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Contribution to the embryology of Leiothrix fluitans (Eriocaulaceae: Poales)
Aquatic Botany 87 (2007) 155–160 www.elsevier.com/locate/aquabot
Short communication
Contribution to the embryology of Leiothrix fluitans (Eriocaulaceae: Poales) Alessandra Ike Coan *, Michele Marcelino Rosa, Vera Lucia Scatena Departamento de Botaˆnica, Instituto de Biocieˆncias, Universidade Estadual Paulista, C.P. 199, Rio Claro, Sa˜o Paulo 13506-900, Brazil Received 2 May 2006; received in revised form 5 April 2007; accepted 6 April 2007 Available online 13 April 2007
Abstract This paper presents a contribution to the understanding of the embryology, especially microsporogenesis, the antipodal cell behavior, and the early stages of the micropylar seed operculum, in Leiothrix fluitans, to elucidate these aspects both within the subgenus Rheocaulon and within the genus in Eriocaulaceae. Contrarily to previous descriptions of this same species, our results show the following: microsporogenesis is of the successive type and results in isobilateral microspore tetrads; the antipodal cells gradually fuse together to form a conspicuous cyst; and the inner integument, which does not develop into an endothelium, shows evidence of the initiation of the seed operculum in its micropylar end. Such features are common to the family as a whole. Evidenced for the first time in the family, the chalazal end of the ovule differentiates into a hypostase closely associated to the antipodal cyst. These overall features of L. fluitans point out previous misinterpretations on some of its embryological aspects, especially those concerning the only report of simultaneous microsporogenesis and proliferation of the antipodal cells. Furthermore, the results presented here allow us to reinforce the uniformity of the embryological aspects within the Eriocaulaceae, strengthening the cystic arrangement of the antipodal cells as a potential autapomorphy of the family within the other Poales (commelinids). # 2007 Elsevier B.V. All rights reserved. Keywords: Antipodal cyst; Commelinids; Microsporogenesis; Pollen grain; Seed operculum
1. Introduction Eriocaulaceae is a well-defined monocot family comprising eleven genera and circa 1200 species distributed throughout the tropics and subtropics (Giulietti et al., 1995; Sano, 2004) and distinguished by its characteristic inflorescence called capitulum. The greatest species diversity for the family is found in South America, especially in the Cadeia do Espinhac¸o Mountain Range, Brazil. Leiothrix Ruhland includes about 37 species distributed exclusively in South America, mainly restricted to Brazil (Giulietti et al., 1995; Stu¨tzel, 1998). According to Giulietti and Pirani (1988), the State of Minas Gerais (Brazil) is the centre of diversity of the genus with 30 species, mostly endemic to small areas. Ruhland (1903) divided the genus Leiothrix into five subgenera: Calycocephalus, Eleutherandra, Psilandra, Rheocaulon, and Stephanophyllum. In their taxonomic studies on the genus, Giulietti (1984) and Giulietti et al. (1995) recognized four of those subgenera were valid, but merged Psilandra into a section of genus Syngonanthus.
* Corresponding author. Tel.: +55 19 35264212; fax: +55 19 35264201. E-mail address: [email protected] (A.I. Coan). 0304-3770/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aquabot.2007.04.001
Although taxonomically important, subgenus Rheocaulon only includes one species, Leiothrix fluitans. It was distinguished by Ruhland (1903) and Giulietti (1978) because of the elongate stem and of the free inner tepals of the staminate flower. Leiothrix fluitans is the only aquatic species in the whole genus and its occurrence is restricted to the State of Minas Gerais (Brazil). It grows on riverbeds and on the rocks of waterfalls (Giulietti, 1984). The species possesses many adaptations to the aquatic environment, mainly represented by the submerged and reduced leaves, with only one vascular bundle, the absence of trichomes and presence of air spaces, the low stomata number, and two root types, specialized in air storage and in substrate fixing (Monteiro et al., 1985; Coan et al., 2002). The embryology of specimens of all the accepted subgenera of Leiothrix has been studied, from pollen morphology (Thanikaimoni, 1965), to embryogeny (Ramaswamy and Arekal, 1982a) and to seed structure and development (Giulietti et al., 1988). Monteiro-Scanavacca and Mazzoni (1978) described the embryology and seed development of L. fluitans, stressing the occurrence of tetrahedral microspore tetrads resulting from simultaneous microsporogenesis, the presence of a well-developed endothelium in the ovule, the proliferation of the antipodal cells in the megagametophyte, and the formation of a reduced caruncle in the seeds. When comparing
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these embryologic data of L. fluitans with those of a related species, L. nubigena, Ramaswamy and Arekal (1981a,b) first pointed out some misinterpretations in the study by MonteiroScanavacca and Mazzoni (1978). According to Ramaswamy and Arekal (1981a,b), the antipodal cells do not proliferate in Leiothrix, but develop into a conspicuous cyst, as is known for all the species from the other genera already studied. Based on the description by Monteiro-Scanavacca and Mazzoni (1978), some embryological aspects of L. fluitans suggest different character states in the family, which are not consistent features when compared to the other species studied (Begum, 1968; Arekal and Ramaswamy, 1980; Ramaswamy and Arekal, 1981a,b,c, 1982b; Ramaswamy and Nagendran, 1996; Giulietti et al., 1988; Scatena and Bouman, 2001; Coan and Scatena, 2004; Coan et al., 2007), mainly those related to the type of microsporogenesis, the endothelium formation in the ovule, the antipodal behavior, and the origin of the caruncle in the seed. Since the embryologic data on Eriocaulaceae have proved uniform in specimens of the different genera already studied, and even in other Leiothrix species, but contrast with those previously presented by Monteiro-Scanavacca and Mazzoni (1978) for L. fluitans, the aim of the present study was to elucidate its embryology, with an emphasis on the microsporogenesis, the ovule integument development, and the antipodal cell behavior. 