Nordic Journal of Botany 30: 215225, 2012 doi: 10.1111/j.1756-1051.2011.01151.x, 2012 The Authors.

. Nordic Journal of Botany 2012 Nordic Society Oikos Subject Editor: Brita Stedje. Accepted 11 March 2011 Date of publication: 20 April 2012

Embryology of the dioecious Woonyoungia septentrionalis (Magnoliaceae)

Lin Fu, Feng-Xia Xu and Qing-Wen Zeng
Lin Fu, Feng-Xia Xu and Qing-Wen Zeng (zengqw@scbg.ac.cn), South China Botanical Garden, the Chinese Academy of Sciences, CN-510650 Guangzhou, PR China.

The embryological characteristics and ovular integument development of the dioecious species Woonyoungia septentrionalis (Dandy) Law (Magnoliaceae), which are poorly understood, were investigated under laser scanning confocal microscope (LSCM) and light microscope (LM). The embryological characteristics conform to most of the previously studied species in Magnoliaceae. The anther has 4 microsporangia, and the anther wall develops according to the dicotyledonous type. Cytokinesis at meiosis of the microspore mother cells follows a modied simultaneous type, giving rise to isobilateral or decussate tetrads, and a cell plate is absent, but a membrane was observed. Mature pollen grains are 2-cellular and have high germination rates. The ovule is anatropous, crassinucellate and bitegmic, and meiotic result in linear tetrads of megaspores, the one at the chalazal end functions directly as an embryo-sac cell. The development of the embryo sac is of the Polygonum-type and endosperm formation is of the nuclear type. The outer integument of the ovule dierentiates into an outer eshy and an inner stony layer while the inner integument is reduced to a tanniniferous layer. The normal embryological development, high germination rates of pollen and high seed set indicate that the primary reason for the decline of the species is not to be found in these developmental processes.

Woonyoungia septentrionalis (Dandy) Law, a rare and critically endangered species (Fu 1992), is the only species of the genus Woonyoungia Law. The species was rst described by Dandy (1931) as a species of Kmeria Dandy, based on the specimen R. C. Ching 5247, which was collected in Guangxi, China. It was seggregated from Kmeria Dandy by Law (1997) whose classication is adopted in this paper, based on its unisexual and dioecious owers, subglobose fruits of connate leathery carpels dehiscing longitudinally on the dorsal side (Law 1997). Although Woonyoungia has been accepted as a genus of its own, there is still disagreement about its systematic position (Nooteboom 2000, Kim et al. 2001, Figlar and Nooteboom 2004, Lin et al. 2005). Kim et al. (2001) found that Kmeria was closely related to Magnolia fraseri based on molecular evidence. Figlar and Nooteboom (2004) assumed that Kmeria (including Woonyoungia septentrionalis) was derived from Magnolia, and did not merit generic rank. Figlar and Nooteboom (2004) followed Kim et al. (2001) and argued that Kmeria should be included the genus Magnolia. Lin et al. (2005) thought W. septentrionalis should not be separated as a distinctive genus, but rather belonged to Kmeria based on morphological characters. Because of limitied knowledge, its systematic position has not yet been well understood.

Being a dioecious species W. septentrionalis is of special interest in the systematics and conservation biology of Magnoliaceae. It has been studied with respect to vascular anatomy (Zhang et al. 2000), pollen morphology (Xi et al. 2000, Zhang 2007, Xu and Kircho 2008), systematic classication (Lin et al. 2005), population characters (Dong et al. 2009), karyology (Meng et al. 2006). Zeng et al. (2003) and Wang et al. (2004) reported that W. septentrionalis showed facultative apomixis, but little is known about its reproductive structures. Embryology has been studied in several species of Magnoliaceae. Megasporogenesis and development of female gametophytes have been examined in the genus Magnolia (7 species), Manglietia (5 species), Michelia (4 species), Liriodendron (1 species), Tsoongiodendron (1 species). Microsporogenesis and development of male gametophytes of Magnolia (10 species), Manglietia (4 species), Michelia (4 species), Liriodendron (1 species), Tsoongiodendron (1 species) have also been described (Maneval 1914, Earle 1938, Padmanabhan 1960, Kaeiser and Boyce 1962, Hayashi 1964, 1966, Kapil and Bhandari 1964, Davis 1966, Fan et al. 1992, Qin and Li 1996, Liao et al. 2000, Pan and Gong 2002, Pan et al. 2003, Tang et al. 2003, Xiao and Yu 2004, Wang et al. 2005, Xiao and Xu 2006, Zhao and Sun 2009). In this paper, the embryology and the

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development of the fruit of W. septentrionalis are investigated to reveal similarities and dierences between it and other magnoliaceous species, and better understand the evolutionary trends.

