Floral morphology and embryo sac development in Burretiodendron kydiifolium Y. C. Hsu et R. Zhuge (Tiliaceae) and their systematic significance

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1 BotunicalJouml ofthe Limean (19981, 128: With 32 figures Article ID: bt Floral morphology and embryo sac development in Burretiodendron kydiifolium Y. C. Hsu et R. Zhuge (Tiliaceae) and their systematic significance YA TANG* Chengdu Institute ofbiology, Chinese Academy of Sciences, F! 0. Box 416, Chengdu, Sichuan , Peoples Republic of China * Received October 1997; accepted for publication April 1998 Floral morphology and development of megaspore and megagametophyte in Burretiodendmn &ydifoliun are described. The ovule is anatropous, crassinucellate and bitegmic. The archesporium is multicellular and usually two cells develop to megasporocytes. The tetrad is linear or very rarely T-shaped. The chalazal megaspore functions and develops into a Polygonumtype of embryo sac. The antipodals degenerate very early and the fertilizable embryo sac consists of only one egg cell, two synergids and two polar nuclei. In a fertilizable embryo sac the micropyle is formed by both outer and inner integuments, which are three to four and five cells thick, respectively. The epidermal cells of the nucellus divide to form a nucellar cap. A group of special cells with large nuclei and less staining are observed below the sporogeneous cells. Comparison of the embryological features of Bumtiodendmn S.S. with those of Tiliaceae indicates that this genus differs greatly from other members of Tiliaceae and no close relationship is established among Burretiodendmn and other tiliaceous taxa. Most embryological features of Bunetiodendmn are shared by the genus Ptmspennum of Sterculiaceae although they differ considerably in gross morphology The Linnean Society of London ADDITIONAL KEY WORDS:-functionally unisexual flowers - Malvales - megagametogenesis ~ Polygonum embryo sac - Pterospenurn. CONTENTS Introduction Material and methods Observations Flowers Ovary and ovule Megasporogenesis and megagametogenesis Integuments Discussion Acknowledgements References * Present address: International Centre for Integrated Mountain Development (ICIMOD), GPO Box 3226, Kathmandu, Nepal. tangya@icimod.org.np /98/ $30.00/ The Linnean Society of London

2 IKTRODUCllON The order Malvales comprises Elaeocarpaceae, Tiliaceae, Sterculiaceae, Bomhacaceae and hlalvaceae (Cronquist, 198 1). These families are similar in floral morphology (van Heel, 1966), pollen morphology (Erdtman, 1952), and wood anatomy (Metcalfe & Chalk, 1950). As traditionally defined, they overlap in numerous characters and in some instances the criteria used to distinguish them from each other are weak (Hutchinson, 1967). Some genera, or even tribes, are placed in different families by different authors. These families are so closely related that the delimitation between families is far from satisfactory. Thus the order should be in\-estigated further, particularly a few key genera. Burretiodmdron Rehder was initially based on a single species, B. aquirolii (Lev1.) Rehder in the Tiliaceae (Rehder, 1936), but now includes six species (Zhuge, 1990). This genus is isolated in terms of morphology (Rehder, 1936; Zhuge, 1990) and pollen morphology (Tang & Gao, 1993). Except for taxonomic revisions (Kosterrnans, 1961; Chang & Miau, 1978; Zhuge, 1990) and palynological investigation (Tang & Gao, 1993), no detailed studies on this genus have been undertaken and its affinities are not well understood. The floral morphology and the development of ovule and embryo sac of B. lydifoolium Y. C. Hsu et R. Zhuge are described here in order to shed some light on its affinities. MATERIAL AND METHODS The hermaphroditic flower buds of B. bdiijilium collected in Yuanjiang County, Yunnan Province, People s Republic of China were fixed in FAA. Flower buds were Lxed every other day for 12 days. A voucher is deposited in the herbarium of Chengdu Institute of Biology (CDBI), Chinese Academy of Sciences, People s Republic of China. The fixed buds were dehydrated through an ethanol series before being infiltrated and embedded in paraffin with a melting point of C. Serial sections were cut 8-10 pm in thickness, stained with safranin and fast green, and permanent preparations were sealed with Canada balsam. OBSER\ ATIONS Flowers Burretiodendron /ydi$lium is polygamous with both staminate and hermaphroditic flowers. There are five calyx segments which are closely cohesive and difficult to separate before anthesis. Petals are five and imbricate. Both staminate and hermaphroditic flowers are enclosed completely by three big epicalyces and are axillary on the branches of the current year. The hermaphroditic flowers have 30 stamens and five staminodes. The stamens of the hermaphroditic flowers are smaller than those of the staminate flowers, and were checked under a microscope but no pollen grains were found inside, so they function as pistillate flowers. Flowers of Burrehodendm bdi@lium are therefore functionally unisexual. Staminate flowers have stamens. Petals are smaller than sepals in flower buds of both staminate and

