex Sims Tillandsia tenuifolia L.

BROM00753

Nova Roma, RS

BROM00610

Nova Palma, RS

BROM00727

Maximiliano de Almeida, RS

BROM00760

Tillandsia Subg. Diaphoranthema Tillandsia usneoides (L.) L.

Tillandsia recurvata (L.) L.

ethanol series (0–100 %) and transferred to 100 % acetone. Then it was submitted to drying by the critical point method (Gersterberger and Leins 1978) using BAL-TEC CPD 030 equipment. After that, the samples were mounted on aluminum stubs and coated with gold in a BALTEC SCD 050 sputtering system. The analysis was performed in a JEOL 6060 scanning electron microscope, under 10 kV. The terminology used for seed coat description follows Corner’s (1976) classification. Thus, the outer seed coat is called the testa, and the inner seed coat is called the tegmen.

Counties

Vriesea Lindl Sect. Vriesea Vriesea carinata Wawra

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Santo Antoˆnio da Patrulha, RS

BROM00211

Rolante, RS

BROM00224

Machadinho, RS Maquine´, RS

BROM00713 BROM00449

Vila Maria, RS

BROM00715

Toropi, RS

BROM00724

The seeds are characterized by yellowish- and white-colored appendages in Vriesea and Tillandsia, respectively. The plumose appendages of both genera arise in the micropylar end of the ovule. Since the ovule is anatropous (Fig. 2a), the micropylar pole is positioned next to the funiculus, facing the placenta (in the proximal portion of the ovary). Thus, the plumose appendage of the mature seed is located at the base of the fruit (Fig. 1a, b), and it is considered basal. Without considering the length of the plumose appendage, the seed length of Vriesea species ranges from 3.4 to 4.8 mm (V. carinata and V. gigantea, respectively) and are filiform. In Tillandsia they range from 2.5 to 4.7 mm (T. aeranthos and T. geminiflora, respectively) and vary in shape from fusiform to narrowly fusiform (Fig. 1c; Table 2). In both genera, the mature seed coat is thin and composed of five cell layers (Fig. 1d). The testa consists of three layers of longitudinally elongated cells with lignified walls, derived from the outer integument of the ovule, added to funicular tissue (Fig. 1e). The tegmen is biseriate and composed of exo- and endotegmen, derived from the inner integument of the ovule. The exotegmen is formed by irregular cells with thickened walls and without content at maturity. Cells with thickened walls that contain phenolic compounds inside, which give this layer a brownish color, form the endotegmen. In Vriesea, near the endostome, cells have irregular shape and size, differing from the rest of the tegmen (Fig. 1f, g). In T. geminiflora, T. recurvata and T. usneoides, the tegmen extends toward the exostome, providing an elongated appearance to these seeds (Fig. 1h). The testa protective mechanical function is transferred to the exotegmen, because the testa cells are responsible for the plumose appendage formation. The basal plumose appendage (micropylar) is formed by exo- and mesotesta,

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Seed morphoanatomy and its systematic relevance to Tillandsioideae

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b Fig. 1 Seeds general aspect and seed coat of Vriesea Lindl. and

endostome. The stem that keeps the first parachute connected to the seed is the funiculus. The second parachute is attached to the seed by some endotesta cells that remain connected to exotegmen.

Tillandsia L. a Vriesea gigantea Gaudich. Yellowish basal plumose appendages. b Tillandsia aeranthos (Loisel.) L.B.Sm. White basal plumose appendages. c Vriesea carinata Wawra, T. aeranthos and Tillandsia geminiflora Brogn. Seed shape: filiform, fusiform and narrowly fusiform, respectively. d Tillandsia tenuifolia L. Transversal section with arrows pointing to the testa cell layers (thin arrow), tegmen (large arrow) and funiculus (asterisk). e Tillandsia recurvata (L.) L. Funicular vascular bundles at the chalazal region. f, g V. carinata. Endostome region (arrow). h T. geminiflora. Tegmen elongation (arrow) and funiculus (asterisk) Table 2 Morphological aspects of Vriesea Lindl. and Tillandsia L. seeds Taxon

Length (mm)

