Implications for Schisandraceae, Austrobaileyales, and the Early Evolution of Flowering Plants

FEMALE GAMETOPHYTE DEVELOPMENT IN KADSURA: IMPLICATIONS FOR SCHISANDRACEAE, AUSTROBAILEYALES, AND THE EARLY EVOLUTION OF FLOWERING PLANTS

William E. Friedman,1 William N. Gallup, and Joseph H. Williams2

Department of Ecology and Evolutionary Biology, University of Colorado, Boulder, Colorado 80309, U.S.A.

Recent phylogenetic analyses of angiosperms have identified a set of “basal” angiosperm lineages (Amborella, Nymphaeales, and a clade that includes Illiciaceae, Schisandraceae, Trimeniaceae, and Austrobaileyaceae) that are central to the study of the origin and early diversification of flowering plants. Prior to this phylogenetic revelation, much of the work on the embryology of ancient angiosperm lineages focused on core magnoliids (e.g., Magnoliales, Winterales). It is now apparent that little is known about the basic embryological features of the most ancient extant lineages of flowering plants, particularly with respect to the nature and development of the female gametophyte and the ploidy and genetics of the endosperm. Here, we report that Kadsura japonica (Schisandraceae) develops a four-celled female gametophyte with an egg cell, two synergids, and a uninucleate central cell. The pattern of free-nuclear divisions in the female gametophyte of Kadsura precisely matches what has recently been reported for four-celled gametophytes in the Nymphaeales. Following the first mitosis, migration of one of the two nuclei to the chalazal pole of the female gametophyte, as in Polygonum- type female gametophytes, does not occur. Rather, both nuclei remain close together in the micropylar domain where they undergo one additional mitotic division to yield four free nuclei before cellularization. Micro- spectrofluorometric analysis of relative DNA content of the central cell nucleus in Kadsura shows that this nucleus is haploid and contains the 1C quantity of DNA prior to fertilization. Thus, the endosperm of Kadsura should be diploid and biparental, as it is in Nuphar and other Nymphaeales. It now appears that four-celled female gametophytes, with consequent production of diploid endosperms, are common among the most ancient lineages of angiosperms, with the sole exception, to date, of Amborella. Finally, based on an analysis of the modular nature of the angiosperm female gametophyte, we provide evidence that four-celled female gametophytes that yield diploid biparental endosperms are likely to be plesiomorphic for flowering plants.

Keywords: angiosperm, evolution, developmental evolution, modularity, gametes, cell cycle, embryology.

Introduction Donoghue 1999, 2000; Parkinson et al. 1999; Qiu et al. 1999, 2000; Soltis et al. 1999, 2000; Barkman et al. 2000; Doyle More than 150 years after “comparative” biology emerged and Endress 2000; Graham and Olmstead 2000; Graham et as a central paradigmatic approach to the study of plant di- al. 2000; Zanis et al. 2002). It is now clear that these taxa are versity and evolution, one could be pardoned for assuming central to the study of the origin and early diversification of that everything about the basic biology and variation in the flowering plants. Prior to these recent phylogenetic inferences, reproductive process of plants has been described and cata- much of the work on the embryology of ancient angiosperm loged. Yet, despite recent progress in analyzing the phyloge- lineages had been focused on members of the Magnoliales, netic relationships of plants, much of the organismic diversity Winterales, and other assorted magnoliid lineages (Maneval of extant plants has never been studied, even at the most ru- 1914; Earle 1938; Hayashi 1965; Bhandari and Venkataraman dimentary level. These “morphological gaps” among plants 1968; Bhandari 1971a, 1971b; Heo et al. 1998). Given the remain as the largest obstacles to deciphering the key evolu- long-standing impression that Amborella, Nymphaeales tionary historical events that gave rise to the remarkable var- (Nymphaeaceae, Cabombaceae), and Austrobaileyales (Illici- iation of extant photosynthetic life. aceae, Schisandraceae, Trimeniaceae, and Austrobaileyaceae) Recent phylogenetic analyses of angiosperms have identified were not the most ancient (or potentially plesiomorphic) line- a set of “basal” angiosperm lineages that include Amborella, ages of flowering plants, relatively little attention was focused Nymphaeales, and Austrobaileyales (Illiciaceae, Schisandra- on these biologically diverse groups. Thus, little is known ceae, Trimeniaceae, and Austrobaileyaceae) (Mathews and about the basic embryological features of members of these flowering plants, particularly with respect to the development 1 Author for correspondence; telephone 303-492-3082; fax 303- and mature structure of the female gametophyte and the ploidy 492-8699; e-mail [email protected]. and genetics of the endosperm (Friedman 2001a, 2001b; Fried- 2 Current address: Department of Botany, University of Tennessee, man and Floyd 2001). Knoxville, Tennessee 37996, U.S.A.; e-mail [email protected]. For more than a century, the Polygonum-type (monosporic Manuscript received January 2003; revised manuscript received April 2003. seven-celled, eight-nucleate) female gametophyte has been as-