Material and methods

Material of W. septentrionalis (Dandy) Law was collected from 29 Mar to 15 Aug 2007, from cultivated material in the Magnolia Garden at SCBG, Guangdong Province, China. Voucher specimens were deposited in IBSC. Floral buds of all developmental stages were collected every 24 days, and bracts were removed. The samples were xed in FAA (70% ethyl alcohol/glacial acetic acid/ formaldehyde 90/5/5%). Both the traditional paran microtome sectioning technique and the whole clearing and staining technique were used. Material treated with the whole clearing and staining technique could be observed under the LSCM directly. It is easier and less time-consuming to treat the material and sample artifacts could be avoided. However, material at later developmental stages was too large to be observed under LSCM, therefore, the paran section technique was used for seeds longer than 5 mm. For LM observation, the material was stained in Ehrlichs haematoxylin, dehydrated in an ethyl alcohol series, inltrated and embedded in paran and sectioned at 310 m on a Leica RM 2016 rotary microtome. All sections were observed and photographed under an Olympus AX70 lighting microscope (LM) with an Olympus DP50 digital camera. For LSCM observation, the material was washed 6 times in double distilled water, dehydrated in an ethanol series (30, 50, 70, 90 and 95%), and stained for 20 h in dye liquid (0.03 ml 50% eosin 0.01 ml 5% orange G 9.96 ml 95% ethanol). The following day, the stained material was washed 6 times in double distilled water, dehydrated in an ethanol series (30, 50, 70, 90, 95 and 100%). Then the material was xed in the solution (pure methyl salicylate/ pure ethanol 1/1) for 3 h, washed in pure methyl salicylate 3 times, and stored in pure methyl salicylate. The material was observed under a Zeiss 510 meta laser scanning confocal microscope (LSCM). For pollen germination tests, male owers were taken into the lab before anther dehiscence. Until pollen was released, they were taken into dierent germination media in Petri dishes: sucrose and boric acid combinations (1) sucrose 10% 20 ppm H3B03; 2) sucrose 15% 20 ppm H3B03; 3) sucrose 20% 20 ppm H3B03; 4) sucrose 30% 20 ppm H3B03). Pollen grain counting was performed after 6 h incubation at 25C and the experiment was repeated 6 times.

SCBG. Tepal numbers of both male and female owers are unxed. In male owers tepals are mostly in 1 whorl, usually 3, but varying from 2 to 6, subequal, white, obovate or elliptic; androecia are creamy white, obovoid; stamens are 80150 with linear and laterally dehiscent anthers (Fig. 1A). Female owers have 24 white tepals in one whorl, which are obovate, 2.53.0 cm long and 2.02.5 cm broad, 613 linear-oblanceolate sterile stamens which previously have been regarded as inner whorl tepals and 49 obovoid carpels, each with 2 (rarely 3) ovules, the stigmas have ligulate short appendages (Fig. 1BD). Microsporogenesis and male gametophyte formation
Formation of anther and anther wall

Results
Floral morphology Flowers are unisexual, dioecious, solitary and terminal, rarely axillary. The proportion of female/male plants is 1/3 in the cultivated population in the Magnolia Garden of