2. Material and methods Inflorescences of L. fluitans (Mart.) Ruhland at different developmental stages were collected in the Serra do Cipo´, district of Santana do Riacho, along the highway MG-010 (Minas Gerais, Brazil), and fixed in FAA 50 (1 formalin:1 glacial acetic acid:18 ethanol, v/v) (Johansen, 1940). The material was then transferred and stored in 70% ethanol with a few drops of glycerin. Vouchers of L. fluitans were deposited at the Herbarium of the Department of Botany, Universidade Estadual Paulista (HRCB 33562, HRCB 39172) and at the Herbarium of the Department of Botany, Universidade de Sa˜o Paulo (SPF 146328). For light microscope (LM) examination, the material was dehydrated through a normal-butyl alcohol series under vacuum (Feder and O’Brien, 1968, with modifications in dehydration times), embedded in (2-hydroxyethyl)-methacrylate, and sectioned at 4–8 mm on a Reichert-Jung Model 2040 Microtome using glass knives. The sections were then stained with periodic acid–Schiff’s reagent (PAS reaction) and toluidine blue (Feder and O’Brien, 1968). For scanning electron microscopy (SEM), fixed mature anthers were dehydrated through an absolute ethanol series, critical point dried, coated with gold, and examined using a JEOL JSM-P15 scanning electron microscope. 3. Results The present study confirms most of the embryological characters Monteiro-Scanavacca and Mazzoni (1978)
described for L. fluitans. To avoid data duplication, we only present the characters differing from this previous work here. In L. fluitans, the cells of the sporogenous tissue differentiate into microsporocytes that undergo meiotic divisions and form dyads (Fig. 1A and B) and isobilateral microspore tetrads (Fig. 1C and D) enclosed in a callose coat. Microsporogenesis is of the successive type. At the tetrad stage (Fig. 1D), the longitudinal sections show two microspores in each tetrad, which forms a row and fills completely the anther locule. The fairly thick callose coat deposited around each microspore (Fig. 1D) dissolves soon after meiosis and releases the four microspores into the anther locule (Fig. 1E). The microspores enlarge, separate, acquire a spherical shape, and accumulate starch in their cytoplasm (Fig. 1E and F). At anthesis stage, pollen grains are shed at the binucleate stage (Fig. 1G). Pollen is spiraperturate (Fig. 1H). The anther wall develops according to the ‘‘monocotyledonous’’ type and comprises epidermis, endothecium, middle layer and tapetum (Fig. 1B). As the anther develops, the epidermal cells shrink, except at the stomium (Fig. 1E and F). The endothecial cells increase in size and develop regular bandlike thickenings on their anticlinal and outer periclinal walls, while the inner periclinal one constitutes the complete and nonperforated baseplate (Fig. 1E and F). The endothecial thickening is of the baseplate type (Fig. 1E and F), following the terminology proposed by Manning (1996). In the ovular primordium, the inner integument forms before the outer one (Fig. 2A) and both are initially dermal and possess two cell layers. The one-layered nucellar epidermis (Fig. 2A and B) is gradually compressed as the ovule develops (Fig. 2C) and leaves remains in the micropylar region (Fig. 2F and G). The inner cell layer of the inner integument of the ovule does not differentiate into an endothelium and shows no cell differentiation or amyloplast accumulation (Fig. 2F and H). The meiosis of the megasporocyte (Fig. 2A) produces a Tshaped tetrad of megaspores in which only the chalazal one is functional (Fig. 2B). During megagametogenesis, the functional chalazal megaspore divides to form a megagametophyte of the Polygonum type (Fig. 2C). The three-celled egg apparatus lies in the micropylar region of the megagametophyte (Fig. 2F). Soon after organization, the antipodal walls dissolve and the three uninucleate protoplasts gradually fuse together resulting in a conspicuous antipodal cyst (Fig. 2D). This cyst acquires a dense cytoplasm and its three nuclei increase in size (Fig. 2E). The three enlarged nuclei of the cyst then fuse together to form a single large nucleus (Fig. 2F). After fertilization and megagametophyte nutrition, the antipodal cyst becomes less conspicuous and recedes (Fig. 2G). Concomitantly with the antipodal cyst formation, some chalazal cells become cutinized to form the hypostase (Fig. 2D and F). As for the antipodal cyst, the hypostase also becomes less conspicuous after fertilization (Fig. 2G). As the megagametophyte develops, the cells of the inner integument that constitute the micropyle divide anticlinally (Fig. 2H, arrow). These cells become cutinized after fertilization (Fig. 2I, arrow) and point out the initiation of the
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Fig. 1. Microsporogenesis and microgametophyte of Leiothrix fluitans. (A) Cross section of young anthers at dyad stage (bar = 30 mm); (B) detail of a dyad, enclosed in a callose coat, in cross section (bar = 10 mm); (C) cross section of an anther locule in isobilateral microspore tetrad stage (bar = 20 mm); (D) longitudinal section of a row of tetrads, with two microspores of each tetrad visible (bar = 20 mm); (E) cross section of a developing anther, with thickened endothecium and separating microspores (bar = 20 mm); (F) longitudinal section of mature anther (bar = 20 mm); (G) detail of a binucleate pollen grain, in longitudinal section (bar = 10 mm); (H) scanning electron micrograph of spiraperturate pollen grains (bar = 10 mm) (c, callose coat; e, epidermis; en, endothecium; ml, middle layer; st, stomium; t, tapetum).
