Ann. Bot. Fennici 49: 55–63 ISSN 0003-3847 (print) ISSN 1797-2442 (online) Helsinki 26 April 2012 © Finnish Zoological and Botanical Publishing Board 2012 Female gametophyte development in Sinopodophyllum hexandrum (Berberidaceae) and its evolutionary significance Heng-Yu Huang & Li Li* Key Laboratory of Plant Resources Conservation and Utilization (Jishou University), College of Hunan Province, Jishou, Hunan, 416000, China (*corresponding author’s e-mail: [email protected]) Received 11 Mar. 2011, final version received 30 Mar. 2011, accepted 19 Apr. 2011 Huang, H. Y. & Li, L. 2012: Female gametophyte development in Sinopodophyllum hexandrum (Berberidaceae) and its evolutionary significance. — Ann. Bot. Fennici 49: 55–63. Female gametophyte development of Sinopodophyllum hexandrum (Berberidaceae) was studied on materials from different altitudes. The plant displays diversity in the megagametogenesis, and two categories of four female-gametophyte development models are observed, including the monosporous Polygonum-type, a new Sinopodo- phyllum type (and its two variants), which is intermediate between the monosporic and bisporic types. The results suggest that the Polygonum type is more primitive than the other types, and that the Sinopodophyllum type is intermediate between the Poly- gonum type and the Allium type. The diversity in the female gametophyte develop- ment in S. hexandrum reveals megagametogenesis may be influenced by altitude and be of great significance to its adaptability and evolution. In the Podophyllum group, Sinopodophyllum is more advanced than Diphylleia and Dysosma, where the female gametophyte development conforms to the Polygonum type. In addition, the results of this study directly provide evidence that the disporous Allium-type is derived from the monosporous Polygonum-type. Introduction In families where some embryological variation occurs, genera are constant on the whole. Embryological information makes great contri- Megagametophyte development may be of butions to an understanding of the phylogenetic major importance in the determination of rela- relationships in plants, and embryological char- tionships within families and genera. Huang and acteristics are at present incorporated in many Sheridan (1994, 1996) stated that embryo forms publications for their value to angiosperm sys- are similar in all systematic groups, and embryo- tematics (Cronquist 1981, 1988). However, the genesis can not provide information of prime systematic value of embryological characteristics importance to the main groups of plant families. has long been persuasively argued (Maheshwari However, they considered that at the generic and 1950, Palser 1975, Heo & Tobe 1995). Palser specific levels, embryogeny is of some system- (1975) concluded that embryological character- atic value. Few investigations have dealt with istics are relatively constant at the family level. variation in embryological characteristics within 56 Huang & Li • ANN. BOT. FENNICI Vol. 49 species. In this study on the female gameto- at three localities: Bi-ta-hai (alt. 3200 m), Zang- phyte development in the Himalayan mayapple ba (alt. 3750 m) and Tian-e-hu (alt. 4200 m), in Sinopodophyllum hexandrum (Berberidaceae) the Xiang-ge-li-la county, Yunnan province, PR we demonstrate that embryological characteris- China. Both entire and partial flower buds and tics are not constant within the species, but vary flowers at different development stages were with different altitudes. collected and fixed in modified FAA (50% alco- Sinopodophyllum hexandrum is a self- hol–glacial acetic acid–formaldehyde = 89:6:5). pollinating perennial herb, found at altitudes The fixed materials were stained in Ehrfich’s from 1500 to 4300 m in the eastern Himalayan hematoxyfin after being washed with water. The regions. There are various habitats in this area materials were embedded in paraffin after being and the environment is harsh in some regions, dehydrated in ten grades of alcohol (mixtures but the Himalayan Mayapple shows great tol- of alcohol and water), cleared with five grades erance and phenotypic plasticity (Ma & Hu of xylene (mixtures of xylene and alcohol), and 1996). Traditionally, the Himalayan mayapple serially sectioned at the thickness of 5–8 µm by and its closely related genera, Diphylleia, Dys- Microm. The sections were mounted on slides in osma and Podophyllum, are members of Berberi- Neutral balsam, and observed and photographed daceae, but in some classification systems, they with the compound microscope Leica DM2000. are treated in a separate family, Podophyllaceae. There is some dispute about their classification and the relationships among these four genera. Results Information from an investigation of female gametophyte development may