Megagametophyte development of Nymphaea nouchali Burm. f. (Nymphaeaceae)

Botanical Journal of the Linnean Society (1998), 126: 339–348. With 13 figures

Megagametophyte development of Nymphaea nouchali Burm. f. (Nymphaeaceae) ISABELLE ORBAN AND JULES BOUHARMONT Department of Plant Science, Cytogenetic Laboratory, University of Louvain, 5 bte 13 place Croix du Sud, B-1348 Louvain-la-Neuve, Belgium Received April 1997; accepted for publication November 1997

The development of reproductive cells has been analysed in Nymphaea nouchali, family Nymphaeaceae, subclass Magnoliidae. Our observations on embryo sac development differ from those in the literature. Megasporogenesis results in a triad of one micropylar cell and two innermost megaspores. Embryo sacs derive from a single functional megaspore and are tetranucleate. Megasporogenesis is monosporic and of the Polygonum type, but megagametogenesis is of Oenothera type.  1998 The Linnean Society of London

ADDITIONAL KEY WORDS:—megasporogenesis – female gametophyte. CONTENTS

Introduction . . . . Material and methods Results . . . . . Megasporogenesis Megagametogenesis Discussion . . . . Megasporogenesis Megagametogenesis Conclusions . . . . Acknowledgements . References . . . .

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INTRODUCTION

The systematic position of the Nymphaeaceae has long been an unsettled question, since the family presents some characters of both monocotyledons and dicotyledons. The reports in the literature that the Nymphaeaceae are monocotyledons or ancestors to the monocotyledons based upon the presence of a single bilobed cotyledon, such as described in Nelumbo, Nymphaea and Nuphar (Lyon, 1901; York, 1904; Cook, 1906, 1909; Haines & Lye, 1976; Philomena & Shah, 1985) are 0024–4074/98/040339+10 $25.00/0/bt970157

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 1998 The Linnean Society of London

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T 1. Classification according to Cronquist (1981) Order

Families

Genus

Nymphaeales

Nelumbonaceae Nymphaeceae

Nelumbo Nymphaea Nuphar Euryale Victoria Ondinea Barclaya Cabomba Brasenia Ceratophyllum

Barclayaceae Cabombaceae Ceratophyllaceae

questioned. The embryo of Ondinea (Schneider & Ford, 1978) is clearly dicotyledonous and its morphology is identical to the described embryos of Nymphaea (Conard, 1905), Nuphar (Chifflot, 1902), Victoria (Khanna, 1967), Euryale (Khanna, 1964b) and Barclaya (Schneider, 1978). Such observations coupled with those of Gupta & Ahuja (1967), who re-examined the embryo of Nelumbo and found it to be dicotyledonous, refute the suggested monocotyledonous nature of the Nymphaeaceae and put seriously in question the family’s placement in evolutionary schemes. The present investigation follows the classification of Cronquist (1981) (Table 1) who considers Nymphaeaceae to be dicotyledonous. Nymphaea, with about 40 species of waterlilies, is the largest and most widely distributed genus in the Nymphaeaceae. These are distributed on all continents except Antarctica. Few botanists have studied megasporogenesis in Nymphaea. Most of the literature is old and interpretations are often obsolete. This paper discusses the megasporogenesis of Nymphaea nouchali Burm. f.. The purpose of the study is to clarify our understanding of the development of the embryo sac in the Nymphaeaceae. This information, moreover, may be expected to aid in the eventual determination of the relationship among members of the Nymphaeaceae. MATERIAL AND METHODS

Flowering material of N. nouchali was obtained from the collection of the National Belgium Botanical Garden, Meise, Belgium. Buds and flowers of various ages were selected. Material was fixed in Carnoy’s solution (70% alcohol, glacial acetic acid, 3:1 vv) for 12 hours and stored in 70% alcohol. The samples were processed through tertiary butyl alcohol series and usual embedding techniques were followed. Sections were cut at 12 lm thickness on a rotary microtome and stained with iron alumhaematoxylin and fast green ( Jensen, 1962). RESULTS

Megasporogenesis A hypodermal archesporial cell cuts off an upper cell, the primary parietal cell and a lower one, the megaspore mother cell (i.e. crassinucellate condition) (Fig. 1).

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Figures 1–3. Fig. 1. Young ovule. Primary sporogenous cell and primary parietal cell. Scale bar= 100 lm. Fig. 2. Young ovule. Part of the nucellus. Megasporocyte. Scale bar=100 lm. Fig. 3. Ovule. Part of the nucellus. Metaphase of meiosis I. Scale bar=100 lm.