micropylar seed operculum. A thick cuticle between the nucellus and inner integument, and between the whole length of the inner and outer integuments (Fig. 2G, arrowheads) indicate the post-fertilization stages. 4. Discussion The present data on Leiothrix fluitans diverge from those found in a previous study by Monteiro-Scanavacca and Mazzoni (1978) with regard to the microspore tetrads, the endothelium, the behavior of the antipodal cells, and the differentiation of cells of the micropylar end of the fertilized ovule. Furness and Rudall (1999) assert microsporogenesis is predominantly successive within monocotyledons and is usually consistent within family level, especially within the commelinids. Our results confirmed that the microsporogenesis of L. fluitans is successive and results in isobilateral tetrads of microspores, as has been reported for all the Eriocaulaceae specimens studied so far, viz. Eriocaulon species (Palm, 1920; Arekal and Ramaswamy, 1980; Ramaswamy and Arekal,
1981c, 1982b; Furness and Rudall, 1999), Syngonanthus nitens (Ramaswamy and Nagendran, 1996), Paepalanthus sect. Actinocephalus species (=Actinocephalus) (Scatena and Bouman, 2001), Blastocaulon scirpeum and P. scleranthus (Coan and Scatena, 2004), and Syngonanthus caulescens (Coan et al., 2007). Among all the authors who have studied the embryology of Eriocaulaceae, Monteiro-Scanavacca and Mazzoni (1978) were the only ones to verify the predominance of tetrahedral tetrads in L. fluitans. Nevertheless, their results did not present any detailed descriptions or illustrations to confirm their observations but only two figures of longitudinal sections of young anthers illustrating the whole microsporogenesis process: Fig. 1 showed the beginning of meiosis, and Fig. 2, the microspore tetrad. Such figures, which are not even sharp, do not suffice to affirm the predominance of tetrahedral tetrads that indicate the occurrence of simultaneous microsporogenesis. As shown by the results of the present work, in L. fluitans, after the organization of a megagametophyte of the Polygonum type, the antipodal cells lose their walls to produce a single protoplast, which gradually merges into a conspicuous cyst.
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Fig. 2. Successive stages of ovule development of Leiothrix fluitans, in longitudinal sections. (A) Tenuinucellate ovule with megasporocyte under division and appearance of the inner and outer integuments (bar = 20 mm); (B) degenerating T-shaped tetrad of megaspores with the functional one in chalazal position (bar = 30 mm); (C) eight-nucleated stage with degenerating nucellar epidermis in micropylar end (bar = 20 mm); (D) detail of the chalazal region of a mature megagametophyte showing the three antipodal nuclei in a single protoplast (bar = 30 mm); (E) detail of the chalazal region showing the antipodals at the time of polar nuclei migration to the center of the central cell (bar = 20 mm); (F) mature megagametophyte of the Polygonum-type, with early stage of antipodal cyst formation (bar = 20 mm); (G) post-fertilization stage, with degenerated antipodal cyst at the chalazal end, and the two synergids at the micropylar end (bar = 20 mm); (H and I) early stages of operculum formation in the micropylar end (arrow), before (H) and after fertilization (I) (bars = 20 mm) (arrowhead, cuticle; a, antipodals; ac, antipodal cyst; fm, functional megaspore; h, hypostase; ii, inner integument; oi, outer integument; ow, ovarian wall).