3 EMBRYO SAC DEVELOPMENT IN BURRETIODLNDRON 151 bisexual flowers. The staminate flowers and functionally pistillate flowers are borne on different individual plants, hence Burretiodendron. S.S. is dioecious. The ratio of plants with staminate flowers to plants with bisexual flowers is about 20: 1. Staminate flowers are usually borne in cymes of 3-7 flowers, while the bisexual flowers are usually solitary or very rarely in small cymes of 2-3 flowers. Bisexual flowers open for a period of about 20 days, while the staminate flowers open for usually only 2-3 days. The style is very short or subsessile while the stigma is greatly swollen. No staminodes are found in the staminate flowers, although rudimentary pistils are sometimes present. Ovary and ovule The ovary is superior, syncarpous, five-carpellary, five-locular and five-ridged (Fig. 1). There are two basal ovules with a septum in each locule on axile placentation. The two ovules differentiate simultaneously and initially lie side by side, but later a septum develops and grows to the axis, separating the two ovules (Figs 2,3). At an early megasporocyte stage, glandular outgrowths develop on the outer epidermis of the ovary, eventually forming scale-like appendages (Figs 23-26), which were also observed on the outer epidermis of anthers and filaments of the same species. The ovules arise as small papillae from the base of the ovary locule. They are bitegmic, anatropous and crassinucellate. At first the ovules do not fill the locules (Fig. 2) but at the late stage of megaspore tetrad they closely fill the locule and there is little space between ovules and the inner wall of the carpels. At the archesporial stage integument initials are not yet demarcated, but initiate at the megasporocyte stage, when the ovules begin to become curved. Ovules become completely anatropous before megasporocyte division (Fig. 30). The primary parietal cell divides periclinally to form two cells of parietal tissue, which then divide both periclinally and anticlinally to form 2-4 layers of parietal tissue at the late megasporocyte stage (Figs 27-30). Meanwhile the nucellar epidermal cells above the nucellus also undergo one periclinal division. A nucellar cap is therefore developed in the mature embryo sac. As a result of division of parietal cells and nucellar epidermal cells, at the late megasporocyte stage the parietal tissue increases to five layers (Fig. 30), seven or eight layers at the tetrad stage (Fig. 31), and eight to ten layers at the stage of binucleate embryo sac. As a result of enlargement of the embryo sac, some layers of parietal tissue are crushed, and the parietal tissue is five to six cells thick in a fertilizable embryo sac. The nucellus is straight and massive but relatively small compared with the ovules (Fig. 32). Megasporogenesis and megagametogenesis The primary archesporium is multicellular, consisting of both hypodermal and subhypodermal cells (Figs 5-7). Usually two, or occasionally one or three cells develop into megasporocytes (Figs ). Each archesporial cell divides periclinally into a primary parietal cell to the outer side and a sporogenous cell to the inner side (Fig. 8). Each sporogenous cell functions directly as a megasporocyte (Fig. 9). Before divisions, the two or three megasporocytes enlarge in volume and attain an

4 Figures Burretiodmdron Fig. 1. Transverse section of ovary. Figs 2,3. Different stages of scptum development in one locule. Fig. 4. Mcropyle of a fertilizable embryo sac. Figs 5-7. Archesporial cells; note group of special cells in 5. Fig. 8. Division of one archesporial cell. Fig. 9. Divisions of parietal cells, and two megasporocytes. Fig. 10. Dermal origin of inner and outer inte,pments. Figs Megasporocytes and parietal tissue. Scale bars; Fig. 1 =400 pm; Figs 2-13 = 20 gm.