The parietal cell divides further periclinically to form a parietal layer. The megaspore mother cell is thus soon one cell deep (Fig. 2). The megasporocyte is characterized by a nucleus at the micropylar side and starch reserves at the chalazal end (Fig. 2). The megaspore mother cell soon divides transversely (Fig. 3), to form two unequal cells: the micropylar one is smaller than the chalazal cell (Fig. 4). The succeeding divisions are also unequal. The micropylar daughter cell undergoes no division and degenerates (Fig. 5). The lower one divides again forming two megaspores (Figs 6 and 7). An axial row of three cells is present after meiosis with one degenerated cell

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Figures 4–6. Ovule, part of the nucellus. Fig. 4. Diad of unequal cells; micropylar dyad member is smaller than the chalazal member. Scale bar=100 lm. Fig. 5. Section showing the chalazal cell and the micropylar crushed cell. Scale bar=100 lm. Fig. 6. Metaphase of meiosis II. Scale bar=100 lm.

below the parietal cells and two innermost megaspores (Fig. 7). The middle cell is smaller than the lower megaspore, which is the embryo sac mother cell. The functionless megaspore then degenerates and disappears and the functional megaspore lengthens toward the micropyle (Fig. 8).

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Figures 7–9. Ovule, part of the nucellus. Fig. 7. Triad stage of megaspore. Scale bar=100 lm. Fig. 8. Functional megaspore enlarging; note the degenerating megaspore and the degenerating micropylar cell. Scale bar=100 lm. Fig. 9. Note the position of the megagametophyte in the nucellus; 2-nucleate megagametophyte. Scale bar=100 lm.

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Megagametogenesis formation of the female gametophyte The functional innermost megaspore develops into the embryo sac at the expense of the other megaspores and the parietal cells. The enlarged cell becomes much vacuolated (Fig. 8). Then the embryo sac mother cell passes rapidly through the two nucleate stage and enlarges in the direction of the longitudinal axis of the ovule (Figs 9, 10 and 11). The early embryo sac further expands toward the micropyle. The sac is fourcelled and contains the egg apparatus (two synergids and an egg) and a ‘central’ cell with the polar nucleus near the middle of the cell (Fig. 12). Antipodals are absent. At the first day of anthesis the embryo sac appears elliptical in section. The egg apparatus is complete. In the upper part of the sac, three similar cells are crowded, with two synergids above and one egg below. The polar nucleus of the central cell can be seen in the middle part of the embryo sac (Fig. 13).

DISCUSSION

Megasporogenesis Data on the megasporogenesis of Nymphaea are sparse and relatively old. Cook (1902, 1906, 1909), Conard (1905) and Seaton (1908) made some observations on N. odorata Willd. and N. advena Sol. which differ from our observations. For these two species, the megasporocyte has four megaspores after meiosis. On the other hand, our observations on N. nouchali show three different cells after meiosis: the degenerate micropylar cell, a middle megaspore and a chalazal megaspore. Our results establish a parallel with data on N. stellata F. Muell. (Khanna, 1967). After the heterotypic division, the megasporocyte produces a dyad composed of a micropylar cell and a chalazal cell, of which only the chalazal cell divides. More recent observations on N. heudelotii Planch. (Van Miegroet & Dujardin, 1992) show the heterotypic division. The micropylar cell does not divide. The chalazal lengthened cell undergoes homeotypic division. In N. nouchali, as in N. stellata and N. heudelotii, the micropylar cell degenerates before the homeotypic division of the chalazal cell. Megasporogenesis of N. nouchali, as in N. stellata (Khanna, 1967) and N. heudelotii (Van Miegroet & Dujardin, 1992), thus conforms to the monosporic Polygonum type ( Johri, 1984). The megasporocyte undergoes meiosis, after which a megaspore remains functional, and only the chalazal megaspore gives rise to the embryo sac. Usually the Polygonum type of megasporogenesis gives a tetrad of megaspores, of which the three upper cells degenerate and the lower megaspore is functional. However, this is not always the case. Variations occur in particular in the number of haploid cells born of the meiosis; so for Amaranthaceae, Araliaceae, Cactaceae, Caryophyllaceae, Orchidaceae, Petiveriaceae and Phytolacaceae, the micropylar cell formed after heterotypic division does not divide and degenerates. Only the chalazal cell gives two megaspores ( Johri, 1984; Law & Yeung, 1989; Yeung & Law, 1989) as in this paper for Nymphaea but the development of the functional megaspore for Nymphaea cannot be compared with a Polygonum type of development.