Such result was initially presented for Leiothrix nubigena and Paepalanthus bifidus by Ramaswamy and Arekal (1981a), who questioned the interpretation by Monteiro-Scanavacca and Mazzoni (1978) and offered a different interpretation of the megagametophyte development, which was subsequently confirmed by the same authors (Ramaswamy and Arekal, 1981b, 1982b), and by Scatena and Bouman (2001), Coan and Scatena (2004), and Coan et al. (2007) for other species of different genera of Eriocaulaceae. The formation of the
antipodal cyst can be interpreted as an autapomorphy of Eriocaulaceae within the order Poales, and corroborates the observation of Ramaswamy and Arekal (1981b), who affirmed that this behavior is constant and exclusive of the Eriocaulaceae. Still according to Ramaswamy and Arekal (1981b), this peculiar behavior of the antipodal cells in Eriocaulaceae is definitely related to the nutrition of the megagametophyte, which receives material from the chalazal region or perhaps
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syntheses some of its own. The report of a hypostase in the ovule of L. fluitans, in the present study, should be verified in other taxa of the family, since it may be an argument in favor of the nutritional role of the antipodal cyst in the megagametophyte. In the present study, the cutinized chalazal cells surrounding the antipodal cells, and later the antipodal cyst, was called a hypostase according the definition proposed by Rudall (1997), in a study on the nucellus and chalaza in monocotyledons, following the nutritional function emphasized by Maheshwari (1950) and Tilton (1980). As shown here, in L. fluitans, the hypostase develops early in the megagametophyte development, it is closely associated with the antipodal cyst and both structures recede as the megagametophyte matures. The presence of an endothelium in the ovules of species of Eriocaulaceae was only evidenced in Eriocaulon quinquangulare by Begum (1968) and in L. fluitans by MonteiroScanavacca and Mazzoni (1978). During the ovule ontogeny of the Eriocaulaceae species already studied, which include L. fluitans, here analyzed, the cells of the inner integument do not represent an endothelium because they do not show any histological or functional change that would characterize a typical endothelium, as the one reported in Tabebuia pulcherrima (Bignoniaceae) by Bittencourt and Mariath (2002), for example, or in many other eudicotyledons. In a review of the endothelium in monocotyledons, Swamy and Krishnamurthy (1970) reported the absence of this structure in Eriocaulaceae. The present study followed up the early modifications in the cells of the inner integument that form the micropyle of L. fluitans. They corresponded to what is already known for other Eriocaulaceae species and point the initiation of the micropylar seed operculum. The micropylar operculum seems to be a widespread character in the seeds of Eriocaulaceae, also reported for species of Eriocaulon (Arekal and Ramaswamy, 1980), Syngonanthus (Scatena et al., 1993; Coan et al., 2007), Paepalanthus (Kraus et al., 1996; Coan and Scatena, 2004), Paepalanthus sect. Actinocephalus (=Actinocephalus) (Scatena and Bouman, 2001), and Blastocaulon (Coan and Scatena, 2004). According to Stevenson et al. (2000) and Bremer (2002), this characteristic of the Eriocaulaceae seeds is a synapomorphy supporting the relationships between Eriocaulaceae, Hydatellaceae, Mayacaceae, and Xyridaceae, which, together, form the xyrid clade within Poales. Monteiro-Scanavacca and Mazzoni (1978) described this same structure in the seeds of L. fluitans as a reduced caruncle, but failed in giving further details about its formation. The confirmation of the occurrence of successive microsporogenesis resulting in isobilateral microspore tetrads, of ovules without endothelium, of the formation of a conspicuous antipodal cyst, and of the formation of a micropylar operculum in L. fluitans allows us to characterize the subgenus Rheocaulon as well as the genus Leiothrix within Eriocaulaceae. Similarly, the presence of these features allows us to reaffirm the embryologic uniformity of the family as a whole, strengthening the occurrence of the antipodal cyst as an autapomorphy of Eriocaulaceae among other Poales.
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Acknowledgements The authors thank Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq 141928/2002-6; 301404/20046; 471809/04-7), and Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP 05/02141-4) for financial support. Thanks are due also to the two anonymous referees for providing valuable comments. References Arekal, G.D., Ramaswamy, S.N., 1980. Embryology of Eriocaulon hookerianum Stapf. and the systematic position of Eriocaulaceae. Bot. Not. 133, 295–309. Begum, M., 1968. Embryological studies in Eriocaulon quinquangulare Linn. Proc. Indian Acad. Sci. B 67, 148–156. Bittencourt Jr., N.S., Mariath, J.E.A., 2002. Ovule ontogeny of Tabebuia pulcherrima Sandwich (Bignoniaceae): megasporogenesis and integument development. Rev. Bras. Bot. 25, 103–115. Bremer, K., 2002. Gondwanan evolution of the grass alliance of families (Poales). Evolution 56, 1374–1387. Coan, A.I., Scatena, V.L., 2004. Embryology and seed development of Blastocaulon scirpeum and Paepalanthus scleranthus (Eriocaulaceae). Flora 199, 47–57. Coan, A.I., Scatena, V.L., Giulietti, A.M., 2002. Anatomia de algumas espe´cies aqua´ticas de Eriocaulaceae