5 EMBRYO SAC DEVELOPMENT IN BURRETIODENDROJV 153 Figures ~u~e~~ffdend~n ~~i~ffl~z~m. Figs 14, 15. Tetrad. Fig. 16. Triple tetrads. Fig. 17. T-shaped tetrad. Fig. 18. Two-nucleate embryo sac. Fig. 19. Four-nucleate embryo sac. Figs Successive sections of a fertilizable embryo sac. Fig. 20. Synergids. Fig. 21. Egg cell. Fig. 22. Two polar nuclei. Scale bars = 20 pm.

6 Y. TANG Figures Bumtiodendmn lydifalium. Figs Development of scale-like appendages on the outer surface of the ovary wall. Figs Development of parietal tissue. Fig. 32. Relative volume of iiucellus to ovule. Scale bars: Figs 23, 25=200 pm. Figs 24, 26, 27-31, 32=20 pm. elongated form, tapering towards the chalazal end. Of the two or three megasporocytes, usually only one undergoes meiosis forming a dyad (Fig. 14) and a linear (Fig. 15) or very rarely T-shaped tetrad of megaspores (Fig. 17). Very rarely two or three megasporocytes develop into double or triple tetrads of megaspores (Fig. 16). In the case of double or triple tetrads, usually only one megaspore develops and the others disintegrate. In all preparations a double embryo sac was observed only once. Of the megaspores the chalazal one functions and develops successively into a two-nucleate (Fig. 18), four-nucleate (Fig. 19) and mature embryo sac (Figs 20-22). In a four-nucleate embryo sac, every two nuclei initially lie transversely at the two ends but later the two micropylar nuclei are arranged transversely, while the two chalazal nuclei are arranged longitudinally. The mature embryo sac consists of bvo synergids, one egg cell, two polar nuclei, and three antipodals. The antipodals degenerate very soon after their formation. In numerous fertilizable/mature embryo sacs, no antipodals or their traces were observed. Therefore the mature embryo sac consists of only two nuclei and three cells: an egg cell, two synergids and two polar nuclei. The synergids are not hooked (Fig. 20). The development of the embryo sac conforms to the Polygonum-type.

7 EMBRYO SAC DEVELOPMENT IN BURRETIODENDRON 155 While the primary parietal cells divide periclinally into two cells, a group of cells in the central region of the ovule, just beneath the sporogenous cells, is observed to lose the capacity for deep staining and takes even less stain than the neighbouring cells of the nucellus. These cells are large in size, and some even larger than sporogenous cells; their nuclei are proportionally large, but show scanty chromatin. These cells do not divide but persist in this condition to the stage when the megasporocytes are quite large. They probably function as nutritive cells, which are consumed by the development of megasporocytes, as they are not observed at and after the megasporocytes stage. Integuments The ovules are bitegmic and the micropyle of the mature embryo sac is formed by both inner and outer integuments, and is zigzag (Fig. 4). Both integuments are initiated almost simultaneously by oblique periclinal divisions of dermal cells in a small ovule primordium (Fig. lo), around the stage when the sporogenous cell is first formed. However, subsequent growth of both integuments is different. The inner integument shows tardy growth. At the early megasporocyte stage, the inner and outer integuments both have two layers of cells, and at the late megasporocyte stage are similar in height. Subsequently the outer integument develops a third layer and grows as high as the nucellus, although the inner integument grows to only a third or half the height of the nucellus. The inner integument becomes three cells thick when the megaspore tetrad is formed. From tetrad to four-nucleate stages the thickness of both inner and outer integuments is three cells. The outer integument grows to form the micropyle at the binucleate embryo sac stage, at which the inner integument extends over only one third or half the height of the nucellus. The inner integument becomes five cells thick at the late stage of mature embryo sac, while the outer integument becomes three or four cells thick. The inner integument does not form the micropyle until the late stage of the mature embryo sac. In a mature embryo sac the inner integument is five cells thick, while the outer integument is three cells thick. At the mature embryo sac stage, the outer integument stains the same as the other ovary tissue, while the inner integument stains similarly to nucellar tissue. The outer integument is easily detached from the inner integument and the nucellus. DISCUSSION Embryological studies often contribute important data in assessing relationships between taxa (e.g. Maheswari, 1950; Johri, 1984; Tobe, 1989). In Burretiodendron &ydi$lium, embryological features prior to fertilization can be summarized as follows: ovules anatropous, bitegmic and crassinucellate; outer integument 34 cells thick and inner integument five cells thick in a mature embryo sac; micropyle formed by both integuments; archesporium multicellular and usually two cells developing to megasporocytes; tetrad usually linear; Polygonum-type of embryo sac development; antipodals degenerating very early; a group of special cells developing during the late stage of sporogenous cells.