Shape

Embryo/ endosperm (%)a

Vriesea Sect. Vriesea V. carinata

3.4

Filiform

28/72

V. corcovadensis V. flammea

4.1 4.0

Filiform Filiform

27/73 27/73

V. incurvata

3.6

Filiform

29/71

V. psittacina

3.4

Filiform

29/71

V. rodigasiana

3.7

Filiform

27/73

In Vriesea, the endotesta cells have irregular secondary thickening (undulations) on their anticlinal walls (Fig. 2d), and the connections between cells may be straight or slightly curved. In Tillandsia there is no irregular thickening, and cellular connections are often inclined (Fig. 2e). In both genera, the connections between the cells that form the parachute of the plumose appendage are bifurcated (Fig. 2f). At the apical end (chalazal) of Vriesea seeds (Fig. 2g), there is a prolongation of the testa, consisting of longitudinally elongated cells. After the basal appendage formation some cells remain attached to the seed and form a cap-like structure at the base of the apical appendage of V. corcovadensis, V. flammea, V. gigantea and V. platynema. In the other Vriesea species, the apical appendage is smaller than in the species mentioned above. In Tillandsia, the apical appendage is shorter than in Vriesea, and there is no cap formation (Fig. 2h).

Vriesea Sect. Xiphion V. gigantea

4.8

Filiform

25/75

V. platynema

4.5

Filiform

23/77

Tillandsia Subg. Anoplophytum T. aeranthos

2.5

Fusiform

T. geminiflora

4.7

Narrowly fusiform

67/33

T. stricta

3.4

Fusiform

73/27

T. tenuifolia

2.7

Fusiform

65/35

66/34

Tillandsia Subg. Diaphoranthema T. usneoides

3.2

Narrowly fusiform

65/35

T. recurvata

3.1

Narrowly fusiform

100/0

Values of 10 mature seeds measured under a stereomicroscope a

Seed portion occupied by the embryo and endosperm

which split up from the other layers. It is the endotesta that remains in contact with the exotegmen, connecting the plumose appendage to the seed. The seed plumose appendage shows two types of structural arrangements (Fig. 2b, c): Type I occurs in all Vriesea species. The exo- and mesotesta split up in the chalazal region and remain attached to each other in the micropylar region, near the exostome, forming a parachute-like structure. The endotesta cells form a stem, which keeps the parachute connected to the seed. Type II occurs in all Tillandsia species. The exo- and mesotesta split up at the chalazal region, forming a parachute-like structure near the exostome. The endotesta splits in the micropylar region (just below the exostome) and forms a second parachute near the

Endosperm The seeds are classified as albuminous, except T. recurvata. The endosperm is situated mainly in the seed chalazal region. Its amount remains constant in Vriesea, representing about 70 % of the seed. In Tillandsia, it may occupy up to 35 % of the seed interior and was completely consumed in T. recurvata (Table 2). The endosperm central region consists of large cells with irregular shape and size, thin walls and inconspicuous nuclei. The endosperm peripheral region consists of a single layer, composed of cells that vary from round to rectangular, with thickened walls and conspicuous nuclei. In the portion of the endosperm adjacent to the cotyledon, there are layers of compressed cells whose contents were consumed by the growing embryo (Fig. 3a, b). In Vriesea, in the micropylar region the endosperm was almost completely consumed, with only the endosperm periphery remaining (Fig. 1g). In Tillandsia the endosperm of this region was completely consumed, with only vestiges of it remaining. The reserves accumulated in the central endosperm vary according to the species analyzed (Fig. 3c–e, f–h). The seeds of all Vriesea species, T. aeranthos, T. geminiflora and T. tenuifolia, showed positive reaction to Lugol’s reagent, demonstrating the presence of starch. In T. stricta and T. usneoides there was no reaction. The amount of starch grains present in Vriesea and also their size are bigger than those found in Tillandsia. In the endosperm periphery of both genera there was no reaction to Lugol’s reagent, demonstrating the absence of

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Seed morphoanatomy and its systematic relevance to Tillandsioideae b Fig. 2