brasileiras. Acta Bot. Bras. 16, 371–384. Coan, A.I., Rosa, M.M., Scatena, V.L., 2007. Embryology and seed development of Syngonanthus caulescens (Poir.) Ruhland (Eriocaulaceae—Poales). Aquat. Bot. 86, 148–156. Feder, N., O’Brien, T.P., 1968. Plant microthechnique: some principles and new methods. Am. J. Bot. 55, 123–142. Furness, C.A., Rudall, P.J., 1999. Microsporogenesis in monocotyledons. Ann. Bot. 84, 475–499. Giulietti, A.M., 1978. Os geˆneros Eriocaulon L. e Leiothrix Ruhland (Eriocaulaceae) na Serra do Cipo´, Minas Gerais. Tese de Doutorado, Instituto de Biocieˆncias, Univ. de Sa˜o Paulo, Brasil. Giulietti, A.M., 1984. Estudos taxonoˆmicos no geˆnero Leiothrix Ruhland (Eriocaulaceae). Tese de Livre Doceˆncia. Instituto de Biocieˆncias, Univ. de Sa˜o Paulo. Giulietti, A.M., Pirani, J.R., 1988. Patterns of geographic distribution of some plant species from the Espinhac¸o Range, Minas Gerais and Bahia. In: Vanzolini, P.E., Heyer, W.R. (Eds.), Proceedings of a Workshop of Neotropical Distribution Patterns. Academia Brasileira de Cieˆncias, Rio de Janeiro, Brazil, pp. 39–69. Giulietti, A.M., Monteiro, W.R., Mayo, S.J., Stephens, J., 1988. A preliminary survey of testa sculpture in Eriocaulaceae. Beit. Biol. Pflanz. 62, 189–209. Giulietti, A.M., Amaral, M.C.E., Bittrich, V., 1995. Phylogenetic analysis of inter- and infrageneric relationships of Leiothrix Ruhland (Eriocaulaceae). Kew Bull. 50, 55–71. Johansen, D.A., 1940. Plant Microthechnique. McGraw-Hill Book Company, New York. Kraus, J.E., Scatena, V.L., Lewinger, M.E., Trench, K.U.S., 1996. Morfologia externa e interna de quatro espe´cies de Paepalanthus Kunth (Eriocaulaceae) em desenvolvimento po´s-seminal. Bolm Bot. Univ. S. Paulo 15, 45–53. Maheshwari, P., 1950. An Introduction to the Embryology of Angiosperms. McGraw-Hill Book Company, New York. Manning, J.C., 1996. Diversity of endothecial patterns in the angiosperms. In: D’Arcy, W.G., Keating, R.C. (Eds.), The anther: Form, Function and Phylogeny. Cambridge University Press, Cambridge, pp. 136–158. Monteiro, W.R., Castro, M.M., Giulietti, A.M., 1985. Aspects of leaf structure of some species of Leiothrix Ruhl. (Eriocaulaceae) from the Serra do Cipo´ (Minas Gerais, Brazil). Rev. Bras. Bot. 8, 109–125. Monteiro-Scanavacca, W.R., Mazzoni, S.C., 1978. Embryological studies in Leiothrix fluitans (Mart.) Ruhl. (Eriocaulaceae). Rev. Bras. Bot. 1, 59–64.
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Short communication
Contribution to the embryology of Leiothrix fluitans (Eriocaulaceae: Poales) Alessandra Ike Coan *, Michele Marcelino Rosa, Vera Lucia Scatena Departamento de Botaˆnica, Instituto de Biocieˆncias, Universidade Estadual Paulista, C.P. 199, Rio Claro, Sa˜o Paulo 13506-900, Brazil Received 2 May 2006; received in revised form 5 April 2007; accepted 6 April 2007 Available online 13 April 2007
Abstract This paper presents a contribution to the understanding of the embryology, especially microsporogenesis, the antipodal cell behavior, and the early stages of the micropylar seed operculum, in Leiothrix fluitans, to elucidate these aspects both within the subgenus Rheocaulon and within the genus in Eriocaulaceae. Contrarily to previous descriptions of this same species, our results show the following: microsporogenesis is of the successive type and results in isobilateral microspore tetrads; the antipodal cells gradually fuse together to form a conspicuous cyst; and the inner integument, which does not develop into an endothelium, shows evidence of the initiation of the seed operculum in its micropylar end. Such features are common to the family as a whole. Evidenced for the first time in the family, the chalazal end of the ovule differentiates into a hypostase closely associated to the antipodal cyst. These overall features of L. fluitans point out previous misinterpretations on some of its embryological aspects, especially those concerning the only report of simultaneous microsporogenesis and proliferation of the antipodal cells. Furthermore, the results presented here allow us to reinforce the uniformity of the embryological aspects within the Eriocaulaceae, strengthening the cystic arrangement of the antipodal cells as a potential autapomorphy of the family within the other Poales (commelinids). # 2007 Elsevier B.V. All rights reserved. Keywords: Antipodal cyst; Commelinids; Microsporogenesis; Pollen grain; Seed operculum
1. Introduction Eriocaulaceae is a well-defined monocot family comprising eleven genera and circa 1200 species distributed throughout the tropics and subtropics (Giulietti et al., 1995; Sano, 2004) and distinguished by its characteristic inflorescence called capitulum. The greatest species diversity for the family is found in South America, especially in the Cadeia do Espinhac¸o Mountain Range, Brazil. Leiothrix Ruhland includes about 37 species distributed exclusively in South America, mainly restricted to Brazil (Giulietti et al., 1995; Stu¨tzel, 1998). According to Giulietti and Pirani (1988), the State of Minas Gerais (Brazil) is the centre of diversity of the genus with 30 species, mostly endemic to small areas. Ruhland (1903) divided the genus Leiothrix into five subgenera: Calycocephalus, Eleutherandra, Psilandra, Rheocaulon, and Stephanophyllum. In their taxonomic studies on the genus, Giulietti (1984) and Giulietti et al. (1995) recognized four of those subgenera were valid, but merged Psilandra into a section of genus Syngonanthus.