8 The relationships of Burretiodendron are controversial. It has been considered to be related to Luehea (Rehder, 1936), Colonu (Kostermans, 1961), Crui a (Chang& Miau, 1978), or Schoutenia and Surea (Zhuge, 1990). It was even divided into two genera, Burretiodendron (s.s.) and Excentrodendron Chang & Miau (Chang & Miau, 1978). While the segregation of Excentrodendron has never been accepted (Mabberley, 1989; Zhuge, 1990; Wu, 1991 ; Brummitt, 1992), pollen of Burretiondendron (d.) comprises two very distinctive types: (1) the rough reticulate type, unique both in Tiliaceae and in the whole Malvales; (2) the spiny type, similar to the pollen type of some taxa of Sterculiaceae and Malvaceae. Of the genera considered related to Burretiodendron, only Schoutenia has pollen grains more or less similar to the spiny pollen type of Burretiodendron (s.s.), and the other genera are clearly distinct from Burretiodendron (s.1.) by their pollen morphology (Tang & Gao, 1993). Pollen of B. kydizjilium is of the spiny type (Tang & Gao, 1993). In the following discussion, the genus Burvetiodendron is used in its strict sense only. A review of the literature reveals that the embryology of Tiliaceae has not received much attention, including all the genera referred to earlier with possible affinities of Burretiodendmn (s.s.). In the family Tiliaceae only five genera-corchorus, Entelea, Spannannia, 7ilia and Tn urnfetta-have been embryologically investigated (Stenar, 1925; Banerji, 1932; Rao & Rao, 1952). Embryological investigations into Tiliaceae prior to 1965 were summarized by Davis (1966), and no addition seems to have been reported since then apart from Dnyansagar & Gaoli on Corchorus triloculairs (1964-5) and Tang on Corchompsis and Plagioptmon (Tang, 1992a, 1994). The latter two genera were earlier placed in Tiliaceae but now are treated as a member of Sterculiaceae and a separate family, respectively (Tang, 1992a, 1994). Burretiodendron S.S. shares some embryological features with these five genera, including anatropous, bitegmic and crassinucellate ovules, tardy growth of the inner intecgument, multicellular megaspore archesporium and linear megaspore tetrad with the chalaza1 one functioning to develop into an embryo sac of Polygonurn-type. Two adjacent megasporocytes lying side by side are also found in members of Corchorus (Stenar, 1925; Banerji, 1932; Rao & Rao, 1952), but not in other members of the family, and three megasporocytes together are reported here for the first time in Tiliaceae, and even in the order Malvales. However, there are also many differences between Burretwdmdron S.S. and other tiliaceous genera. The integuments of Burretiodendron S.S. are more massive than those of other genera of Tiliaceae. The outer integument in Burretiodendmn S.S. is 34 cell-layered, and the inner integument is five cells thick, while the outer integument of other tiliaceous genera is two cells thick, and the inner integument is three layered. In particular, in the mature ovule the three layered outer integument is stained in the same way as the ovary wall, and distinct from the inner integument. At this stage, superficial observation would produce a false impression of a unitegmic ovule. After maturation of the embryo sac, the outer integument is easily separated from the inner integument and the nucellus. These two features have not been reported earlier in Tiliaceae but have been observed in Cruigiu (Tang, unpublished observation). In a mature embryo sac, the micropyle is formed by both inner and outer integuments. This condition has been reported only for 7iliu (Stenar, 1925). In Corchorus and Triumjtta the micropyle is formed only by the outer integument, while in Enteleu it is formed only by the inner integument (Davis, 1966). The division of nucellar epidermal cells, their contribution to the organization of parietal tissue, and the development of a nucellar cap have not been reported in other members of Tiliaceae.