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Figures 10–12. Ovule. Fig. 10. Part of the nucellus, note the position of the megagametophyte in the nucellus; 2-nucleate megagametophyte. Scale bar=100 lm. Fig. 11. Part of the nucellus, note the position of the megagametophyte in the nucellus; 2-nucleate megagametophyte. Scale bar=100 lm. Fig. 12. Ovule at mature embryo sac stage. Maturing embryo sac; polar nucleus migrated to the centre, egg cell beneath synergids. Scale bar=100 lm.

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Figure 13. Ovule at mature stage. Mature female gametophyte prior to fertilization with a central cell, egg cell and two synergids. Scale bar=100 lm.

Megagametogenesis and embryo sac In N. nouchali and N. heudelotii (Van Miegroet & Dujardin, 1992) the functional megaspore and bi-nucleate coenocyte enlarge toward the micropyle at the expense of the middle megaspore and the micropylar cell, and then of the parietal cells. The bi-nucleate and then tetra-nucleate coenocyte and the embryo sac are placed below the nucellar epidermis as for N. odorata (Cook, 1902). For N. advena the functional megaspore is below the nucellar epidermis and then gives rise to a 2-, 4- and 8nucleate coenocyte (Cook, 1906). On the other hand, Seaton (1908) noted for N. advena that the embryo sac mother cell and then the bi-nucleate coenocyte enlarge toward the micropyle. The 4-nucleate coenocyte lengthens but both it and the mature embryo sac are again separated from the nucellar epidermis by 8, 6 or 4 parietal cells. When mature, the embryo sac of N. nouchali, N. heudelotii (Van Miegroet & Dujardin, 1992), N. gigantea Hook. and N. alba (Winter & Shamrov, 1991) occurs below the nucellar epidermis. The embryo sac has four cells: two synergids, one egg and one central cell. These observations do not conform with those of Conard (1905), Cook (1902, 1906, 1909), Seaton (1908), Khanna (1967) and Batygina, Kravtsova & Shamrov (1980) on N. odorata, N. advena, N. stellata, Victoria cruziana Orb. of Nymphaeaceae or for other species of related families (Table 1) such as Barclaya longifolia Wall. (Schneider, 1978), Brasenia schreberei J.F. Gmel. (Khanna, 1965), Cabomba caroliniana A. Gray (Ramji & Padmanabhan, 1965; Schneider & Jeter, 1982), Euryale ferox (Khanna, 1964a, b, Batygina et al., 1980) and Nelumbo nucifera Gaertn. (Khanna,

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1965; Gupta & Ahluwalia, 1977). The ovules for those species have an 8-nucleate embryo sac and Polygonum type of development. In our material, there are always four cells in the mature embryo sac. The two synergids and the egg form the egg apparatus at the micropylar pole. The central cell occupies the greatest part of the sac with the nucleus in the middle of the female gametophyte. This type of embryo sac is found in Oenothera ( Johri, 1984). The female gametophyte has a monosporic development and gives rise to a 4-nucleate embryo sac. The functional megaspore undergoes only two mitoses during megagametogenesis and gives rise to a 4-nucleate and 4-celled embryo sac. Galati (1985) also observed a 4-celled embryo sac with four nuclei in Cabomba australis Speg. as opposed to Polygonum type in Cabomba caroliniana (Ramji & Padmanabhan, 1965; Schneider & Jeter, 1982). Megagametogenesis includes two mitoses, and is a variant of the Oenothera type. In Oenotheraceae, megasporogenesis gives rise to a functional megaspore at the micropylar pole. However, the functional megaspore is at the chalazal end of the tetrad in Cabomba australis (Galati, 1985), N. advena, N. odorata and of the triad in N. nouchali (this paper) and N. heudelotii (Van Miegroet & Dujardin, 1992). We have described what may be regarded as a Polygonum variant with only two mitoses. The young embryo sac has four cells: two synergids and an agg at micropylar end and the central cell with her nucleus in the middle of the female gametophyte.

CONCLUSIONS

Our observations represent the most complete survey of megasporogenesis and megagametogenesis in Nymphaea to date. Megasporogenesis results in a triad of one micropylar cell and two innermost megaspores. During megagametogenesis, the chalazal functional megaspore undergoes two mitoses. The embryo sac has four cells: the egg cell, the two synergid cells and the central cell. Megasporogenesis is monosporic and of the Polygonum type and megagametogenesis is of the Oenothera type. The development of the Nymphaea embryo sac is unusual because it has not been previously observed in the angiosperms. The development of the megagametophyte has been erroneously recorded in Nymphaea by several authors who have described it as having an 8-nucleate embryo sac of the Polygonum type.

ACKNOWLEDGEMENTS

We are greatly indebted to Prof. C. Evrard and to the National Belgium Botanical Garden for providing plant samples.