* Corresponding author. Tel.: +55 19 35264212; fax: +55 19 35264201. E-mail address: [email protected] (A.I. Coan). 0304-3770/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aquabot.2007.04.001
Although taxonomically important, subgenus Rheocaulon only includes one species, Leiothrix fluitans. It was distinguished by Ruhland (1903) and Giulietti (1978) because of the elongate stem and of the free inner tepals of the staminate flower. Leiothrix fluitans is the only aquatic species in the whole genus and its occurrence is restricted to the State of Minas Gerais (Brazil). It grows on riverbeds and on the rocks of waterfalls (Giulietti, 1984). The species possesses many adaptations to the aquatic environment, mainly represented by the submerged and reduced leaves, with only one vascular bundle, the absence of trichomes and presence of air spaces, the low stomata number, and two root types, specialized in air storage and in substrate fixing (Monteiro et al., 1985; Coan et al., 2002). The embryology of specimens of all the accepted subgenera of Leiothrix has been studied, from pollen morphology (Thanikaimoni, 1965), to embryogeny (Ramaswamy and Arekal, 1982a) and to seed structure and development (Giulietti et al., 1988). Monteiro-Scanavacca and Mazzoni (1978) described the embryology and seed development of L. fluitans, stressing the occurrence of tetrahedral microspore tetrads resulting from simultaneous microsporogenesis, the presence of a well-developed endothelium in the ovule, the proliferation of the antipodal cells in the megagametophyte, and the formation of a reduced caruncle in the seeds. When comparing
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these embryologic data of L. fluitans with those of a related species, L. nubigena, Ramaswamy and Arekal (1981a,b) first pointed out some misinterpretations in the study by MonteiroScanavacca and Mazzoni (1978). According to Ramaswamy and Arekal (1981a,b), the antipodal cells do not proliferate in Leiothrix, but develop into a conspicuous cyst, as is known for all the species from the other genera already studied. Based on the description by Monteiro-Scanavacca and Mazzoni (1978), some embryological aspects of L. fluitans suggest different character states in the family, which are not consistent features when compared to the other species studied (Begum, 1968; Arekal and Ramaswamy, 1980; Ramaswamy and Arekal, 1981a,b,c, 1982b; Ramaswamy and Nagendran, 1996; Giulietti et al., 1988; Scatena and Bouman, 2001; Coan and Scatena, 2004; Coan et al., 2007), mainly those related to the type of microsporogenesis, the endothelium formation in the ovule, the antipodal behavior, and the origin of the caruncle in the seed. Since the embryologic data on Eriocaulaceae have proved uniform in specimens of the different genera already studied, and even in other Leiothrix species, but contrast with those previously presented by Monteiro-Scanavacca and Mazzoni (1978) for L. fluitans, the aim of the present study was to elucidate its embryology, with an emphasis on the microsporogenesis, the ovule integument development, and the antipodal cell behavior. 2. Material and methods Inflorescences of L. fluitans (Mart.) Ruhland at different developmental stages were collected in the Serra do Cipo´, district of Santana do Riacho, along the highway MG-010 (Minas Gerais, Brazil), and fixed in FAA 50 (1 formalin:1 glacial acetic acid:18 ethanol, v/v) (Johansen, 1940). The material was then transferred and stored in 70% ethanol with a few drops of glycerin. Vouchers of L. fluitans were deposited at the Herbarium of the Department of Botany, Universidade Estadual Paulista (HRCB 33562, HRCB 39172) and at the Herbarium of the Department of Botany, Universidade de Sa˜o Paulo (SPF 146328). For light microscope (LM) examination, the material was dehydrated through a normal-butyl alcohol series under vacuum (Feder and O’Brien, 1968, with modifications in dehydration times), embedded in (2-hydroxyethyl)-methacrylate, and sectioned at 4–8 mm on a Reichert-Jung Model 2040 Microtome using glass knives. The sections were then stained with periodic acid–Schiff’s reagent (PAS reaction) and toluidine blue (Feder and O’Brien, 1968). For scanning electron microscopy (SEM), fixed mature anthers were dehydrated through an absolute ethanol series, critical point dried, coated with gold, and examined using a JEOL JSM-P15 scanning electron microscope. 3. Results The present study confirms most of the embryological characters Monteiro-Scanavacca and Mazzoni (1978)
described for L. fluitans. To avoid data duplication, we only present the characters differing from this previous work here. In L. fluitans, the cells of the sporogenous tissue differentiate into microsporocytes that undergo meiotic divisions and form dyads (Fig. 1A and B) and isobilateral microspore tetrads (Fig. 1C and D) enclosed in a callose coat. Microsporogenesis is of the successive type. At the tetrad stage (Fig. 1D), the longitudinal sections show two microspores in each tetrad, which forms a row and fills completely the anther locule. The fairly thick callose coat deposited around each microspore (Fig. 1D) dissolves soon after meiosis and releases the four microspores into the anther locule (Fig. 1E). The microspores enlarge, separate, acquire a spherical shape, and accumulate starch in their cytoplasm (Fig. 1E and F). At anthesis stage, pollen grains are shed at the binucleate stage (Fig. 1G). Pollen is