In the male owers, the stamens are initiated in the last ten days of March (Fig. 1A). The stamens are tightly spaced so that they form regular hexagons in transverse section (Fig. 2A). The hypodermal layer of each of the four young anther lobes contains a large archesporial cell with a big nucleus and homogeneous cytoplasm (Fig. 2B). It divides periclinally into a primary parietal cell and a primary sporogenous cell (Fig. 2C). The primary sporogenous cell gives rise to several secondary sporogenous cells by successive mitosis (Fig. 2D, E), and the secondary sporogenous cells divide into microspore mother cells (Fig. 2F). The primary parietal cell divides periclinally into two secondary parietal layers (Fig. 2D), of which the outer layer divides again, giving rise to the middle layer and the endothecium (Fig. 2D, E), while the inner layer transforms into the tapetum layer directly (Fig. 2E). The cells of the rst middle layer divide 24 times to form 35 middle layers (Fig. 2E, F). All parietal cells are similar in size during the early development stage. Anther wall structure is uniform among the studied samples, including an epidermis, an endothecium, 35 middle layers, and one tapetum layer (Fig. 2G). Thus, the anther wall formation conforms to the dicotyledonous type. At meiosis the innermost cell layer of the anther wall adjacent to the tapetum degenerates and becomes compressed (Fig. 2F). The tapetum cells enlarge with irregular size, dense cytoplasm and big nucleus (Fig. 2G, H). When the microspores are released from the tetrads, the innermost cell layer of the anther wall disappears or remains inconspicuous, while the endothecium cells enlarge and develop brous thickenings (Fig. 2I, L). The cytoplasm of the tapetum cells remains in its original position and thus the tapetum is of the glandular type. Towards pollen maturity, the tapetum disappears and only the endothecium and small-celled epidermis remain as anther wall layers.
Microsporogenesis and formation of the male gametophyte

In the stamens, about 13 mm long, the chromosomes align in a single row in the equatorial plate in the center of the microspore mother cells (Fig. 3A), then they move to the

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Figure 1. Flowers and fruits of Woonyoungia septentrionalis. (A) male ower with 3 tepals, (B) female ower with 3 tepals, (C) young fruit, (D) mature fruit with the red seeds hanging on.

poles and undergo a second replication (Fig. 3B, C). After replication, the two groups of chromosomes at the poles each align in a single row again (Fig. 3D, E). The second separation of the chromosomes varies with respect to the relative position of the two spindles. If the two spindles are arranged perpendicularly to each other in the two planes (Fig. 3D, F), the result is a decussate tetrad (ca 42%, Fig. 3L, M). If they are arranged in parallel in the same plane (Fig. 3GI), the result is an isobilateral tetrad (ca 58%, Fig. 3J, K). The quadripartition of the microspore mother cells is by furrowing, a process rarely found in dicotyledons. After the rst separation of the chromosomes, callose produces a furrow in the center of the microspore mother cells without formation of a cell plate (Fig. 3BF), while in a few cases, a membrane forms at the position where the cell plate would be expected (Fig. 3B, G). The furrowing continues develop until the four nuclei are formed, and nally lead to the quadripartition of the microspore mother cell. Cytokinesis is of the modied simultaneous type which was rst reported by Farr (1918).

When released from a tetrad, the young microspore has no conspicuous vacuole but dense cytoplasm and irregular shape (Fig. 2I, 3N). But later it becomes vacuolated and nally developes a large vacuole (Fig. 3O). The dense cytoplasm is then conned to the cell wall (Fig. 3P). The chromosomes rst arrange in the plate (Fig. 3Q), and then move to the poles (Fig. 3R), resulting in a large vegetative nucleus and a small reproductive nucleus. An arch-shaped cell wall is formed between the two nuclei, resulting in a large vegetative cell and a small reproductive cell (Fig. 3S), which gradually enters the cytoplasm of the former (Fig. 3T). The generative cell does not divide into two sperms and the pollen is 2-celled before anther dehiscense.
Pollen germination rates

To investigate the pollen viability as well as pollen germination capability, in vitro pollen germination were tested. Results show that the germination rate varies between 75.5 and 91.2%, with an average of 82.8%. The germination rate is highest in the 20% sucrose medium and lowest in the 10% sucrose medium (Table 1). The high pollen germination 217

Figure 2. Microsporogenesis of Woonyoungia septentrionalis. (A) transverse section of group of young hexagonal stamens (10 Mar), (B) archesporial cell (arrow, 10 Mar), (C) archesporial cell divided into primary sporogenous cell and primary parietal cell (arrows, 10 Mar), (D) primary sporogenous cell divided into secondary sporogenous cell, and primary parietal cell divides periclinally into two secondary parietal layers (arrows, 15 Mar), (E) outer layer of secondary parietal cells divided into middle layer and endothecium, while the inner layer transforms into the tapetum (arrows, 15 Mar), (F) mature microspore mother cells before meiosis (22 Mar), (G) anther wall of tetrad stage, showing tapetum and degenerated middle layer (22 Mar), (H) degenerated tetrads (5 Apr), (I) microspores released from tetrads (16 Apr), (J) young microspores (16 Apr), (K) degenerated microspores, and large tapetum cells (1 May), (L) mature anther, showing the dehisced theca and the anther wall (10 May). All panels are transverse sections of the stamens. Scale bars 50 m. ARC archesporial cell; BT binucleate tapetum cell; DP degenerated pollen; DTE degenerated tetrad; EN endothecium; ML middle layer; MMC microspore mother cell; MP mature pollen; PP primary parietal cell; PS primary sporogenous cell; SP secondary parietal cell; SS secondary sporogenous cell; TA tapetum; TE tetrad.