S293 This content downloaded from 128.103.149.052 on April 11, 2016 12:08:58 PM All use subject to University of Chicago Press Terms and Conditions (http://www.journals.uchicago.edu/t-and-c). This content downloaded from 128.103.149.052 on April 11, 2016 12:08:58 PM All use subject to University of Chicago Press Terms and Conditions (http://www.journals.uchicago.edu/t-and-c). FRIEDMAN ET AL.—FEMALE GAMETOPHYTE DEVELOPMENT IN KADSURA S295 sumed to be a defining feature and ancestral condition of the ology is likely to be plesiomorphic among flowering plants reproductive biology of flowering plants. Recently, however, (Friedman and Williams 2003). it was predicted (Friedman 2001b), and then confirmed (Wil- liams and Friedman 2002; Friedman and Williams 2003), that members of some of the most ancient lineages of flowering Material and Methods plants produce mature female gametophytes with only four uninucleate cells (an egg cell, two synergids, and a central cell with a single haploid nucleus) and that the endosperm of these Plant Collection lineages is genetically biparental and diploid. Kadsura japonica (L.) Dunal is an evergreen woody vine Among basal angiosperms, Nuphar polysepala (and perhaps native to Korea, Japan, and Taiwan (Saunders 1998). Kadsura all members of the Nymphaeaceae and Cabombaceae) pro- species are generally monoecious; however, K. japonica is oc- duces a four-celled female gametophyte (two synergids, an egg, casionally dioecious (Okada 1971). Staminate flowers bear nu- and a uninucleate central cell), and double fertilization in this merous spirally arranged cream-colored tepals and bright red taxon yields a diploid zygote and diploid endosperm (Williams stamens; carpellate flowers also bear numerous spirally ar- and Friedman 2002; Friedman and Williams 2003). It is also ranged cream-colored tepals and green apocarpous carpels. evident (based on an extensive analysis of the embryological Carpellate buds and flowers at various stages of development literature) that most basal angiosperms may yield four-celled were collected in July 2001 and July and September 2002 from female gametophytes and diploid endosperms (Friedman and specimens in the University of California Botanical Garden in Williams 2003; Williams and Friedman, in press). The only Berkeley, California (accession number 90.0667; Honshu, known currently documented exception to this generalization Japan). is found in Amborella, which has recently been shown to produce a Polygonum-type female gametophyte similar to most angiosperms (Tobe et al. 2000). Brightfield and Fluorescence Microscopy In this article, we report on the developmental embryology of the female gametophyte of Kadsura japonica, a member of Flowers were fixed for 24 h either in 50 mM PIPES buffer Schisandraceae and the broader Austrobaileyales clade that containing 5 mM EGTA and 1 mM MgSO4 with 4% acrolein also includes Illiciaceae, Trimeniaceae, and Austrobaileyaceae. (pH 6.8) and stored in PIPES buffer or in a solution of 3 : 1 Little is known about female gametophyte structure in Aus- (95% ethanol : acetic acid) and stored in 70% ethanol. Spec- trobaileyales. Illicium and Schisandra have been studied em- imens were dehydrated through an ethanol series and infil- bryologically (Yoshida 1962; Hayashi 1963a, 1963b; Kapil trated and embedded with glycol methacrylate (JB-4 embed- and Jalan 1964; Swamy 1964; Solntseva 1981), but there is ding kit, Polysciences, Warrington, Pa.). Embedded flowers considerable ambiguity about the specifics of female gameto- were serially sectioned into 5-mm-thick ribbons. Sectioned phyte structure in these taxa (Battaglia 1986). flowers were stained with either 0.1% toluidine blue or with The female gametophyte of Kadsura has been examined once 0.25 mg/mL of DAPI (4,6-diamidino-2-phenylindole) in 0.05 (Hayashi 1963b) and was reported to be of the Polygonum- M TRIS (pH 7.2). Digital imaging was performed on a Zeiss type. Given the prediction that four-celled female gameto- Axiophot microscope equipped with a Zeiss Axiocam digital phytes are potentially prevalent among basal angiosperms, we camera using both brightfield and fluorescence optics. Fluo- decided to reinvestigate the development of the female ga- rescence was visualized with an HBO 100 W burner (Carl metophyte in Kadsura. This study provides clear evidence that Zeiss, Oberkochen, Germany), using a UV filter set (model the female gametophyte of Kadsura is four-celled and should, 48702) with excitation filter (365 nm, band pass 12 nm), di- on double fertilization, yield a genetically biparental diploid chroic mirror (FT395), and barrier filter (LP397). Images were endosperm. These data, when viewed within the context of processed with Adobe Photoshop 6.0. Image manipulations the developmental evolution of the female gametophyte, were restricted to operations that were applied to the entire strengthen the conclusion that this pattern of reproductive bi- image except as noted in specific figure legends.

Fig. 1 Megasporogenesis and megagametogenesis in Kadsura. A, The megasporocyte features a micropylar-positioned nucleus and a cytoplasmically dense zone in the chalazal region. B, At the dyad stage, the megasporocyte becomes subdivided by a cell wall after meiosis I. C, Chalazal nucleus of the dyad at metaphase of meiosis II (the micropylar nucleus of the dyad may lag in the cell cycle). The cytoplasmically dense zone can be seen in the chalazal region. D, After meiosis II and cytokinesis, the chalazal-most megaspore cell becomes the functional megaspore, or the one-nucleate female gametophyte. At this stage, the cytoplasmically dense zone no longer appears in the chalazal region. The remaining megaspores degenerate and become crushed (one of three degenerate megaspores [arrow] is visible in this section). E, At the two-nucleate stage of female gametophyte development, the female gametophyte is highly vacuolate, and the two nuclei are positioned within the micropylar domain, typically in perpendicular orientation to the longitudinal axis of the female gametophyte. This image is a composite of two images from different focal planes of the same section (improved resolution). F, G, The four-celled, four-nucleate female gametophyte is composed of two synergids with ﬁliform apparatus (arrows) (F), an egg (G), and the large, vacuolated central cell containing a single central cell (polar) nucleus (F). The central cell nucleus is typically positioned near the center or the chalazal third of the mature female gametophyte. All sections were stained with toluidine blue. For all ﬁgures, the micropylar pole is at top and the chalazal pole at bottom.c p cytoplasmically dense zone;ccn p central cell nucleus;e p egg cell;fm p functional megaspore (one-nucleate female gametophyte);s p synergid . Scale bar p 10 mm.