spiraperturate (Fig. 1H). The anther wall develops according to the ‘‘monocotyledonous’’ type and comprises epidermis, endothecium, middle layer and tapetum (Fig. 1B). As the anther develops, the epidermal cells shrink, except at the stomium (Fig. 1E and F). The endothecial cells increase in size and develop regular bandlike thickenings on their anticlinal and outer periclinal walls, while the inner periclinal one constitutes the complete and nonperforated baseplate (Fig. 1E and F). The endothecial thickening is of the baseplate type (Fig. 1E and F), following the terminology proposed by Manning (1996). In the ovular primordium, the inner integument forms before the outer one (Fig. 2A) and both are initially dermal and possess two cell layers. The one-layered nucellar epidermis (Fig. 2A and B) is gradually compressed as the ovule develops (Fig. 2C) and leaves remains in the micropylar region (Fig. 2F and G). The inner cell layer of the inner integument of the ovule does not differentiate into an endothelium and shows no cell differentiation or amyloplast accumulation (Fig. 2F and H). The meiosis of the megasporocyte (Fig. 2A) produces a Tshaped tetrad of megaspores in which only the chalazal one is functional (Fig. 2B). During megagametogenesis, the functional chalazal megaspore divides to form a megagametophyte of the Polygonum type (Fig. 2C). The three-celled egg apparatus lies in the micropylar region of the megagametophyte (Fig. 2F). Soon after organization, the antipodal walls dissolve and the three uninucleate protoplasts gradually fuse together resulting in a conspicuous antipodal cyst (Fig. 2D). This cyst acquires a dense cytoplasm and its three nuclei increase in size (Fig. 2E). The three enlarged nuclei of the cyst then fuse together to form a single large nucleus (Fig. 2F). After fertilization and megagametophyte nutrition, the antipodal cyst becomes less conspicuous and recedes (Fig. 2G). Concomitantly with the antipodal cyst formation, some chalazal cells become cutinized to form the hypostase (Fig. 2D and F). As for the antipodal cyst, the hypostase also becomes less conspicuous after fertilization (Fig. 2G). As the megagametophyte develops, the cells of the inner integument that constitute the micropyle divide anticlinally (Fig. 2H, arrow). These cells become cutinized after fertilization (Fig. 2I, arrow) and point out the initiation of the
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Fig. 1. Microsporogenesis and microgametophyte of Leiothrix fluitans. (A) Cross section of young anthers at dyad stage (bar = 30 mm); (B) detail of a dyad, enclosed in a callose coat, in cross section (bar = 10 mm); (C) cross section of an anther locule in isobilateral microspore tetrad stage (bar = 20 mm); (D) longitudinal section of a row of tetrads, with two microspores of each tetrad visible (bar = 20 mm); (E) cross section of a developing anther, with thickened endothecium and separating microspores (bar = 20 mm); (F) longitudinal section of mature anther (bar = 20 mm); (G) detail of a binucleate pollen grain, in longitudinal section (bar = 10 mm); (H) scanning electron micrograph of spiraperturate pollen grains (bar = 10 mm) (c, callose coat; e, epidermis; en, endothecium; ml, middle layer; st, stomium; t, tapetum).
micropylar seed operculum. A thick cuticle between the nucellus and inner integument, and between the whole length of the inner and outer integuments (Fig. 2G, arrowheads) indicate the post-fertilization stages. 4. Discussion The present data on Leiothrix fluitans diverge from those found in a previous study by Monteiro-Scanavacca and Mazzoni (1978) with regard to the microspore tetrads, the endothelium, the behavior of the antipodal cells, and the differentiation of cells of the micropylar end of the fertilized ovule. Furness and Rudall (1999) assert microsporogenesis is predominantly successive within monocotyledons and is usually consistent within family level, especially within the commelinids. Our results confirmed that the microsporogenesis of L. fluitans is successive and results in isobilateral tetrads of microspores, as has been reported for all the Eriocaulaceae specimens studied so far, viz. Eriocaulon species (Palm, 1920; Arekal and Ramaswamy, 1980; Ramaswamy and Arekal,
1981c, 1982b; Furness and Rudall, 1999), Syngonanthus nitens (Ramaswamy and Nagendran, 1996), Paepalanthus sect. Actinocephalus species (=Actinocephalus) (Scatena and Bouman, 2001), Blastocaulon scirpeum and P. scleranthus (Coan and Scatena, 2004), and Syngonanthus caulescens (Coan et al., 2007). Among all the authors who have studied the embryology of Eriocaulaceae, Monteiro-Scanavacca and Mazzoni (1978) were the only ones to verify the predominance of tetrahedral tetrads in L. fluitans. Nevertheless, their results did not present any detailed descriptions or illustrations to confirm their observations but only two figures of longitudinal sections of young anthers illustrating the whole microsporogenesis process: Fig. 1 showed the beginning of meiosis, and Fig. 2, the microspore tetrad. Such figures, which are not even sharp, do not suffice to affirm the predominance of tetrahedral tetrads that indicate the occurrence of simultaneous microsporogenesis. As shown by the results of the present work, in L. fluitans, after the organization of a megagametophyte of the Polygonum type, the antipodal cells lose their walls to produce a single protoplast, which gradually merges into a conspicuous cyst.