REFERENCES

Batygina TB, Kravtsova TI, Shamrov II. 1980. The comparative embryology of some representatives of the orders Nymphaeales and Nelumbonales. Botanicheskii Zhurnal SSSR 65: 1071–1087. Chifflot JBJ. 1902. Contributions a` l’e´tude de la classe des Nympheine´es. Annales de l’Universite´ de Lyon, Sciences Me´decine 40: 1–294.

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Conard HS. 1905. The Water-lilies: a monograph of the genus Nymphaea. Publication Carnegie Institute 4: 1–229. Cook MT. 1902. Development of the embryo sac and embryos of Castalia odorata, Nymphaea advena. Bulletin of the Torrey Botanical Club 29: 211–220. Cook MT. 1906. The embryogeny of some Cuban Nymphaeaceae. Botanical Gazette 42: 376–392. Cook MT. 1909. Notes on the embryogeny of the Nymphaeaceae. Botanical Gazette 48: 56–61. Cronquist A. 1981. An integrated system of classification of flowering plants. New York: Columbia University Press. Galati BG. 1985. Estudios embriologicos en Cabomba australis (Nymphaeaceae). I. La esporogenesis y las generaciones sexuales. Boletin De La Sociedad Argentina De Botanica 24: 29–47. Gupta SC, Ahluwalia R. 1977. The carpel of Nelumbo nucifera. Phytomorphology 27: 274–282. Gupta SC, Ahuja R. 1967. Is Nelumbo a monocot? Naturwissenschaften 54: 498. Haines RW, Lye KA. 1976. Seedlings of Nymphaeaceae. Botanical Journal of the Linnean Society 70: 255–265. Jensen WA. 1962. Botanical histochemistry. Principles and practice. San Francisco: Freeman. Johri BM. 1984. Embryology of Angiosperms. Springer-Verlag, Berlin, Heidelberg, New York and Tokyo. Khanna P. 1964a. Some interesting observations in Euryale ferox Salisb. Current Science 33: 152. Khanna P. 1964b. Morphological and embryological studies in Nymphaeaceae. I. Euryale ferox Salisb. Proceedings of the Indian Academy of Sciences 59: 237–243. Khanna P. 1965. Morphological and embryological studies in the Nymphaeaceae. II. Brasenia schreberei Gmel and Nelumbo nucifera Gaertn. Australian Journal of Botany 13: 379–387. Khanna P. 1967. Morphological and embryological studies in Nymphaeaceae. III. Victoria cruziana D’Orb. and Nymphaea stellata Willd. Botanical Magazine 80: 305–312. Law SK, Yeung EC. 1989. Embryology of Calypso bulbosa. I. Ovule development. American Journal of Botany 76: 1668–1674. Lyon HL. 1901. Observations on the embryology of Nelumbo. Minnesota Botanical Studies 2: 643–655. Philomena PA, Shah CK. 1985. Unusual germination and seedling development in two monocotyledonous dicotyledons. Proceedings of the Indian Academy of Sciences 95: 221–225. Ramji MN, Padmanabhan DP. 1965. Developmental studies on Cabomba caroliniana Gray. I. Ovule and carpel. Proceedings of the Indian Academy of Sciences 62: 215–223. Schneider EL. 1978. Morphological studies of the Nymphaeaceae. IX. The seed of Barclaya longifolia Wall. Botanical Gazette 139: 223–230. Schneider EL, Ford EG. 1978. Morphological studies of the Nymphaeaceae. X. The seed of Ondinea purpurea Den Hartog. Bulletin of the Torrey Botanical Club 105: 192–200. Schneider EL, Jetter JM. 1982. Morphological studies of the Nymphaeaceae. XII. The floral biology of Cabomba caroliniana. American Journal of Botany 69: 1410–1419. Seaton S. 1908. The development of the embryo sac of Nymphaea advena. Bulletin of the Torrey Botanical Club 35: 283–290. Van Meigroet F, Dujardin M. 1992. Cytologie et histologie de la reproduction chez Nymphaea heudelotii. Canadian Journal of Botany 70: 1991–1996. Winter AN, Shamrov II. 1991. Megasporogenesis and embryo sac development in representatives of the genera Nymphaea and Victoria (Nymphaeaceae). Botanicheskii Zhurnal SSSR 76: 1716–1728. Yeung C, Law SK. 1989. Embryology of Epidendrum ibuguense. I. Ovule development. Canadian Journal of Botany 67: 2219–2226. York HH. 1904. The embryo sac and embryo of Nelumbo. Ohio Naturalist 4: 167–176.