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Figure 3. Meiosis and male gametophyte formation of Woonyoungia septentrionalis. (A) metaphase of meiosis I, chromosomes aligning in a single row in the metaphase plate, (B) anaphase of meiosis I, separation of chromosomes and membrane formation (arrowhead), (C) prophase and metaphase of meiosis II, showing asynchronous meiosis, (D)(F) telophase of meiosis II, showing furrowing of the cytoplasm and the formation of decussate tetrads, (G) metaphase of meiosis II, chromosomes in the plates and membrane, (H)(I) telophase of meiosis II, furrowing of cytoplasm and the formation of isobilateral tetrads, (J)(K) isobilateral tetrads, (L)(M) decussate tetrads, (N) microspore without large vacuole, (O) microspore with large vacuole, nucleus clinging to the cell wall, (P) microspore before mitosis, dispersion of the chromatin, (Q) mitosis metaphase of nucleus, (R) mitosis anaphase of nucleus, (S) bicellular pollen grain with cell plate, (T) mature pollen grain. Scale bars 20 m.

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Table 1. Pollen germination rates in Woonyoungia septentrionalis. Concentration of sugar (%) (W/V) 10 15 20 30 Germination rate (%) 75.5 5.6 83.4 6.4 91.2 4.9 80.8 5.8

rates show that pollen germination capacity is not a reproductive barrier. Megasporogenesis and female gametophyte formation
Megagametogenesis

The ovules are initiated as dome-like protuberances on the lateral margin of the carpellary wall (Fig. 5A). A hypodermal cell of the nucellus tip dierentiates into an archesporial cell in early April (Fig. 4A), which is easy to distinguish by its large size and conspicuous nucleolus (Fig. 5B). The archesporial cell divides periclinally at rst and forms an outer parietal cell and an inner sporogenous cell (Fig. 4B). The inner sporogenous cell functions as a megaspore mother cell (Fig. 4C). Meiosis results in a linear tetrad (Fig. 4D). Only the chalazal megaspore is functional and the other three degenerate and disappear (Fig. 4D). The functional megaspore enlarges quickly and undergoes the rst nuclear division (Fig. 5E). The two daughter nuclei move apart to the two ends of the embryo sac and divide once again to form a four-nucleate embryo sac (Fig. 4F), and a third round of divisions leads to an eight-nucleate embryo sac with four nuclei in each end. Two nuclei, each from one end, move into the center of the embryo sac and fuse to form a secondary nucleus (Fig. 4G, L). Among the remaining six nuclei, three nuclei at the micropylar end constitute the egg apparatus including an egg cell and two synergid cells (Fig. 4H, I), while the other three at the chalazal end become antipodal cells, which degenerate before fertilization (Fig. 4J, K).
Fertilization, embryogeny and endosperm development

relatively long and forms a stalk (Fig. 5B, C). The outer integument is initiated on the lateral portions of the primordium by periclinal cell divisions in the subdermal layer, while the inner integument is initiated later (Fig. 4A). The inner integument becomes annular around the nucellus (Fig. 5C, D). The elongating outer integument overgrows the inner integument gradually and the ovule becomes curved at the megaspore mother cell stage (Fig. 5E). The ovule becomes anatropous with a zigzag micropyle (Fig. 5F). At this stage, the ovule is heart-shaped with two distinct integuments in longitudinal view (Fig. 5G). The outer integument consists of more than 10 layers of parenchymatous cells, of which the inner epidermal cells are more compressed and more densely stained than the outer ones, while the inner integument consists of 23 layers of compressed cells (Fig. 5H). Most notably, the inner epidermis of the inner integument is composed of tanniniferous cells, which have not been reported in other Magnoliaceae species (Fig. 5H). After fertilization, the integuments develop into a seed coat. The inner integument, except for the tanniniferous cells, becomes crushed (Fig. 5I, J), persisting only in the micropylar region, where it forms a tissue that plugs the micropyle (Fig. 5K). In some seeds, the inner integument at the micropylar region is crushed rst, and the embryo sac protrudes into the micropylar region (Fig. 5L). The cells of the inner epidermis of the outer integument stretch greatly and divide periclinally (Fig. 5M), giving rise to a multi-layered lignied stony tissue. The other layers of the outer integument are parenchyma and develop into a eshy layer including the vascular bundle (Fig. 5N, O). After the disappearence of the outer epidermis of the inner integument, the tanniniferous cells keep enlarging (Fig. 5M, O), But are nally crushed at the dicotyledonous embryo stage, leaving only a dark line (Fig. 5P). At this time, the endosperm is full of starch grains.
Seed set