Seed appendages aspect of Vriesea Lindl. and Tillandsia L. a Anatropous ovule. Micropylar region (thin arrow), funiculus (asterisk) and chalazal region (large arrow). b Vriesea carinata Wawra. Exostome region (thin arrow) and stem (large arrow). c Tillandsia geminiflora Brogn. Endostome region (thin arrowabove), exostome (thin arrow-below) and stem (large arrow). d Vriesea incurvata Gaudich. Undulated anticlinal walls of endotesta cells (arrow). e T. geminiflora. Inclined cells connections of endotesta (arrow). f Tillandsia usneoides (L.) L. Bifurcated cell end of the plumose appendage. g Vriesea gigantea Gaudich. Apical appendage (arrow) and testa cells (asterisk). h Tillandsia aeranthos (Loisel.) L.B.Sm. Apical appendage (arrow)

starch. There was a positive reaction to Coomassie blue, showing the presence of proteins (in protein bodies). However, in the region adjacent to the embryo, the cell content is an amorphous protein mass, differing from the other cells. Embryo In the embryo of all species, it is possible to distinguish a shoot portion, consisting of one cotyledon and hypocotyl, and also a root portion. In Vriesea, the embryo occupies about 30 % of the seed interior, while in Tillandsia it occupies 65–100 % (Fig. 4c). In Vriesea, the cotyledon epidermis is uniseriate, consisting of pavement cells with thin walls, usually elongated in longitudinal section. The mesophyll is composed of polygonal cells with thin walls, which are bigger than the other embryo cells. In addition to the cotyledon, there are lateral expansions involving the embryo shoot apical meristem. The shoot apical meristem is formed by isodiametric cells with thin walls, dense cytoplasm and conspicuous nuclei (Fig. 5a, b). The root portion (Fig. 5c) of these embryos is made of isodiametric cells with thin walls that have lipid and protein contents. The provascular meristem consists of longitudinally elongated cells with thin walls, dense cytoplasm and conspicuous nuclei. The cells that compose the epidermis and the mesophyll of Tillandsia embryo are similar to those described in Vriesea. Lateral expansions involving the shoot apical meristem were also observed. The cotyledon may present variations in its shape and in the amount of reserves. In T. recurvata and T. usneoides the cotyledon apex is rounded, whereas in the other species the apex is truncated. The shoot apical meristem is formed by isodiametric cells with thin walls, dense cytoplasm and conspicuous nuclei. In some cases, as in T. recurvata, T. usneoides and T. stricta, there is a leaf primordium under development (Fig. 5d). In T. geminiflora, the leaf primordium is well developed, composed of isodiametric epidermal cells with thin walls and polygonal mesophyll cells, which are larger than the epidermal cells (Fig. 5f).

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The root portion of Vriesea and Tillandsia embryos differs considerably. The median portion of the hypocotylradicle axis of Tillandsia embryo consists of cells that vary from isodiametric to rectangular in longitudinal section. There is lipid deposition on these walls, leading to the separation of the radicle from the rest of the embryo. Such changes occur centripetally. Thus, the peripheral cells have thicker walls, which react strongly to Sudan black B (Fig. 5g). The central cells have different levels of lipid deposition on their walls, soluble substances in the cytoplasm and, in some cases, conspicuous nuclei. This region also reacts to Toluidine blue, indicating the presence of phenolic compounds (Fig. 5h). Under this region, the root portion is formed by large polygonal cells with thickened walls. Thus, it forms an area that separates the shoot and the root portion. The vascular bundles of this region are formed by tracheids with thickenings that vary from annular to reticulated (Fig. 5i), and they seem to mobilize substances toward the shoot portion (Fig. 5h). When observed in longitudinal section, in T. aeranthos, T. stricta and T. tenuifolia the median region of the hypocotyl-radicle axis is slightly dislocated and composed of six to ten cell layers. Their shapes are usually rectangular at the periphery and isodiametric at the center (Fig. 6c). In T. geminiflora, T. recurvata and T. usneoides, the median region of the hypocotyl-radicle axis is straight and composed of four to nine layers of isodiametric cells (Fig. 6d–f). From all analyzed species, only T. aeranthos embryo showed a positive reaction to Lugol’s reagent, demonstrating the presence of starch. As for proteins, they are present in all species, occurring as masses associated with globoids and crystalloids, which may be surrounded by lipid bodies (Fig. 7d).

Discussion Shape, size and seed coat Tillandsioideae seeds arise from anatropous and bitegmic ovules (Johri et al. 1992). At maturity they are characterized by the presence of plumose appendages in apical or basal position (Gross 1988; Palacı´ et al. 2004; Smith and Downs 1977). According to our results, the most significant differences are in the seed shape (Vriesea—filiform; Tillandsia—fusiform and narrowly fusiform), in the plumose appendage structural arrangement (Vriesea—type I; Tillandsia—type II) and in its color (Vriesea—yellowish; Tillandsia—white). In addition, the apical appendage is long in Vriesea and short in Tillandsia. Many authors (Billings 1904; Gross 1988; Morra et al. 2002) affirmed

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that the basal plumose appendages of Vriesea and Tillandsia are formed by the split of the testa and are adapted to wind dispersal. According to Smith and Downs (1977),

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the apical appendages of Vriesea and Tillandsia are usually not as developed as the basal one, but increase seed flotation.