provide some Under natural conditions, S. hexandrum repro- clues toward settling their phylogenic relations. duces mainly through seeds. There is an interval Sinopodophyllum hexandrum and its relative of about five or six years from seed germination Podophyllum peltatum are the major sources of to flower development. The plant forms flower podophyllotoxin, an effective medicine used to buds in autumn when the vegetative growth is treat genital warts (condyloma acuminatum). It complete. At this time, the morphological devel- is also used as the precursor in the manufacture opment of all flower parts is well underway, of the semi-synthetic derivatives etoposide (VP- but the mega- and microsporocytes have not 16), teniposide, and etopophos. Since 1995, CPH undergone meiosis. In late autumn, the vegeta- 82, a semi-synthetic derivative of podophyllo-podophyllo- tive shoot withers; the flower buds cease devel- toxin, is in its third phase of clinical trials for opment, become dormant, and are enveloped by the treatment of rheumatoid arthritis (Lerndal & two succulent bracts. In the following late spring Svensson 2000). Although many pharmaceuti- or early summer, when the temperature rises cal and biological studies on S. hexandrum have to a threshold, the dormant flower buds rejuve- been reported, the embryological information is nate again. The Himalayan mayapple grows at insufficient. ground level. The flowers and leaves of mature plants develop through growth at the base of the plant. When the flower buds just emerge from Material and methods the ground, the microsporocytes initiate meiosis. There is an interval of about five or six days Sinopodophyllum hexandrum is a short-day plant. from the emergence of flowers to the completion The flower buds begin to form in the autumn and of pollination. remain dormant through the winter and early The flowering phenology depends on tem- spring. In late spring and early summer, the perature, but not much on precipitation. The flower buds resume activity and undergo anthe- earlier the temperature increases, the earlier the sis. Materials for embryological studies should flowering occurs. At the altitude of 3200 m, be collected from the middle of May to the the Himalayan mayapple usually flowers in the beginning of June. In the present study, the mate- middle of May. The higher the altitude, the later rials were collected in June 2007 and May 2008 the flowering takes place. Generally, when the ANN. BOT. FENNICI Vol. 49 • Female gametophyte development in Sinopodophyllum hexandrum 57 Fig. 1. Female game- tophyte development in Sinopodophyllum hexan- drum: Polygonum-type. — A: Female arche- sporium (arrow). — B: Megaspore mother cell. — C: Megasporocyte at prophase of meiosis. — D: Megasporocyte at telophase of meiosis. — E: Megaspore dyad, note similar shape and size. — F: Linear tetrad of megaspores (1–4). — G and H: A functional megaspore at chalazal end (F) and three degen- erated megaspores at micropylar end (2–4). — I: A functional megaspore at chalazal end (F) and traces of three degen- erated megaspores at micropylar end (arrow). — J: Uninucleate female gametophyte and traces of three degenerated megaspores at micro- pylar end (arrow). — K: Late stage of uninucleate female gametophyte and traces of three degen- erated megaspores at micropylar end (arrow). — L: Two-nucleate female gametophyte just formed. Scale bars: 10 µm. altitude increases by 100 m, the blossoming time locule develop asynchronously. When the carpel will delay by three to five days. margins coalesce, the cytoplast of the cells in The flower is solitary in a terminal position the basal part of the ovary thickens and grows on the aerial stem. It is composed of six sepals rapidly toward the locule to form a conic ovule in two whorls of three each, six petals in two primordial. Each primordium further develops, whorls of three each, six stamens, and a single and its apex forms the nucellus above the funicu- terminal pistil. The three outer sepals are vari- lus. Beneath the epidermis at the apex of the able in size and shape. The three inner sepals are nucellus, an archesporial cell differentiates. The elliptic and with a well-developed venation. The cell contains dense cytoplasm and an enlarged six petals are obovate, membranous and have a nucleus with a large nucleolus (Fig. 1A). The well developed venation. The carpel has a long archesporial cell does not divide, but enlarges style, and it enlarges to assume a sac-like shape. and directly becomes a megasporocyte. The Many ovules grow on the enlarged marginal megasporocyte is much larger than any of the placenta of the ocular ovary. Ovules in the same surrounding nucellar cells, and it contains dense 58 Huang & Li • ANN. BOT. FENNICI Vol. 49 At this time, the