Microspectrofluorometry within the female gametophyte of Kadsura yields four free nuclei, all of which are located in the micropylar domain. The Sections from flowers fixed in 3 : 1 (95% ethanol : acetic syncytial four-nucleate stage is apparently quite transitory be- acid) were stained with DAPI (see “Brightfield and Fluores- cause we never observed sectioned ovules in this developmental cence Microscopy”) for 1 h in the dark at room temperature. phase, although both young and mature four-celled gameto- Microspectrofluorometric measurements of relative DNA lev- phytes were common (fig. 1F,1G). However, one female ga- els of DAPI-stained nuclei were performed within 2 h. Mea- metophyte yielded the transitional stage from four-nucleate to surements were made with a Zeiss MSP 20 microspectropho- four-celled female gametophyte, with clear evidence of a phrag- tometer with digital microprocessor coupled to a Zeiss moplast depositing a cell plate between the egg cell and the Axioskop microscope equipped with epifluorescence (HBO central cell (fig. 2). 100 W burner). A UV filter set (model 48702) with excitation Cellularization of the syncytial female gametophyte yields filter (365 nm, band pass 12 nm), dichroic mirror (FT395), four uninucleate cells: two synergids, an egg cell, and a central and barrier filter (LP397) was used with a Zeiss Plan Neofluar cell. The synergids are extremely prominent, with an identi- #40 objective. Before each recording session, the photometer fiable filiform apparatus (fig. 1F). The egg cell abuts the two was standardized by taking a reading of fluorescence emitted synergids (fig. 3A,3B). Initially, the central cell nucleus is lo- from a fluorescence standard (GG 17), and this reading was cated very close to the egg apparatus (its original position at taken to represent 100 relative fluorescence units (RFU). At the conclusion of the second round of free-nuclear mitosis; fig. the completion of each session, an additional reading was made 2C), but following cellularization, it migrates near to the mid- of the fluorescence standard to confirm that little or no de- point of the length of the female gametophyte (fig. 1F,1G). viation in the relative fluorescence value obtained from the None of the more than 300 cellularized female gametophytes fluorescence standard had occurred during the period of data that we examined showed any evidence of the presence of recording. Relative DNA content for each nucleus was deter- antipodal cells or any nuclei at the chalazal pole. mined by summation of individual fluorescence values of each On two occasions (out of a total of 303 mature ovules ex- of the serial sections through that nucleus. A net photometric amined), we observed six nuclei within the female gameto- value for each section of a nucleus was obtained by recording phyte. The “extra” nuclei were found in the central cell. In an initial reading of the nucleus and subtracting a background one case, the three nuclei of the central cell were clustered value obtained from cytoplasm proximal to the nucleus. Thus, together, and in the other case (fig. 4A–4E), two nuclei were background fluorescence from the glycol methacrylate was re- clustered while the third nucleus was found in a different lo- moved from the photometric analysis of relative DNA content. cation. Given the rarity (in essence, teratological nature) of this event, we were unable to determine with absolute certainty Results the developmental preludes to this situation. Neither of these female gametophytes had been fertilized (no pollen tubes in Megasporogenesis micropyle; no degenerate synergid), indicating that the extra Megasporogenesis typically yields a linear tetrad of mega- nuclei in the central cell were not derived from sperm. As with spores of which the chalazal-most cell will develop into the all of the other mature female gametophytes observed, neither functional megaspore. A cytoplasmically dense zone is prom- of these female gametophytes showed any evidence of the pres- inent in the megasporocyte (in a position chalazal to the nu- ence (or past presence) of antipodals. cleus of this cell; fig. 1A) and is ultimately transmitted to the We can provide two possible explanations for these rare six- functional megaspore during megasporogenesis (fig. 1C), nucleate female gametophytes. Some angiosperms are known where it continues to reside at the chalazal pole. The functional to initiate female gametophytes in which the functional mega- megaspore contains countless small vacuoles that begin to co- spore is binucleate (a result of the lack of cytokinesis after alesce during the growth of the one-nucleate female gameto- meiosis II of the chalazal dyad cell). The more micropylar phyte (fig. 1D). Prior to the first mitosis of the female game- nucleus of this bisporic cell goes on to produce four nuclei, tophyte, the cytoplasmically dense zone disappears (fig. 1D). while the chalazal megaspore nucleus may degenerate, remain The degenerate megaspores are quickly crushed and are often intact without dividing, or divide once to yield two nuclei difficult to identify. (Haig 1990). In the latter case, female gametophytes with six nuclei will be formed. In our material, we only saw typical uninucleate megaspore tetrads. However, given the rarity of Female Gametophyte Development six-nucleate female gametophytes in Kadsura japonica (!1%), As noted, the one-nucleate stage of female gametophyte de- our histological sampling of megasporogenesis could easily velopment shows considerable vacuolation compared with have missed rare aberrations. stages of megasporogenesis. The first mitotic division in the We believe that a second hypothesis is the more likely ex- female gametophyte results in the formation of two free nuclei planation for these two anomalous female gametophytes. that are situated either at the extreme micropylar end of the Twenty-one of the total of 303 (ca. 7%) mature ovules con- female gametophyte (fig. 1E) or as much as a quarter of the tained a second female gametophyte in addition to the normal distance in length from the micropylar pole. At no time did four-celled female gametophyte. Of these 21 ovules with two we observe any evidence of migration of one of the two free female gametophytes, the second female gametophyte in 15 nuclei to the chalazal end of the female gametophyte, as would ovules was binucleate and appears to have arrested develop- occur in a typical Polygonum-type female gametophyte during ment at this stage (fig. 4F–4H). The other six ovules contained the two-nucleate stage. The second and final round of mitosis two four-celled female gametophytes. It is unclear whether the

