茜草科的个体发育

Ovule ontogeny in Rubiaceae (Juss.): Chomelia obtusa (Cinchonoideae–Guettardeae) and Ixora coccinea (Ixoroideae–Ixoreae) K. L. G. De Toni,1 J. E. A. Mariath2 1 Rio de Janeiro Botanical Garden, Rio de Janeiro, Brazil 2 Department of Botany, Biosciences Institute, Federal University of Rio Grande do Sul, Porto Alegre, Brazil Received 4 December 2006; Accepted 12 November 2007; Published online 21 April 2008 � Springer-Verlag 2008 Summary. Ovule development and gynosporogene- sis (megasporogenesis) of two Rubiaceae, Chomelia obtusa (Cinchonoideae–Guettardeae) and Ixora coc- cinea (Ixoroideae–Ixoreae) were studied. Based on the new results it is proposed that the ‘Ixora type’ of ovule development is established because of the convex nucellus and the presence of several arche- sporial cells. This result supports the close relation- ships of Cinchonoideae and Ixoroideae established by molecular systematic studies. Keywords: Rubiaceae; Chomelia obtusa; Ixora coc- cinea; integument; gynosporogenesis; nucellus; ontogeny; ovule Introduction The Rubiaceae, one of the four largest among the angiosperms, consists of 637 genera and 10,700 species (Robbrecht 1988). With the exception of certain groups of Rubieae, Rubiaceae have a basically tropical distribution. The first classification for the family was made by De Candolle (1830). Robbrecht (1988) indicated many gaps in taxonomic knowledge regarding the delimitation of the family and proposed a new classification divided into four subfamilies and 44 tribes. However, because Bremer (1996) recognized the Antirhoideae (sensu Robbrecht 1988) as a paraphyletic group, the present paper considers Chomelia to be in the Cinchonoideae sensu Bremer (1996). Recently, Andreasen and Bremer (2000) have suggested modifications in the Ixoroideae, mainly concerning the position of genera and establish- ment of new tribes, such as Cremasporeae and Ixoreae. In addition to supporting molecular phyloge- netic results, the use of comparative embryology has been important in revealing the relationships among taxa at all levels. Historically, embryological study in Rubiaceae began with Schleiden (1837) and Lloyd (1899, 1902). Fagerlind (1937) made the most precise and significant study in this field, analyzing the evolution of ovule characteristics in this family. In addition, other important works were done by e.g. Andronova (1977), Galati Correspondence: K. L. G. De Toni, Rio de Janeiro Botanical Garden, Rio de Janeiro, Brazil e-mail: karen@jbrj.gov.br Pl Syst Evol 272: 39–48 (2008) DOI 10.1007/s00606-007-0635-x Printed in The Netherlands Plant Systematics and Evolution (1991), Mariath and Cocucci (1997), and De Toni and Mariath (2004). The genus Chomelia has approximately 76 pantropical species (Andersson 1992), of which 50 are located in South America (Steyermark 1974). Chomelia obtusa has been recorded in the northeast, southeast and south of Brazil, espe- cially the Amazon region and the Brazilian Plains (Andersson 1992). The genus Ixora, considered monophyletic by Bremer et al. (1995) and Andreasen et al. (1999), consists of ornamental shrubs and small trees distributed throughout all tropical and subtropical regions of the world (Husain and Paul 1988), includes probably between 200 and 300 species, a few in American tropics, but many more in the Old World tropics, as Ixora coccinea (Fosberg and Sachet 1989). The purpose of this paper is to examine the ovule development and gynosporogenesis of C. obtusa and I. coccinea and discuss the evolution of embryological characters in the Cinchonoideae and Ixoroideae. Materials and methods Floral buds and flowers of C. obtusa Cham. & Schltdl. and I. coccinea L. at different stages of development were collected at the Botanical Gardens of Porto Alegre/RS – Brazil (ICN 120232–120233). The material was first fixed in glutaraldehyde 1% and formaldehyde 4% (McDowell and Trump 1976), in sodium phosphate