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Fig. 2. Successive stages of ovule development of Leiothrix fluitans, in longitudinal sections. (A) Tenuinucellate ovule with megasporocyte under division and appearance of the inner and outer integuments (bar = 20 mm); (B) degenerating T-shaped tetrad of megaspores with the functional one in chalazal position (bar = 30 mm); (C) eight-nucleated stage with degenerating nucellar epidermis in micropylar end (bar = 20 mm); (D) detail of the chalazal region of a mature megagametophyte showing the three antipodal nuclei in a single protoplast (bar = 30 mm); (E) detail of the chalazal region showing the antipodals at the time of polar nuclei migration to the center of the central cell (bar = 20 mm); (F) mature megagametophyte of the Polygonum-type, with early stage of antipodal cyst formation (bar = 20 mm); (G) post-fertilization stage, with degenerated antipodal cyst at the chalazal end, and the two synergids at the micropylar end (bar = 20 mm); (H and I) early stages of operculum formation in the micropylar end (arrow), before (H) and after fertilization (I) (bars = 20 mm) (arrowhead, cuticle; a, antipodals; ac, antipodal cyst; fm, functional megaspore; h, hypostase; ii, inner integument; oi, outer integument; ow, ovarian wall).
Such result was initially presented for Leiothrix nubigena and Paepalanthus bifidus by Ramaswamy and Arekal (1981a), who questioned the interpretation by Monteiro-Scanavacca and Mazzoni (1978) and offered a different interpretation of the megagametophyte development, which was subsequently confirmed by the same authors (Ramaswamy and Arekal, 1981b, 1982b), and by Scatena and Bouman (2001), Coan and Scatena (2004), and Coan et al. (2007) for other species of different genera of Eriocaulaceae. The formation of the
antipodal cyst can be interpreted as an autapomorphy of Eriocaulaceae within the order Poales, and corroborates the observation of Ramaswamy and Arekal (1981b), who affirmed that this behavior is constant and exclusive of the Eriocaulaceae. Still according to Ramaswamy and Arekal (1981b), this peculiar behavior of the antipodal cells in Eriocaulaceae is definitely related to the nutrition of the megagametophyte, which receives material from the chalazal region or perhaps
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syntheses some of its own. The report of a hypostase in the ovule of L. fluitans, in the present study, should be verified in other taxa of the family, since it may be an argument in favor of the nutritional role of the antipodal cyst in the megagametophyte. In the present study, the cutinized chalazal cells surrounding the antipodal cells, and later the antipodal cyst, was called a hypostase according the definition proposed by Rudall (1997), in a study on the nucellus and chalaza in monocotyledons, following the nutritional function emphasized by Maheshwari (1950) and Tilton (1980). As shown here, in L. fluitans, the hypostase develops early in the megagametophyte development, it is closely associated with the antipodal cyst and both structures recede as the megagametophyte matures. The presence of an endothelium in the ovules of species of Eriocaulaceae was only evidenced in Eriocaulon quinquangulare by Begum (1968) and in L. fluitans by MonteiroScanavacca and Mazzoni (1978). During the ovule ontogeny of the Eriocaulaceae species already studied, which include L. fluitans, here analyzed, the cells of the inner integument do not represent an endothelium because they do not show any histological or functional change that would characterize a typical endothelium, as the one reported in Tabebuia pulcherrima (Bignoniaceae) by Bittencourt and Mariath (2002), for example, or in many other eudicotyledons. In a review of the endothelium in monocotyledons, Swamy and Krishnamurthy (1970) reported the absence of this structure in Eriocaulaceae. The present study followed up the early modifications in the cells of the inner integument that form the micropyle of L. fluitans. They corresponded to what is already known for other Eriocaulaceae species and point the initiation of the micropylar seed operculum. The micropylar operculum seems to be a widespread character in the seeds of Eriocaulaceae, also reported for species of Eriocaulon (Arekal and Ramaswamy, 1980), Syngonanthus (Scatena et al., 1993; Coan et al., 2007), Paepalanthus (Kraus et al., 1996; Coan and Scatena, 2004), Paepalanthus sect. Actinocephalus (=Actinocephalus) (Scatena and Bouman, 2001), and Blastocaulon (Coan and Scatena, 2004). According to Stevenson et al. (2000) and Bremer (2002), this characteristic of the Eriocaulaceae seeds is a synapomorphy supporting the relationships between Eriocaulaceae, Hydatellaceae, Mayacaceae, and Xyridaceae, which, together, form the xyrid clade within Poales. Monteiro-Scanavacca and Mazzoni (1978) described this same structure in the seeds of L. fluitans as a reduced caruncle, but failed in giving further details about its formation. The confirmation of the occurrence of successive microsporogenesis resulting in isobilateral microspore tetrads, of ovules without endothelium, of the formation of a conspicuous antipodal cyst, and of the formation of a micropylar operculum in L. fluitans allows us to characterize the subgenus Rheocaulon as well as the genus Leiothrix within Eriocaulaceae. Similarly, the presence of these features allows us to reaffirm the embryologic uniformity of the family as a whole, strengthening the occurrence of the antipodal cyst as an autapomorphy of Eriocaulaceae among other Poales.
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