Fig. 3 Kadsura egg apparatus. In this oblique view of the micropylar end of a mature female gametophyte, the egg cell (A) contains a nucleus near its junction with the central cell. The egg cell abuts the two synergids (B) whose nuclei lie near the micropylar wall of the female gametophyte. All sections were stained with DAPI and visualized with ﬂuorescence and DIC optics.cc p central cell; e p egg cell;s p synergid . Scale bar p 10 mm.

two female gametophytes derive from the initial formation of two megasporocytes or from the development of two functional megaspores from a single megasporocyte. In one of the two six-nucleate female gametophytes described above, clear evidence of a second (subordinate) female gametophyte was found (fig. 4A–4E); however, there were no nuclei present in this structure, and there appears to be a clear break in the cell wall material separating the two female gametophyte chambers (fig. 4D). We suspect that, occasionally, the degeneration of the subordinate supernumerary two-nucleate female gametophyte may result in the passage of its nuclei into the central cell of the dominant female gametophyte, thus yielding a six-nucleate structure. It is unknown whether these extra nuclei participate in the second fertilization event and thus produce a polyploid endosperm. During the free-nuclear and cellularization phases of development, the female gametophyte continues to expand in length and in width. At the one-nucleate stage, female gametophytes ranged from 55 to 90 mm long and from 20 to 23 mm wide. By the early four-celled stage, gametophytes ranged from 150 to 195 mm long and from 35 to 50 mm wide. During the final phases of maturation of the female gametophyte, the chalazal domain significantly enlarges in girth to yield a bulbous structure that may be up to 110 mm wide, while the entire gametophyte ranges from 215 to 230 mm long. Often, the central cell nucleus is positioned near the transition zone between the narrower micropylar domain and wider chalazal domain of

Fig. 2 Cellularization of the four-nucleate female gametophyte in Kadsura. Four sequential sections show the two synergid nuclei directly above one another (A, D) and the egg and central cell nuclei (B, C). The egg and central cell nuclei are in the process of being partitioned into separate cells by the phragmoplast (B–D; arrows). All sections were stained with toluidine blue. For all ﬁgures, the micropylar pole is at top and the chalazal pole at bottom.ccn p central cell nucleus; en p egg nucleus;sn p synergid nucleus. Scale bar p 10 mm.