Seed morphoanatomy and its systematic relevance to Tillandsioideae b Fig. 3

Endosperm of Vriesea Lindl. and Tillandsia L. a, b Endosperm general aspect. a Vriesea carinata Wawra. b Tillandsia aeranthos (Loisel.) L.B.Sm. c–e V. carinata. c Detail of endosperm central cells. d Positive reaction to Lugol’s reagent in the central endosperm cells and absence in the endosperm periphery. e Absence of reaction to Coomassie blue in the endosperm central cells and positive reaction in the endosperm periphery. f–h T. aeranthos. f Detail of endosperm cells. g Positive reaction to Lugol’s reagent in the central cells and absence of reaction in the periphery. h Absence of reaction to Coomassie blue in the central cells and positive reaction in the periphery. Endosperm central cells (asterisk), endosperm periphery (thin arrow) and embryo (large arrow)

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The endotegmen of all species is formed by cells with phenolic compounds. Nakamura et al. (2009) suggested that the presence of these compounds in the endotegmen of mature

seeds may be another distinctive feature of Poales. If its value is confirmed in further studies, the presence of phenolic substances in the seeds would be considered another indication of the correct placement of Bromeliaceae in Poales. The seed coat development provides several distinctive features. Its knowledge is extremely important to the correct interpretation of the seed coat of mature seeds. The plumose appendages have been considered homoplasic characters in Tillandsioideae (Benzing 2000, Palacı´ et al. 2004), and in the last stage of seed maturation there may be a compression of the seed coat because of water loss, which complicates the interpretation of these structures (Johri 1984). Thus, to understand species evolutionary trends, considering only the use of the seed coat, it would be

Fig. 4 Endosperm/embryo ratio. a–c General appearance of Vriesea Lindl. and Tillandsia L. seeds. a Vriesea carinata Wawra. Small embryo. Large endosperm amount. b Tillandsia aeranthos (Loisel.) L.B.Sm. Embryo occupying more than half of the seed. The rest is

filled by endosperm. c Tillandsia recurvata (L.) L. Embryo occupying the entire seed interior. Endosperm entirely consumed. Embryo (large arrow); endosperm central cells (asterisk); endosperm periphery (thin arrow)

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necessary to analyze its development to avoid incorrect inferences about them. Endosperm and embryo Considering the classification of Cocucci (2005), the seed formula of Tillandsia and Vriesea species is 325. This classification includes seeds having uninodal embryos (3), with reserves accumulating in the endosperm (2) and in the embryo (5). The only exceptions occur in T. recurvata, whose formula is 35, because it has a uninodal embryo (3) and the reserves are stored only in the embryo (5); T. geminiflora, with the formula 425, because of the multinodal embryo (4), is characterized by the development of a leaf primordium with reserves accumulated in the endosperm (2) and in the embryo (5). The endosperm periphery of many monocot species forms an epidermis-like layer, which is called the aleurone layer (Kumamaru et al. 2007; Larkins and Vasil 1997). According to our results, the cells from the endosperm periphery form an aleurone layer. In cereals, this layer is involved in the accumulation of phytic acid, capable of chelating minerals. It is an important site for the storage of phosphate, magnesium, potassium and calcium. The proteins present in this layer are stored in specialized organelles, the protein bodies (Becraft 2007; Larkins and Vasil 1997). During the initial stage of germination, the aleurone layer takes on a digestive function, secreting enzymes to break down the starch and proteins of the central endosperm. These activities are controlled by gibberellins, produced by the growing embryo, which induce the expression of genes that lead to the formation and secretion of amylase. Abscisic acid acts as an antagonist suppressing these activities (Becraft 2007). According to Becraft (2007), after the release of hydrolytic enzymes, the aleurone layer undergoes programmed cell death. This would explain the absence of the aleurone layer in the region adjacent to the Tillandsia embryo and its presence around the central endosperm. In Vriesea, the aleurone layer is adjacent to the embryo. However, cells of this region show different contents from those observed in cells in contact with the starchy endosperm. This difference probably occurs because the hydrolytic enzymes have already been released, and the aleurone layer has performed its function and will go into the process of programmed cell death. The seed portion occupied by the embryo is a good feature for distinguishing both genera. The embryo of Vriesea species is small and occupies 27–33 % of the seed. In Tillandsia, the embryo is large and occupies 65–100 %. However, classical works make reference to embryos that occupy 1/4–1/3 of the seed (Benzing 2000; Billings 1904; Johri et al. 1992). The size variation of the embryos