9 EMBRYO SAC DEVELOPMENT IN BlJRRETIODEhDRON 157 Apart from Tiliaceae, a review of embryological literature in the order Malvales reveals that Pterospermum (Sterculiaceae) shares most embryological features with Burretiodendron S.S. (Rao, 1949, 1952). Common features include: anatropous, bitegmic and crassinucellate ovules, multicellular archesporium, development of a nucellar cap, linear tetrad, Polygonum-type of embryo sac, outer integument three cells thick and inner integument 4-5 cells thick, development of a group of special cells below sporogenous cells, both integuments forming the micropyle, development of double and triple embryo sacs, and tardy growth of the inner integument. Pollen of Pterospermum is also similar to that of Burretiodendron S.S. (Rao, 1950, Tang, 1992b, Tang & Gao, 1993), but F terospemzum is considerably different from Burretiodendmn S.S. in gross morphology. Based on these comparisons it becomes clear that only some embryological features of Burretiodendron S.S. are common with other genera of the Tiliaceae. These features, however, commonly occur in the dicotyledonous plants and are regarded as plesiomorphic. However, most embryological and pollen features of Burretiodendmn S.S. are shared by the genus Pterospermum (Sterculiaceae). Embryologically and palynologically, Burretiodendmn S.S. is closely related to Pterospennum although they are clearly different in floral morphology. Within Sterculiaceae Pterospennum is isolated in terms of combined characteristics of gross morphology, anatomy, pollen morphology, basic chromosome number, and embryology (Tang, ). This investigation once again underlines the fact that the order Malvales needs further systematic research (Tang, 1990). The combined embryological and palynological features of the genus Burretiondendron (s.s.) confirm its isolated position within Tiliaceae. More investigations are needed to elucidate the affinity of the genus. ACKNOWLEDGEMENTS This study is supported by a grant from the Special Support to Biofloristics of the Chinese Academy of Sciences. The author thanks an anonymous referee for useful suggestions for improvement of the paper. REFERENCES Banerji I The development of the embryo sac and fertilization in jute. Journal ofthe Indian Botanical Sociep 11: Brummitt RK Vascular plant families and genera. Kew: Royal Botanical Gardens, Kew. Chang HT, Miau RH The taxonomy of excentrodendroideae, Tiliaceae. Journal ofthe {hangshun Universip, Natural Science Edition 1978:(3) (In Chinese) Cronquist A An integrated system ofclass$ication ofj lowuingplants. New York: Columbia University Press. Davis GL Systematic emblyology ofthe angiosperm New YorkJohn Wiley & Sons. Inc. Dnyansagar VP, Gaoli HP Embryology of Carchorus trilocularis Linn. Journal of Uniuersib of Bombay, B 33: Erdtman G Pollen mo@holoq andplant taxonomy. Angiosperms. Stockholm: Almquist and Wiksell. Hutchinson J ?he genera ofthej lozua ng plants Vol. 11. Oxford: Clarendon Press. Hesse M Entwicklungsgeschichte und Ultrastructur des Pollenkitts bei Elk (Tiliaceae). Plant systematics and Evolution 129: Johri BM, ed EmblyoloQ of angiosperms Berlin, Heidelberg, New York, Tokyo: Springer-Verlag. Kostermans AJGH The genus Burretiodendron Rehder (Tiliaceae). Reinzuardtia 6: 1-16.

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