buffer 0.1 M, pH 7.2. Afterwards, the material was dehydrated in an ethanol series and included in hydroxyethylmethacrylate (Gerrits and Smid 1983). The material was then cut longitudinally with a glass knife in a Zeiss Mikron Microtome, at 2 lm thickness. After the sections had adhered to they in a Leica EMMP heating plate, the slices were stained with Toluidine Blue 0.05% (O’Brien and McCully 1981). Photomicrographs were taken utilizing a Leitz Dialux 20 EB microscope. Results Chomelia obtusa and I. coccinea have a bicar- pellate gynoecium with a bilocular ovary, with one ovule per locule (Fig. 1a, b). In both species the placentation is axial, in C. obtusa the ovule is apically attached to the basal septum (Fig. 1a), and in I. coccinea it is attached in the middle region (Fig. 1b). At maturation, the ovule of these two species is unitegmic and tenuinucellate, with a funicular obturator (Fig. 1b, c). In C. obtusa the ovule is anatropous, while in I. coccinea is hemi-anatropous (Fig. 1b, c). Carpel formation begins after the establish- ment of sepals, petals and stamens (Fig. 1d, e). At this stage, the reproductive apex is slightly flattened (Fig. 1e), and, on the elevated margins, there are periclinal cell divisions, which favor the emergence of the carpel wall (Fig. 1f). Style and stigma are formed, and the placentae with the ovule primordia become pronounced (Fig. 1g, h). Based on the pattern of cell divisions, the placenta has a tri-zonate structure, consisting of the dermal, subdermal and central layers (Fig. 2a). The development of the ovule begins with the periclinal and anticlinal cell divisions of the placental central layer. The dermal and subder- mal layers anticlinal divisions (Fig. 2a). In each ovule, up to eight cells of the subdermal layer are differentiated into archespo- rial cells, with a dense cytoplasm and prominent nucleus (Fig. 2b, d). However, only one of them will mature as a gynospore mother cell (Fig. 2e). At this time, the dermal layer (Fig. 2b, c) under- goes periclinal cell divisions adjacent to the nucellar epidermis (Fig. 2b, d, f), resulting in the formation of the only integument (Fig. 2g–i). The nucellus of C. obtusa and I. coccinea has a flat surface during early development (Fig. 2c) and becomes dome-shaped at maturity, with up to ten elongated cells (Fig. 2d–i). During integument formation the ovule becomes anatropous (Fig. 2h) in C. obtusa, and hemi-anatropous in I. coccinea (Fig. 2e). The integument becomes unusually thick by anticli- nal and periclinal epidermal cell divisions (Fig. 1b, c, 2i). A conspicuous obturator (Fig. 1b, c) covers a considerable part of the ovule. The development of the obturator begins early in ovule develop- ment with a cellular proliferation at the funiculus; it occurs concurrently with integument formation, but originates from central cell layers (Fig. 2b, h). The obturator consists of a tissue with large, 40 K. L. G. De Toni, J. E. A. Mariath: Ovule ontogeny of Chomelia and Ixora (Rubiaceae) Fig. 1. Carpel development in Chomelia obtusa (A, B, D, G) and Ixora coccinea (C, E, F, H). A, C Inferior ovary; the arrows indicate the obturator. B Ovule detail, with obturator indicated by arrow. D, E Early floral stages, calyx (ca), corolla (co), stamens (st) and carpels (cp). F Detail of carpel wall (cw) and placenta (pl). G, H Carpel closure, the carpel wall, septum (spt) and placenta. Scale bar 100 lm; all sections are longitudinal K. L. G. De Toni, J. E. A. Mariath: Ovule ontogeny of Chomelia and Ixora (Rubiaceae) 41 42 K. L. G. De Toni, J. E. A. Mariath: Ovule ontogeny of Chomelia and Ixora (Rubiaceae) thin-walled, polygonal cells, and is thus easily distinguished from the placenta (Fig. 2b). After the integument has been installed, the gynospore mother cell begins the meiotic process (Fig. 3a–e). During meiosis, the volume of the gynospore mother cell increases, presenting a dense cytoplasm and