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R. I. Magalha˜es, J. E. A. Mariath Fig. 5 Characteristics of Vriesea Lindl. and Tillandsia L. embryos. c a–c Vriesea carinata Wawra. a Lateral expansions involving the apical meristem (asterisk) and procambium (arrow). b Shoot apex. c Embryo radicle (asterisk), endosperm peripheral layer (large arrow) and seed coat remnants (thin arrow). d Tillandsia recurvata (L.) L. Lateral expansions (asterisk) and leaf primordium under development (large arrow). e Tillandsia geminiflora Brogn. Lateral expansions (asterisk) involving the shoot apex (thin arrow) and leaf primordium (large arrow). f Detail of the apical shoot meristem (thin arrow) and leaf primordium (large arrow). g–h Tillandsia recurvata (L.) L. g Lipid deposition on cell wall (arrow) in the hypocotyl-radicle axis median region (Sudan black-positive reaction). h Substance mobilization (asterisk) from root to shoot portion. Presence of phenolic compounds on the cell walls. i Tracheids with reticulate thickening (arrow)

corroborates with the results of Gross (1988), who found that Tillandsioideae seeds vary much more than described in the literature. Barfuss et al. (2005) suggested that Tillandsia is an early divergent genus, and our results support that. According to Takhtajan (1991), in early divergent taxa, embryos are large, the endosperm is reduced or even absent, and the seed reserves are stored in the embryo itself. All of these features were found in the mature seeds of Tillandsia. They may be considered adaptations that allowed the colonization of different environments. Despite being a good distinctive feature, the seed portion occupied by the embryo is not the main difference between the analyzed species. The most remarkable difference is related to the presence/absence of series of modifications of the embryo hypocotyl-radicle axis. In all Vriesea species, the embryo does not show modified cells between the shoot and the root portion. However, in Tillandsia embryo there is a region made of structurally distinct cells that separate the root region from the rest of the embryo. From the characteristics of this region, it is possible to organize Tillandsia species into two groups: T. aeranthos, T. stricta and T. tenuifolia, with a slightly dislocated axis; T. geminiflora, T. recurvata and T. usneoides with a straight axis. These features corroborate with the report of Barfuss et al. (2005), who demonstrated the close relationship of T. aeranthos, T. tenuifolia and T. stricta. Billings (1904) noted that in T. usneoides, when the embryo reached a given size, the epidermal and cortical cells of the hypocotyl-radicle axis became distinct. CecchiFiordi et al. (1996) observed the same sort of change on Tillandsia flabellata Back. and Tillandsia schiedeana Steud. embryos and termed this region the ‘‘constriction zone.’’ Its presence was related to the atmospheric species of the genus. Morra et al. (2002) analyzed the seed development of Tillandsia tricholepis Baker and noted the presence of this constriction as well. According to the authors, the presence of a constriction zone between the shoot and root portion is the reason for primary root absence in the germination process of Tillandsia species.


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According to Mantovani and Iglesias (2005), bromeliads that inhabit extreme environments face several problems germinating and establishing themselves as autotrophic

organisms. Such information, combined with the fact that in many monocots the primary root has no function in water uptake and degenerates, leads us to conclude that the

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investment of resources in its formation is disadvantageous. So, avoiding primary root development and reusing the cellular content of a region that will be later aborted seem to be a great advantage for Tillandsia species. This may also have promoted the colonization of new environments. The presence of vascular bundles in Tillandsia embryos, in contrast to the presence of procambium in Vriesea, corroborates our inferences about the use of available resources in the embryo itself. Moreover, it indicates that Tillandsia embryos are in an advanced stage of development at the moment the fruit is ripened.