This content downloaded from 128.103.149.052 on April 11, 2016 12:08:58 PM All use subject to University of Chicago Press Terms and Conditions (http://www.journals.uchicago.edu/t-and-c). This content downloaded from 128.103.149.052 on April 11, 2016 12:08:58 PM All use subject to University of Chicago Press Terms and Conditions (http://www.journals.uchicago.edu/t-and-c). FRIEDMAN ET AL.—FEMALE GAMETOPHYTE DEVELOPMENT IN KADSURA S299 the female gametophyte or may be found in the bulbous chal- Three of the values (137.60, 160.15, and 186.83; mean p azal end of the female gametophyte (figs. 1F,5F). 161.52) are very close to half of the RFU readings of the other three female gametophytes (306.09, 305.02, and 320.39; DNA Content and Ploidy of Central Cell mean p 310.50), indicating that the set of lower values closely approximates the 1C DNA content (haploid nuclei in G of It is possible that the four-celled mature female gametophyte 1 the cell cycle) and that the higher RFU values represent the of K. japonica could arise from a Polygonum-type female ga- 2C DNA content (haploid nuclei in G of the cell cycle). Im- metophyte where the three antipodals have degenerated and 2 portantly, as shown below, the lower RFU values for syncytial the two polar nuclei have fused into a secondary nucleus (dip- stage female gametophytes are very close to the average RFU loid central cell nucleus); this would yield a four-celled struc- values for egg and central cell nuclei, and the higher RFU values ture. To rule out this possibility, we measured relative DNA for syncytial stage female gametophytes are approximately contents of egg and central cell nuclei in mature female ga- double the average RFU values for egg and central cell nuclei. metophytes. If the central cell contains only a single haploid nucleus, the DNA content of this nucleus should be identical To determine whether the central cell of the female game- to that of the haploid egg nucleus, assuming that they occupy tophyte of Kadsura contains a single haploid nucleus or a similar positions within the cell cycle. If, however, the nucleus diploid fusion product of two polar nuclei (secondary nucleus of the central cell is the product of an unobserved fusion of of a Polygonum-type female gametophyte), we examined 26 two haploid polar nuclei, the ratio of DNA content of the unfertilized ovules (no pollen tubes present in micropyle; fig. central cell nucleus to the egg nucleus should be 2 : 1. 5F,5G). The mean fluorescence of the egg nuclei was ע ע Although large numbers of female gametophytes were ex- 165.0 27.8( mean SD ) RFU, strongly indicating that these amined with fluorescence microscopy, mitotic figures were nuclei are in G1 of the cell cycle and contain the 1C amount never observed; thus, we were unable to directly calibrate the of DNA because egg nuclei in sexual plants are haploid. If the relative fluorescence per C unit of DNA. However, the nuclei central cell nucleus contains a single haploid polar nucleus of a syncytial female gametophyte (fig. 5A,5B) are haploid whose cell cycle is synchronized with the egg nucleus, it should and vary in DNA content by a factor of 2 over the course of have a mean DNA fluorescence value of ca. 165 RFU, whereas if the central cell nucleus is a cryptic fusion product of two the cell cycle (1C during anaphase, telophase, and G1; between polar nuclei, it will have a mean DNA fluorescence value of 1C and 2C during the S phase; and 2C during G2, prophase, and metaphase). Measurements made on two one-nucleate fe- ca. 330 RFU. Kadsura central cell nuclei expressed a mean SD, which is clearly more similar to the 25.7 ע male gametophytes (fig. 5A) yielded values of 186.83 and RFU of152.4 320.39 relative fluorescence units (RFU) per nucleus for each 1C value of 165 RFU than to the 2C value of ca. 330 RFU. of the gametophytes. Measurements made on three two- The slight difference in egg nucleus and central cell nucleus nucleate female gametophytes (fig. 5B) yielded average RFU means (paired t-test:P p 0.014 , two-tailed test,n p 26 pairs) values of 306.09, 305.92, and 160.15 per nucleus for each of is probably not an artifact and may reflect subtle, yet real, the gametophytes. Finally, in the one four-nucleate female ga- differences in chromatin conformation between the two types metophyte undergoing cellularization (phragmoplast evident of nuclei (for discussion of these staining issues, see Narayan between incipient egg cell and central cell; figs. 2A–2D,5C– and Rees 1974; Santisteban et al. 1992; Kapuscinski 1995). 5E), the central cell nucleus emitted 137.60 RFU (the other Importantly, the DNA fluorescence values obtained from nuclei were not measurable). Given that the nuclei of this ga- central cell nuclei are completely inconsistent with the hy- metophyte have just emerged from mitosis, they should be in pothesis of a diploid central cell nucleus (or a haploid nucleus

G1 of the cell cycle and thus approximate the amount of fluo- in G2). The mean fluorescence of Kadsura central cell nuclei rescence associated with the 1C content of DNA. was significantly different from the predicted diploid 2C DNA The RFU values for the six free-nuclear-stage female ga- fluorescence level (paired t-test:P p 7.8 # 10Ϫ17 , two-tailed metophytes are distributed in a distinctly bimodal pattern. test,n p 26 pairs). These microspectrofluorometric data in-

Fig. 4 Ovules with two female gametophytes (a dominant mature female gametophyte and a subordinate partially developed female gametophyte) that may yield anomalous six-nucleate female gametophytes. A–E, An ovule in which the walls between a subordinate partially developed female gametophyte and a dominant female gametophyte with a mature egg apparatus have been breached. The contents of the subordinate female gametophyte (two free nuclei) appear within the central cell of the mature dominant female gametophyte. In this oblique view of a mature female gametophyte (fg1), the egg (A, B) and synergids (B) occupy the micropylar pole, and the central cell nucleus is in its typical central position surrounded by a denser zone of cytoplasm (D). A cell wall (arrows) indicates the presence of the subordinate female gametophyte (fg2) (C), which has a large rupture in it (D). This subordinate female gametophyte contains no nuclei (other sections not shown). Two small nuclei are, however, found in the chalazal portion of the mature female gametophyte (E), and these nuclei are not surrounded with cytoplasm, as is typical of a central cell nucleus or fusing polar nuclei (in taxa with two or more polar nuclei). For comparison, most mature ovules that contained two female gametophytes possessed a four-celled female gametophyte alongside a subordinate two-nucleate female gametophyte (F–H). The mature four-celled female gametophyte contains four nuclei: an egg (F), two synergids (G), and a single central cell nucleus (G). Directly adjacent to the mature female gametophyte is the subordinate female gametophyte (F–H). Two small nuclei (H), similar to those in E, remain within the subordinate female gametophyte. H, Composite of two sections (the upper of the two nuclei, designated with an arrow, is from a separate section, shown with arrow in inset). All sections were stained with toluidine blue. For all ﬁgures, the micropylar pole is at top and the chalazal pole at bottom.ccn p central cell nucleus;e p egg cell; fg1 p dominant female gametophyte; fg2 p subordinate female gametophyte; s p synergid. Scale bar p 10 mm.