prominent nucleus (Fig. 3a). However, after the first meiotic division, the dyad cells do not increase to the initial volume of the mother cell (Fig. 3b). The cell wall between the dyad cell is impregnated by callose (Fig. 3b). A linear tetrad (Fig. 3c) is formed by the simulta- neous division of the dyad cells. At the end of meiosis, cytokinesis is again accompanied by the impregnation of callose of the transversal and radial gynospore cell (Fig. 3d), except for the chalazal cell, which is functional. The chalazal gynospore matures, while the others degenerate (Fig. 3d, e). Gametophyte development follows the Polygonum type. Discussion As in most Rubiaceae, C. obtusa and I. coccinea have inferior, bicarpellate and bilocular ovaries, with one ovule per locule. The carpel formation pattern of C. obtusa and I. coccinea is the same as that of other uniovulate Rubiaceae. In these the initial concavity of the gynoecium becomes bilocular by the formation of a septum (Fagerlind 1937; Svoma 1991). This uniform development of the gynoecium in the Fig. 3. Chomelia obtusa (A, E) and I. coccinea (B–D) gynosporogenesis. a Prophase I of gynospore mother cell (asterisk). B Dyad; the asterisks indicate the gynospores, and arrow indicates callose in the cell wall. C Linear tetrad (asterisks indicate the gynospores). D, E Functional gynospore (asterisk) and micropylar gynospore degeneration; callose is indicated by arrows. Scale bar 100 lm; all sections are longitudinal Fig. 2. Ovule development in C. obtusa (B, G–I) and I. coccinea (A, C–F). a Young ovule – tri-zonate structure (layers I, II and III). B–D Detail of ovule; the black arrows indicate the nucellar epidermis, the white arrows the integument establishment and the asterisk the archesporial cells. E Ovule at hemi-anatropous stage, evidencing the obturator (obt) and the gynospore mother cell (asterisk). F Gynospore mother cell (asterisk) and the early integument (white arrows). G, H Ovule at anatropous stage, with integument (white arrows), gynospore mother cell (asterisk) and obturator. I Nucellus detail, with nucellar epidermis (black arrow), gynospore mother cells (asterisk) and integument (white arrows). Scale bar 100 lm; all sections are longitudinal b K. L. G. De Toni, J. E. A. Mariath: Ovule ontogeny of Chomelia and Ixora (Rubiaceae) 43 family rules out the use of this character for taxonomic and phylogenetic analysis at infrafa- milial level (Svoma 1991). Most Rubiaceae have bicarpellate ovaries, but in some, one of the locules is partially or completely reduced. The presence of unilocular ovaries was reported in Otiophora (Robbrecht and Puff 1981), Calanda (Puff and Robbrecht 1989), Rutidea (De Block 1995) and Theligonum (Rutishauser et al. 1998). In these genera, there are variations in the degree of reduction of the second carpel: in Otiophora and Calanda, with remnants of it, and in Theligonum, as an incomplete septum forma- tion; i.e. the ovary is bilocular in the lower region. Knowledge on ovule development in Rubia- ceae is fragmentary. There are only a few data about some species of the Cinchonoideae and Rubioideae. The ovule primordium is formed through periclinal divisions of the second or third layer of the placenta, depending on whether the placenta has two or three cell layers: bi-zonate or tri- zonate (Bouman 1984). According to Bouman (1984), there is probably a relationship between the placental structure and the size of the ovules and seeds: tri-zonate primordia forming large ovules, with a large funiculus and nucellus, as compared to di-zonate primordia (Bouman 1984). However, this is not the case in Rubiaceae, in which there is a trend towards smaller ovules and seed structures in tri-zonate ovules, as in Relbu- nium hypocarpium (Mariath and Cocucci 1997). The integuments of different taxa of angio- sperm ovules show great diversity in their origin and development (Bouman 1984). According to