Fertilization occurs in mid-May at the end of the owering period. Pollen tubes enter the ovule through the micropyle. In the embryo sac, one of the two gametes fuses with the egg cell to form the zygote, the other one fuses with the central cell nucleus to form the primary endosperm nucleus (Fig. 4M, N). The primary endosperm nucleus divides quickly to form many free nuclei before cell walls are laid down between the free nuclei (Fig. 4O, 5I). Thus, endosperm development is of the nuclear type. The zygote divides transversely at a time when already many endosperm cells are formed, resulting in an apical cell and a basal cell (Fig. 4P, Q). The apical and basal cell divide several times (Fig. 4R), forming a globular-shaped proembryo (Fig. 4S), before an embryo with two cotyledons is formed (Fig. 5P). The embryo development is of the Onagrad-type.
Development of the ovular integuments

Twenty fruits from each of four individual trees were studied. The seed set of the fruits varies between 33.3 and 81.9%, with an average of 65.4%.

Discussion
Comparison of megasporogenesis and female gametophyte formation Megasporogenesis and female gametophyte formation in W. septentrionalis is similar to those of other previously studied species in Magnoliaceae: the ovule is anatropous, crassinucellate and bitegmic; meiosis results in a linear tetrad of megaspores, of which the one at the chalazal end functions as a megaspore mother cell and develops into a Polygonum-type embryo sac (Maneval 1914, Earle 1938, Kaeiser and Boyce 1962, Hayashi 1964, Pan and Gong 2002, Pan et al. 2003, Tang et al. 2003). The only exception is an Allium-type embryo sac in Manglietia decidua (Xiao and Xu 2006).

The ovule primordia are initiated as dome-like protuberances on the carpel margins (Fig. 5A). The funiculus is 220

Figure 4. Development of the ovule, embryo sac and embryo of Woonyoungia septentrionalis. (A) Young ovule with an archesporial cell and two young integuments (10 Mar), (B) young ovule with a primary parietal cell and a primary sporogenous cell (10 Mar), (C) young ovule with a megaspore mother cell and developing integuments (15 Mar), (D) linear tetrad of megaspores including a functional megaspore and three degenerated megaspores on the micropylar side (22 Mar), (E) two-nucleate stage of embryo sac, showing two cells, one at the chalazal end and one at the micropylar end (22 Mar), (F) four-nucleate stage of embryo sac, showing two cells at the chalazal end and two cells at the micropylar end (5 Apr), (G) eight-nucleate stage of embryo sac, the two polar nuclei moving to the center of the embryo sac (13 Apr), (H) eight-nucleate stage of embryo sac, showing the egg and two synergid cells (16 Apr), (I) eight-nucleate stage of embryo sac, showing the degenerating synergid cells (16 Apr), (J) eight-nucleate stage of embryo sac, showing three antipodal cells at the chalazal end (16 Apr), (K) eight-nucleate stage of embryo sac, showing three degenerating antipodal cells (20 Apr), (L) eight-nucleate stage of embryo sac, showing the two polar nuclei in the process of fusion (20 Apr), (M) fertilization of the secondary nucleus (27 Apr), (N) detail of M (27 Apr), (O) the early development of the endosperm (10 May), (P) two-celled proembryo (15 May), (Q) two-celled proembryo (15 May), (R) four-celled proembryo (20 May), (S) globular proembryo (1 Jun). All panels are vertical sections of the ovules. The micropylar side is to the right or downwards in all gures. Scale bars 20 m. ac antipodal cells; arc archesporial cell; ec egg cell; e embryo; emb embryo sac; end endosperm; eshy layer; fm functional megaspore; ii inner integument; mmc megaspore mother cell; ob obturator; oi outer integument; pen primary endosperm nucleus; pn polar nuclei; pp primary parietal cell; tc tanniniferous cell; sc synergid cells; sl stony layer; sp sporogenous cell; z zygote.