This content downloaded from 128.103.149.052 on April 11, 2016 12:08:58 PM All use subject to University of Chicago Press Terms and Conditions (http://www.journals.uchicago.edu/t-and-c). This content downloaded from 128.103.149.052 on April 11, 2016 12:08:58 PM All use subject to University of Chicago Press Terms and Conditions (http://www.journals.uchicago.edu/t-and-c). FRIEDMAN ET AL.—FEMALE GAMETOPHYTE DEVELOPMENT IN KADSURA S301 dependently confirm our developmental observations derived they contain four nuclei and four cells (a uninucleate central from light microscopy: the Kadsura female gametophyte is cell, an egg cell, and two synergids) (Galati 1985; Winter and four-celled with a uninucleate haploid central cell (fig. 1F,1G; Shamrov 1991a, 1991b; Van Miegroet and Dujardin 1992; fig. 5F,5G). Moreover, the data strongly indicate that the egg Orban and Bouharmont 1998; Williams and Friedman 2002; and central cell remain in G1 before fertilization (see Friedman Friedman and Williams 2003). These findings stand in contrast 1999 for discussion of different patterns of cell cycle expression with the conclusions of earlier studies that reported seven- in plants during gametogenesis and fertilization). celled, eight-nucleate female gametophytes in Nymphaeales We also measured relative fluorescence values of synergid (Cook 1902, 1906; Khanna 1965, 1967; Ramji and Padma- nuclei in four-celled female gametophytes. The mean RFU of nabhan 1965; Schneider 1978). It is now evident that these SD. This indicates that sim- earlier reports are likely to be in error (Friedman and Williams 23.57 ע these nuclei was160.85 ilar to the egg and central cell nuclei the synergids are haploid 2003). and remain stationed in G1, at least until the time of There have been no recent studies of the female gametophyte fertilization. in any member of the basal angiosperm clade Austrobaileyales, which includes Illiciaceae, Schisandraceae, Trimeniaceae, and Discussion Austrobaileyaceae. Recent molecular systematic analyses indicate that Schisandraceae is apparently monophyletic and For the entire twentieth century, it was a central paradigm comprises members of Kadsura and Schisandra. However, nei- of flowering plant reproductive biology that the seven-celled, ther of these genera has been shown to be monophyletic, and eight-nucleate monosporic female gametophyte (Polygonum- it may be that both taxa are paraphyletic with respect to each type) was characteristic of the most ancient angiosperm line- other (Liu et al. 2000; Hao et al. 2001). Thus, for the purposes ages and indeed a synapomorphy of the entire angiosperm of our discussion, we will treat the entire Schisandraceae, with clade (Schnarf 1931; Maheshwari 1950; Johri 1963; Davis an explicit acknowledgement that members of Kadsura and 1966; Foster and Gifford 1974; Stebbins 1974; Palser 1975; Schisandra are potentially phylogenetically intermingled. Takhtajan 1976; Favre-DuChartre 1984; Cronquist 1988; Bat- Embryological studies of female gametophyte structure in taglia 1989; Haig 1990; Donoghue and Scheiner 1992; Johri the Schisandraceae were first undertaken in the early 1960s. et al. 1992). However, much has changed in light of the recent Yoshida (1962) published a short article in which he noted phylogenetic discovery that Amborella, Nymphaeales, and a that mature female gametophytes in Schisandra chinensis were clade that includes the Illiciaceae, Schisandraceae, Trimeni- four-celled and four-nucleate. Yoshida reported that in S. chi- aceae, and Austrobaileyaceae (Austrobaileyales) constitute a nensis, the single megaspore nucleus divides only twice to yield basal grade of the most ancient flowering plant lineages. Unlike a total of four free nuclei within the female gametophyte before other magnoliids that were carefully and frequently studied cellularization and sexual maturation. He also clearly showed embryologically (reviews by Hayashi 1965; Bhandari 1971a, (in drawings) that at the two-nucleate stage, the two nuclei 1971b), members of these recently identified most ancient an- are not separated by a large vacuole to the chalazal and mi- giosperm lineages were only rarely examined during the last cropylar ends, as is the case in the Polygonum-type female century. gametophyte two-nucleate stage. Rather, in S. chinensis, the Recent work on female gametophyte development in basal two free nuclei remain close together in the micropylar end of angiosperms has involved Amborella (Tobe et al. 2000) and the female gametophyte, as do the four free nuclei following members of the Nymphaeaceae and Cabombaceae (Galati the next and final round of mitotic division. The result is a 1985; Winter and Shamrov 1991a, 1991b; Van Miegroet and four-celled female gametophyte with an egg cell, two synergids, Dujardin 1992; Orban and Bouharmont 1998; Williams and and a central cell with a single polar nucleus (Yoshida 1962). Friedman 2002; Friedman and Williams 2003). In Amborella, Swamy (1964) also found that the female gametophyte of Tobe et al. (2000) have clearly shown that the female game- S. chinensis develops into a four-celled structure at maturity. tophyte is seven-celled and eight-nucleate at maturity, thus con- However, in addition to finding monosporic development, he forming to the Polygonum-type. In contradistinction to Am- suggested that 50% of the female gametophytes in this taxon borella, the most current studies of the mature female were bisporic. As Battaglia (1986) first pointed out, this report gametophytes of members of the Nymphaeales all indicate that of bisporic development in S. chinensis is likely to be based