Bouman, the inner integument in bitegmic ovules, and the single integument in unitegmic ones, generally has a dermal origin, except in some Euphorbiaceae, in which their origin is subdermal. The origin of the outer integument is a taxonomic character at the order or family level in some groups. Most Monocotyledons, Saxifra- gales and Geraniales have an outer integument which is dermal in origin. Warming (1878) states that, in sympetals, the single integument is mainly derived from the dermal layer. However, Bouman and Schier (1979) claim that, in uniteg- mic sympetals, the integument does not always originate only from the dermal zone, but the subdermal cell layer may participate. In C. obtusa and I. coccinea, as in other Rubiaceae the integument is of dermal origin. The integument is formed by periclinal cell divisions of the dermal layer, forming a ring around the nucellus, such as in the following species: Callipeltis cucullaris, Rubia tinctorum (Lloyd 1899, 1902); Asperula humifusa, A. molluginoides, A. odorata, A. prostrata, A. setosa, Galium boreale, G. pa- lustre, G. uliginosum, G. verum, Phuopsis styl- osa, Cruciata laevipes (Andronova 1988); Borreria brachystemonoides, B. eryngioides, B. nelidae, B. spinosa, B. sulcata, Diodia brasil- iensis, D. dasycephala, D. schumannii, Galianthe fastigiata, G. laxa, Mitracarpus hirtus, M. mega- potamicus, Richardia brasiliensis, Spermacoce tenuior, Spermacoceodes glabrum, Staelia thymoides (Galati 1991); Relbunium hypocarpi- um (Mariath and Cocucci 1997) and Borreria verticillata (De Toni and Mariath 2004). Chomelia obtusa and I. coccinea have a funicular outgrowth, a structure which is com- mon among bitegmic and anatropous ovules (Bouman 1984; Johri et al. 1992). In Prismato- merideae–Rubioideae, a similar structure has been described as a ring-shaped obturator that covers most of the ovule (Igersheim and Robbrecht 1993). In Rubiaceae, the terms obtu- rator, strophiole and aril, however, are not uniformly used. Strophiole and aril are used for the same structure, and several authors consider them structures homologous to the obturator (Lloyd 1899; Netolitzky 1926; Hayden 1968). This indiscriminate use of different terms, when referring to the same structure, makes it difficult to understand the morphology and ontogeny of the organ. This structure is found in Richardsonia and Diodia (Lloyd 1899, 1902), Borreria hispida (Farooq 1959), Hydro- phylax maritima (Ganapathy 1956), and Guet- tarda speciosa (Raghavan and Srinivasan 1941). In Richardia brasiliensis covers the whole funicular side of the ovule and extends to the micropyle (Inamuddin and Farooq 1984). In I. burundiensis and Pavetta oliveriana, analyzed by De Block (1995), the ovule is similar to that found in I. coccinea, in which the obturator is 44 K. L. G. De Toni, J. E. A. Mariath: Ovule ontogeny of Chomelia and Ixora (Rubiaceae) proeminent and developed near the micropylar end and the chalazal side of the ovule. With respect to the tenuinucellate ovules, two variations in the nucellar epidermis are recog- nized: typical, when the epidermis embraces the gynospore mother cells (as in most of the asterids); or reduced, when the nucellar epidermis does not embrace the gynospore mother cell and consists of only a few cells, as seen in the Olacaceae, Rubiaceae and Apocynaceae (Shamrov 1998). Fagerlind (1937) proposed three types of nucellus for Rubiaceae: ‘typical’, ‘reduced’, and ‘naked’ (i.e. ategmic). In C. obtusa and I. coccinea, as in species of Phyllis, Psychotria, Cephalanthus, Hoffmannia and Chiococca (Fagerlind 1937), Coffea (Faber 1912), Ophiorrhiza, Mussaenda, Fadogia and Guettarda (Raghavan and Srinivasan 1941), Rondeletia (Shivaramiah and Dutt 1964) and Knoxia (Shivaramiah and Ganapathy 1961), the nucellus is typical, in contradiction to Shamrov (1998), who classified Rubiaceae as belonging to the reduced nucellus type. The morphological variation in the