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Figure 5. Development of the ovule, embryo sac and the integuments of Woonyoungia septentrionalis. (A) ovule primordia forming dome-like protuberances on the carpel margins (10 Mar), (B) young ovule with a large archesporial cell (10 Mar), (C) young ovules, showing the two developing integuments (10 Mar), (D) young ovule, with the two integuments (15 Mar), (E) young ovule, showing the outer integument overgrowing the inner one (15 Mar), (F) the outer integument reaches the obturator, forming the micropyle (5 Apr), (G) heartshaped ovule, showing the two integuments (27 Apr), (H) detail of G (27 Apr), (I) development of the seed coat, showing the taniniferous cell layer, two-layered inner integument and thick outer integument (10 May), (J) detail of I, (K) inner integument remains at the micropylar end (10 May), (L) inner integument crushed in the micropylar area (10 May), (M) the two-layered inner integument disappearing while the cells of the tanniniferous layer and inner epidermis of the outer integument become larger and compressed (10 Jun), (N) tanniniferous cells remain in one layer while the inner epidermis of the outer integument divide periclinally, giving rise to the multicell-layered stony layer (10 Jul), (O) cells in the tanniniferous layer and the stony layer enlarged (20 Jul), (P) embryo with two developing cotyledons (15 Aug). All panels are vertical sections of the ovules. Micropylar side is to the right or upwards or downwards in all gures. Scale bars 50 m. ac antipodal cells; arc archesporial cell; ec egg cell; e embryo; emb embryo sac; end endosperm; eshy layer; fm functional megaspore; ii inner integument; mmc megaspore mother cell; ob obturator; oi outer integument; pen primary endosperm nucleus; pn polar nuclei; pp primary parietal cell; tc tanniniferous cell; sc synergid cells; sl stony layer; sp sporogenous cell; z zygote.

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Endosperm formation In most examined Magnoliaceae species, the endosperm formation is of cellular type in which a transverse wall divides the endosperm into two chambers after the rst division of the primary endosperm nucleus (Maneval 1914, Padmanabhan 1960, Hayashi 1964, 1984, Kapil and Bhandari 1964, Davis 1966, Xiao and Xu 2006). However, studies in Magnolia grandiora (Earle 1938), Manglietia glauca and Michelia guangxiensis (Liao et al. 2000) found a nuclear endosperm but with a comparatively small number of free nuclei before the onset of wall formation (Earle 1938). The observation that Woonyoungia septentrionalis has a nuclear endosperm conrms that both cellular- and nuclear-type endosperm developments occur in this family. As the two endosperm formation types coexist in Magnolia, Manglietia and Michelia, further studies should be carried out to determine the dominating type. Formation of microspore tetrads In Magnoliaceae, quadripartition of microspore mother cells is the result of furrowing, and the tetrads are formed by a modied simultaneous type, which is rarely found in basal angiosperms and eudicots and considered to be a type intermediate between the simultaneous and successive types (Hayashi 1960). Farr (1918) rst reported that cytokinesis takes place by furrowing in Magnoliaceae, but Yasui (1937) argued that there is no important dierence between cytokinesis by cell plate formation and by furrowing, the only point of dierence being that in the former the thickening of the cell plate wall is conspicuous, but not in the latter. From the present study, there is a membrane formed during the furrowing of tetrads in some cases (Fig. 3B, G), indicating that furrowing does not exclude the formation of a cell plate during the cytokinesis. Hayashi (1960) also reported membrane formation in M. liliora. Membrane formation in W. septentrionalis corroborates the view of Yasui (1937). It also shows that cytokinesis in W. septentrionalis is similar to that of Magnolia species and more dierent from Manglietia and Michelia. As most of the embryological characters of W. septentrionalis are similar to those of the previously studied Magnolia species, W. septentrionalis is considered to be closely related to Magnolia. Initiation and development of the ovular integuments In angiosperms, ovules of most species are bitegmic, but some species have ovules with only an inner integument, or an uncompleted (hood-shaped) outer integument, e.g. in Cabombaceae, Austrobaileyaceae, Trimeniaceae, Schisandraceae, Winteraceae, Degeneriaceae, Annonaceae (Endress and Igersheim 1997, Igersheim and Endress 1997, 1998). Eames (1961) and Cronquist (1988) viewed the cupular outer integument as primitive and the hood-shaped outer integument as derived. In contrast, Matsui et al. (1993) and Umeda et al. (1994) interpreted the anatropus ovule with hood-shaped outer integument to be primitive.