Fig. 5 Fluorescence views of the development of Kadsura female gametophytes associated with DNA quantitation and analysis of the cell cycle. A, Early one-nucleate female gametophyte with a nucleus positioned at the micropylar end (arrow). Photometry indicates that this nucleus contains the 2C DNA content and is thus in G2 of the cell cycle. B, Two-nucleate stage with both nuclei (arrows) found oriented perpendicular to the longitudinal axis and in the micropylar domain of the female gametophyte. These nuclei possess the 1C DNA content, consistent with their position in G1 of the cell cycle. C–E, Three of four adjacent sections through a single female gametophyte that show the end of the four- nucleate stage during cellularization (same gametophyte as in fig. 2A,2C,2D). The two synergid nuclei can be seen in C and E after their mitosis, perpendicular to the longitudinal axis. The central cell nucleus and egg nucleus (D) lie parallel to the longitudinal axis. Photometry confirms that the central cell nucleus is in G1 of the cell cycle, as should be the case for a nucleus that has just completed mitosis. The mature female gametophyte (F, G) contains an egg nucleus (F), two synergid nuclei (G), and a central cell nucleus that has migrated to the chalazal region (F). The egg and central cell nuclei are both in G1 of the cell cycle (see “Results”). All sections were stained with DAPI. For all figures, the micropylar pole is at top and the chalazal pole at bottom.ccn p central cell nucleus;en p egg nucleus;sn p synergid nucleus. Scale bar p 10 mm.

dra; they assumed that Yoshida, like themselves, had missed witnessing a developmental stage prior to the supposed degeneration of the antipodals and the fusion of the two polar nuclei into a secondary nucleus. In light of the contradictory reports of four-celled and seven- celled female gametophytes in Schisandraceae, we decided to resolve the characteristics of female gametophyte development in K. japonica through a combination of developmental analysis and microspectrofluorometric analysis of the ploidy of the central cell. Our results clearly and unambiguously show that the female gametophytes of K. japonica are monosporic and produce four-nucleate, four-celled female gametophytes with an egg cell, two synergids, and a haploid uninucleate central cell. Moreover, the pattern of free-nuclear divisions precisely matches the pattern reported for four-celled gametophytes in the Nymphaeales (Friedman and Williams 2003). Following the first mitosis, there is no migration of one of the two nuclei to the chalazal pole of the female gametophyte (as first reported correctly for Schisandraceae by Yoshida 1962), as would occur in a Polygonum-type female gametophyte. Rather, both nuclei remain close together in the micropylar domain, where they Fig. 6 Comparative development of Kadsura female gametophytes undergo one additional mitotic division to yield four free nuclei and Polygonum-type female gametophytes. The female gametophytes (fig. 2). These four nuclei are confined to the micropylar end of angiosperms are fundamentally modular structures in which indi- of the female gametophyte. Only after cellularization does the vidual developmental modules consist of quartets of nuclei. Each mod- central cell nucleus migrate to a more chalazal position within ule (outlined in a gray box) is initiated from a single nucleus that the female gametophyte (figs. 1F,5F). undergoes two free-nuclear mitoses to yield four nuclei. Cellularization Given our very large sample size of four-celled female ga- of a four-nucleate module involves partitioning of three uninucleate metophytes (1300) and that no one, including Hayashi (1963b) cells, while the fourth nucleus is contributed to the central cell of the female gametophyte. The lack of migration of a nucleus to the chalazal and Kapil and Jalan (1964), has ever presented photomicro- pole at the two-nucleate stage of development in Kadsura japonica graphic evidence, as opposed to drawings, of an actual Polyg- results in the establishment of a single modular quartet. Conversely, onum-type seven-celled, eight-nucleate stage in any species of in angiosperms with Polygonum-type female gametophytes, migration Schisandraceae, it seems very likely that Hayashi (1963b) and of one of the nuclei to the chalazal pole at the two-nucleate stage Kapil and Jalan (1964) misinterpreted their embryological results in the establishment of a second (chalazal) developmental mod- data. Thus, we conclude that, like the Nymphaeales (Williams ule (in addition to the micropylar module) that ultimately forms three and Friedman 2002; Friedman and Williams 2003), all evi- p p antipodal cells and a polar nucleus.a antipodal cell; c central dence points to the presence, and prevalence, of monosporic p p cell nucleus (or polar nucleus);e egg cell;s synergid cell. four-celled, four-nucleate female gametophytes in the Schisandraceae. on a misinterpretation of some of the early stages of megasporogenesis and female gametophyte development. Our ex- Evolution of Female Gametophyte Ontogeny in amination of Swamy’s plates also finds no evidence of bisporic Ancient Angiosperm Lineages development in his drawings of megasporogenesis. Contemporaneous with the above two reports (Yoshida Only two known female gametophyte ontogenetic sequences 1962; Swamy 1964), Hayashi (1963b) reported Polygonum- are present in the most ancient clades of extant angiosperms: type female gametophytes in Schisandra repanda and Kadsura the monosporic four-celled, four-nucleate sequence character- japonica. Importantly, however, Hayashi noted that he typi- istic of Schisandraceae, Nymphaeales, and possibly the Illici- cally saw only the egg apparatus (three cells) and what he aceae (further study of Illicium is needed to resolve contra- described as the secondary nucleus derived from the fusion of dictory reports in the earlier literature) and the monosporic the two polar nuclei in mature female gametophytes. Never- seven-celled, eight-nucleate sequence of Amborella (Tobe et al. theless, he presented drawings of syncytial, eight-nucleate fe- 2000). Although the polarity of evolutionary transition is, on male gametophytes and antipodal cells in Polygonum-type ga- strict grounds of parsimony, unresolved, Friedman and Wil- metophytes for both taxa. liams (2003) argued that four-celled, four-nucleate female ga- Kapil and Jalan (1964) analyzed female gametophyte de- metophytes are likely to be plesiomorphic among angiosperms, velopment in Schisandra grandiflora. Like Hayashi, these with seven-celled, eight-nucleate female gametophytes derived. workers reported that the female gametophyte is eight-nucleate As recently (and long ago) argued, the female gametophyte and of the Polygonum-type. Ironically, all of their figures of angiosperms is best viewed as a fundamentally modular (drawings) show four-celled female gametophytes with a three- structure in which individual developmental modules consist celled egg apparatus and a central cell with a single nucleus. of “quartets” of nuclei. Each module can be characterized by In addition, they stated (incorrectly) that Yoshida (1962) had a common developmental pattern: (1) positioning of a single also found Polygonum-type female gametophytes in Schisan- nucleus within a cytoplasmic domain (pole) of the female ga-