three nucel- lar types of Rubiaceae suggest a derivation from typical tenuinucellate ovules towards reduced, and then naked ones (Mariath and Cocucci 1997). Ixora coccinea and C. obtusa have a convex nucellus with a relatively high number of epidermal cells. An example with ‘naked’ ovules is Houstonia (Fagerlind 1937). ‘Reduced’ type ovules are pres- ent in Relbunium (Mariath and Cocucci 1997) and Borreria (De Toni and Mariath 2004). Fig. 4. Diagrams of ovule evolution proposed for the Rubiaceae (based on Fagerlind 1937, with additions) K. L. G. De Toni, J. E. A. Mariath: Ovule ontogeny of Chomelia and Ixora (Rubiaceae) 45 Rubiaceae are considered as monophyletic by several authors (Verdcourt 1958; Bremer 1996). The molecular analyses performed by Bremer et al. (1995), Bremer (1996), Bremer and Thulin (1998) and Andersson and Rova (1999) established three subfamilies – Cincho- noideae, Ixoroideae and Rubioideae. Molecular and morphological data suggest two derived lines from a common ancestor, one of them representing Rubioideae and the other one Cinchonoideae and Ixoroideae. The ovule of the Rubiaceae ancestor was, probably, bitegmic and tenuinucellate, with a nucellar dome-shaped epidermis, a multi-cellular archesporium and a funicular obturator (Fig. 4). During the course of the differentiation process, starting with the hypothetical ancestral species, successive reductions of the ovule occurred. These include the following changes: disappear- ance of the outer integument; flattening of the nucellar epidermis (i.e. from dome-shaped to a flat surface); reduction of the archesporium; elongation of the nucellar epidermis; substitution of the epidermis consisting elongated cells by parenchymatous tissue; and, finally, variations in the placental pattern. When we compare the ovule of C. obtusa and I. coccinea with the species already studied by Fagerlind (1937), Andronova (1977), Galati (1991) and Mariath and Cocucci (1997), is somewhat similar to the Phyllis type (sensu Fagerlind 1937). However, in the lack of an integument and an enhanced number of arche- sporium cells, it differs from the Phyllis type. We propose to establish a new type, the Ixora type, for the species here studied, C. obtusa and I. cocci- nea. These species show similar ovule ontogeny characteristics, as described above, except for the placentation. In C. obtusa the placenta is attached to the top of the basal septum, and in I. coccinea the insertion is lower, characterizing an interme- diate level between the apical and basal insertion. De Block (1998), for the African species of Ixora, mentions that the placenta is attached to the top of the basal septum and is therefore inserted above the middle of the locule, and in I. coccinea the position is not the same, because the placenta is localized in the middle of the septum. Notwithstanding the evidence, as noted above, the ovule ontogeny characteristics are still insuf- ficient to enable differentiation between the Ixoroideae and Cinchonoideae subfamilies, unless the placentation and obturator characters are considered. Thus, a more detailed and probing analysis of related genera is required and could very well clarify the weight of these two characters in schemata describing new evolutionary trends. The authors thank CAPES for the graduate grant provided to the first author and CNPq for the research grant provided to the second author. In addition, we thank the Plant Anatomy Laboratory, Botany Department/UFRGS, for research support. References Andersson L (1992) A provisional checklist of neotropical Rubiaceae. Scr Bot Belg 1: 1–199 Andersson L, Rova JHE (1999) The rps 16 intron and the phylogeny of the Rubioideae (Rubiaceae). Pl Syst Evol 214: 161–186 Andreasen K, Bremer B (2000) Combined phyloge- netic analysis in the Rubiaceae–Ixoroideae: mor- phology, nuclear and chloroplast DNA data. Amer J Bot 87: 1731–1748 Andreasen K, Baldwin BG, Bremer