Some characteristics of the plumose appendages, such as being formed by two parachutes (which improves fixation in the substrate), the white color (which reflects the excess of solar radiation) and the early abortion of the radicle (which improves the use of energetic resources), are considered distinctive for the analyzed Tillandsia. Barfuss et al. (2005) suggested the occurrence of a second distribution center of Tillandsia in arid regions. These regions are known to be more difficult to colonize and require more adapted species. Thus, these features may have contributed to the exploration of new niches, increasing the distribution area of such plants.

Fig. 6 Embryo radicle of Tillandsia L. species. a–c Slightly dislocated hypocotyl-radicle axis (arrow). a Tillandsia aeranthos (Loisel.) L.B.Sm. b Tillandsia stricta Sol. ex. Sims. c Tillandsia

tenuifolia L. d–f Straight hypocotyl-radicle axis (arrow). d Tillandsia geminiflora Brogn. e Tillandsia recurvata (L.) L. f Tillandsia usneoides (L.) L. Seed coat (asterisk)

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Fig. 7 Embryo storage products. a–b Tillandsia aeranthos (Loisel.) L.B.Sm. a Positive reaction to Coomassie blue. b Positive reaction to Lugol’s reagent. c–d Vriesea incurvata Gaudich. c Positive reaction

to Coomassie blue. d Absence of reaction to Lugol’s reagent. Lipid body (thin arrow); protein body (asterisk); starch (large arrow)

According to Cecchi-Fiordi et al. (2001), as Tillandsia species are extreme epiphytes, morphophysiological changes were very important to their success. Billings (1904) noted that, in addition to the cotyledon, a lateral expansion was involved in the shoot apical meristem of the T. usneoides embryo and suggested it was a vestigial cotyledon. However, Gross (1988) stated that the cotyledon was lobed and such structures would be part of the cotyledon. The lateral expansions observed here are expansions of the cotyledon itself, which agree with the observations of Gross (1988), and they probably have a protective function in the meristematic region. Besides morphological features, the embryo and endosperm reserves are also important adaptations for environment colonization. Cecchi-Fiordi et al. (2001) suggested that, because of the difficulty of absorbing water and minerals, the presence of storage proteins is essential for seedling survival until the foliar trichomes are fully developed.

Gross (1988) suggested that species belonging to Diaphoranthema and Anoplophytum subgenera (to which the Tillandsia species analyzed here belong) were phylogenetically younger. This information was subsequently confirmed by the phylogeny generated by Barfuss et al. (2005), and it is consistent with our results. In Vriesea, the seeds do not show differences on the embryos that could be used for infrageneric delimitation. Genera that show relatively constant characteristics are considered phylogenetically older than those with great variability (Gross, 1988). Due to the constancy in embryo size and shape and the type and amount of reserves stored in the endosperm of Vriesea species, our data do not indicate differences that support the separation into sections suggested by Smith and Downs (1974) and Smith and Till (1998). Although both genera are traditionally characterized only by the presence or absence of petal appendages, this

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study provides seed morphological and anatomical characters that are able to help in distinguishing Vriesea and Tillandsia. Among them are: the structural arrangement and color of the plumose appendage; the seed shape; the ratio of embryo size and endosperm amount; the types of products stored in the endosperm; the presence/absence of the aleurone layer in the region in contact with the embryo; the presence/absence of a constriction zone separating the shoot and root portion; and the presence/absence of vascular bundles. There are also some more subtle features, which contribute to the distinction between the analyzed tillandsias, namely: the seed shape (fusiform 9 narrowly fusiform) and the shape of the hypocotyl-radicle axis median portion (straight 9 slightly dislocated). It has to be emphasized that further studies focusing on the ontogeny of these structures will probably increase the number of informative characters and will clarify possible homologies. Even not including all species belonging to Vriesea and Tillandsia, the information presented here about the seed coat, endosperm and embryo is promising for the comprehension of the phylogenetic relationships of Tillandsioideae and deserves further work to increase the number of species. Here, it was possible to recognize taxonomic groups, highlighting the richness of information that seeds can provide. Additionally, some of the features highlighted throughout this study are easily observed under a stereomicroscope and can be used by taxonomists in the delimitation of the genera and some species. Acknowledgments We thank the Plant Anatomy Laboratory at Universidade Federal do Rio Grande do Sul (UFRGS) for technical support; CNPq for the scholarship granted to the first author; Fundaçaõ Zoobotaˆnica do Rio Grande do Sul for permission to collect from the Bromeliaceae Collection; CAPES and FAPERGS for financial support.

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