This content downloaded from 128.103.149.052 on April 11, 2016 12:08:58 PM All use subject to University of Chicago Press Terms and Conditions (http://www.journals.uchicago.edu/t-and-c). FRIEDMAN ET AL.—FEMALE GAMETOPHYTE DEVELOPMENT IN KADSURA S303 metophyte, (2) two free-nuclear mitoses to yield four nuclei Johri 1963; Davis 1966; Foster and Gifford 1974; Stebbins within that domain, and (3) partitioning of three uninucleate 1974; Palser 1975; Takhtajan 1976; Favre-DuChartre 1984; cells adjacent to the pole such that the fourth nucleus is con- Cronquist 1988; Battaglia 1989; Haig 1990; Donoghue and fined to the central cell of the female gametophyte (Friedman Scheiner 1992; Johri et al. 1992; Tobe et al. 2000). It is now and William 2003). Angiosperm female gametophytes may in- evident that this type of female gametophyte may be rare itiate one (most basal angiosperms), two (most angiosperms), among basal angiosperms (namely, Nymphaeales and Austro- or even four modules (e.g., Penaea-type female gametophyte) baileyales), while the four-celled, four-nucleate type of female (Porsch 1907; Schnarf 1936; Maheshwari 1950; Swamy and gametophyte characteristic of Schisandraceae, Nymphaeaceae, Krishnamurthy 1975; Favre-DuChartre 1976; Battaglia 1989; and Cabombaceae may be typical of the most ancient angio- Haig 1990; Friedman and Williams 2003). In K. japonica and sperm lineages as well as plesiomorphic for flowering plants other basal angiosperm taxa with four-celled female gameto- as a whole. Although we did not observe fertilization in K. phytes, all nuclei are confined to the micropylar domain during japonica, the clear documentation of a uninucleate haploid free-nuclear development, and this results in the establishment central cell indicates that if double fertilization occurs in this of a single modular quartet (fig. 6). Conversely, in Amborella taxon (and in other members of the Schisandraceae), the en- and other angiosperms with Polygonum-type female gameto- dosperm will be diploid with a 1 : 1 maternal to paternal ge- phytes, migration of one of the nuclei to the chalazal pole at nome ratio. This would stand in marked contrast with the the two-nucleate stage results in the establishment of a chalazal endosperms of flowering plants with Polygonum-type female developmental module (in addition to the micropylar module) gametophytes in which the endosperm is triploid and contains that ultimately forms three antipodal cells and a (second) polar a 2 : 1 maternal to paternal genome ratio. nucleus (fig. 6). A careful investigation, and reinvestigation, of the most ba- The evolutionary developmental analysis of Friedman and sic embryological features of early divergent angiosperm line- Williams (2003) posited that the first angiosperm female ga- ages is clearly needed. The female gametophytes of Austro- metophytes were composed of a single four-nucleate and four- baileya have never been studied. Embryological studies of the celled module that, on double fertilization, yielded a diploid female gametophytes of Illicium are contradictory (one report endosperm. Early in angiosperm history, ectopic expression of of a four-celled female gametophyte [Solntseva 1981] and one this basic developmental module resulted in the initiation of report of a seven-celled female gametophyte [Hayashi 1963a]). two developmental modules within the female gametophyte Moreover, even a cursory examination of the literature on and the formation of a seven-celled, eight-nucleate structure female gametophyte structure and fertilization biology of basal that yields a triploid endosperm with the 2 : 1 maternal to monocots (e.g., Acorus, diverse alismatids) indicates that vir- paternal genome ratio characteristic of most flowering plants. tually nothing can be inferred with any certainty. Until the The means of establishing a duplicate module was the devel- reproductive biology of these phylogenetically key basal an- opmental insertion of a cytoskeletal apparatus after the first giosperm taxa is analyzed anew, the “morphological gaps” that mitotic division that resulted in the positioning of the nuclei are the result of our own ignorance will continue to prevent (at the two-nucleate stage) in distinct cytoplasmic domains at us from fully reconstructing the major evolutionary historical opposite poles of the female gametophyte. The net effect of events that have produced the remarkable diversification of this novel nuclear migration process was the parallel initiation flowering plants during the last 130 million years. and development of two modular quartets and the creation of a seven-celled and eight-nucleate Polygonum-type female gametophyte (Friedman and Williams 2003). Acknowledgments

Conclusions We thank Holly Forbes for assistance with the collection of plant materials at the University of California Botanical Gar- The monosporic seven-celled, eight-nucleate Polygonum- den, Berkeley. This research was supported by grants from the type female gametophyte has long served as the baseline for National Science Foundation (IBN 9816107) and the Com- analysis of the origin and subsequent evolution of the angio- mittee on Research and Creative Works at the University of sperm female gametophyte (Schnarf 1931; Maheshwari 1950; Colorado.

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