The ovules in Magnoliaceae are bitegmic and anatropous, which is a common pattern in basal angiosperms and is usually considered to be primitive in owering plants as a whole (Stebbins 1974, Cronquist 1988, Takhtajan 1991, Endress and Igersheim 2000, Endress and Doyle 2009), but the initiation sequence of the two integuments appear to dierer among species. In Magnolia liliiora, Magnolia championii, Manglietia glauca var. sumatrana, Michelia guangxiensis and Michelia fuscata, the inner integument is initiated before the outer one (Hayashi 1964, Liao et al. 2000, Wang et al. 2005). In Liriodendron tulipifera, the situation is either the same or both integuments are initiated almost simultaneously (Maneval 1914, Kaeiser and Boyce 1962, Matsui et al. 1993). In contrast, in Magnolia grandiora the outer integument is initiated earlier than the inner one (Umeda et al. 1994). Thus, Woonyoungia septentrionalis resembles M. grandiora in this respect. Further comparative studies are needed to obtain a more comprehensive understanding of integument formation. Structure of the seed coat Seed coats of Magnoliaceae have been described as threelayered, including exotesta, mesotesta and endotesta, but there are dierent views on their formation. Maneval (1914), Earle (1938), Kapil and Bhandari (1964) assume that the outer integument dierentiates into outer eshy and inner stony layers, while the inner integument is reduced to a membranous layer. Xiao and Xu (2006) support this interpretation of the fate of the outer integuments, but assume that the inner integument does not contribute to the formation of the seed coat, but is crushed and persists only at the micropylar end. However, according to Fan et al. (1992) the inner integument becomes the hard layer, together with the inner epidermis of the outer integument. Our present results agrees with Maneval (1914), Earle (1938), Kapil and Bhandari (1964), and shows that there is a membranous layer derived from the inner layer of the inner integument (Fig. 5IP). Law et al. (2004) also reports a membranous structure around the embryo sac, but does not state that the membrane is tanniniferous. The layer derived from the inner integument contains large tanniniferous cells and protect the embryo sac together with the stony layer. This tanniniferous layer persists as an independent layer up to seed maturity, and should thus be considered as a distinct layer of the seed coat. The degeneration phenomenon Several studies have shown that degeneration of reproductive cells, including the microspore mother cells, megaspore cells, tetrads, embryo sac and pollen is common in the embryonic development in many magnoliaceous species. Fan et al. (1992) and Qin and Li (1996) reported that sterility of the ovule and female gametophyte were important factors limiting the reproduction of Liriodendron chinense, and good environmental conditions increased fertility and seed set. Liao et al. (2000) found that in Michelia guangxiensis and Manglietia glauca var. sumatrana, abnormal 223

development is very common in the development of the dyad, tetrad, functional megaspore and ovule. In Manglietia insignis and Manglietia aromatica, the degeneration ratio of egg cells is up to 79.1% and 80%, respectively, and the degeneration ratio of functional megaspores is 27.9% in Manglietia aromatica (Pan et al. 2002, 2003). There is also often degeneration during the development of the tetrad, mature embryo sac and ovules in Tsoongiodendron odorum (Tang et al. 2003), Magnolia biloba (Wang et al. 2005) and Michelia coriacea (Zhao and Sun 2009). All those studies assume that thisdegeneration may be the primary reason for the species being endangered. There is only one species, Manglietia decidua, that showed no degeneration in material collected from the wild (Xiao and Xu 2006). Also in W. septentrionalis some degeneration was found in the development of the embryo sac, microspore mother cells, tetrads and microspores, but the high germination rate of pollen and high seed set indicate that degeneration is not the primary reason of the decline of the species. The degree of degeneration varies between species, but it is not clear whether degeneration is related to environmental stress or other factors. Further studies on both the ecology and the detailed mechanism of degeneration phenomena are certainly needed.
Acknowledgements This work was nancially supported by the National Natural Science Foundation of China (31070305, 31100233, 30770140) and the Doctoral Startup Found of South China Botanical Garden, the Chinese Academy of Sciences (200928). The authors thank Mr. Ke-Ming Yang for help collecting some samples, and Ms Xin-Lan Xu and Ms Yun Shao for lab work support.

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