MODULARITY OF THE ANGIOSPERM FEMALE GAMETOPHYTE AND ITS BEARING ON THE EARLY EVOLUTION OF ENDOSPERM IN FLOWERING Author(s): William E. Friedman and Joseph H. Williams Source: Evolution, 57(2):216-230. Published By: The Society for the Study of Evolution DOI: http://dx.doi.org/10.1554/0014-3820(2003)057[0216:MOTAFG]2.0.CO;2 URL: http://www.bioone.org/doi/full/10.1554/0014-3820%282003%29057%5B0216%3AMOTAFG %5D2.0.CO%3B2

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BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research. Evolution, 57(2), 2003, pp. 216±230

MODULARITY OF THE ANGIOSPERM FEMALE GAMETOPHYTE AND ITS BEARING ON THE EARLY EVOLUTION OF ENDOSPERM IN FLOWERING PLANTS

WILLIAM E. FRIEDMAN1,3 AND JOSEPH H. WILLIAMS2,3 Department of Environmental, Population and Organismic Biology, University of Colorado, Boulder, Colorado 80309 1E-mail: [email protected]

Abstract. The monosporic seven-celled/eight-nucleate Polygonum-type female gametophyte has long served as a focal point for discussion of the origin and subsequent evolution of the angiosperm female gametophyte. In Polygonum- type female gametophytes, two haploid female nuclei are incorporated into the central cell, and fusion of a sperm cell with the binucleate central cell produces a triploid endosperm with a complement of two maternal and one paternal genomes, characteristic of most angiosperms. We document the development of a four-celled/four-nucleate female gametophyte in polysepala (Engelm.) and infer its presence in many other ancient lineages of angiosperms. The central cell of the female gametophyte in these taxa contains only one haploid nucleus; thus endosperm is diploid and has a ratio of one maternal to one paternal genome. Based on comparisons among ¯owering plants, we conclude that the angiosperm female gametophyte is constructed of modular developmental subunits. Each module is charac- terized by a common developmental pattern: (1) positioning of a single nucleus within a cytoplasmic domain (pole) of the female gametophyte; (2) two free-nuclear mitoses to yield four nuclei within that domain; and (3) partitioning of three uninucleate cells adjacent to the pole such that the fourth nucleus is con®ned to the central region of the female gametophyte (central cell). Within the basal angiosperm lineages and Illiciales, female game- tophytes are characterized by a single developmental module that produces a four-celled/four-nucleate structure with a haploid uninucleate central cell. A second pattern, typical of Amborella and the overwhelming majority of eumag- noliids, monocots, and eudicots, involves the early establishment of two developmental modules that produce a seven- celled/eight-nucleate female gametophyte with two haploid nuclei in the central cell. Comparative analysis of onto- genetic sequences suggests that the seven-celled female gametophyte (two modules) evolved by duplication and ectopic expression of an ancestral Nuphar-like developmental module within the chalazal domain of the female gametophyte. These analyses indicate that the ®rst angiosperm female gametophytes were composed of a single developmental module, which upon double fertilization yielded a diploid endosperm. Early in angiosperm history this basic module was duplicated, and resulted in a seven-celled/eight-nucleate female gametophyte, which yielded a triploid endosperm with the characteristic 2:1 maternal to paternal genome ratio.

Key words. ANITA grade, developmental evolution, heterotopy, module, , ontogenetic sequence.

Received July 17, 2002. Accepted October 1, 2002.

Fertilization in angiosperms occurs at the intersection of cell with the central cell produces the triploid endosperm char- ®ve distinct ontogenies: the male gametophyte, female ga- acteristic of the overwhelming majority of angiosperms. metophyte, maternal sporophyte, embryo, and endosperm. For most of the twentieth century, biologists interested in The unique double fertilization process of ¯owering plants the early evolution of angiosperms worked with putatively produces an embryo, and an endosperm that serves as a nour- ``primitive'' groups, such as Magnoliaceae, Winteraceae, and ishing tissue for the embryo. In all angiosperms the male other magnoliid lineages. Embryologists documented the gametophyte produces two haploid sperm cells, one of which widespread occurrence of the Polygonum-type female ga- contributes to the embryo and the other to the endosperm. metophyte in diverse magnoliids and considered its ubiquity The ¯owering female gametophyte also produces two one of the strongest indications of the monophyly of angio- sexual cells that participate in the process of double fertil- sperms (Strasburger 1905; Maneval 1914; Chiarugi 1927; ization: a uninucleate egg cell, and a central cell that may Schnarf 1931; see Gerassimova-Navashina 1961 for an ex- initially contain from one to 14 nuclei, depending on the ception). Subsequent studies have not modi®ed this view. taxon (Johri et al. 1992). Thus, the ploidy level and the ratio Bhandari (1971) used an expanded concept of Magnoliales of maternal:paternal genomes of endosperm are directly re- (sensu Cronquist 1968) as ``primitive'' but still found little lated to the ploidy of the central cell. Variation in the ploidy variation in female gametophyte ontogeny. More recently, of the central cell is, in turn, a consequence of variation in Donoghue and Scheiner (1992) examined female gameto- female gametophyte ontogeny. phyte types within an explicitly phylogenetic context. These Most angiosperms produce a mature female gametophyte authors performed a character analysis with many alternative with eight nuclei (a product of three mitoses of a single func- rootings of angiosperms (Magnoliales, Laurales, Winterales, tional megaspore nucleus) that are partitioned into seven cells. tricolpates, and paleoherbs) and concluded that regardless of This is referred to as the ``normal type'' or Polygonum type the rooting, a seven-celled/eight-nucleate female gameto- (Strasburger 1879). In such female gametophytes, two nuclei phyte (and inferred triploid endosperm) is plesiomorphic. are incorporated into the central cell, and fusion of the sperm Thus, throughout the twentieth century, irrespective of the phylogenetic hypothesis favored for basal angiosperms, it has been a paradigm of plant embryology that the Polygonum- 2 Present address: Department of Botany, University of Tennes- type female gametophyte and triploid endosperm are ances- see, Knoxville, Tennessee 37996; E-mail: [email protected]. tral features of ¯owering plants. 3 The authors contributed equally to the work. Within the last four years, molecular phylogenetic analyses 216 ᭧ 2003 The Society for the Study of Evolution. All rights reserved. DEVELOPMENTAL EVOLUTION OF ANGIOSPERMS 217 have dramatically altered hypotheses of angiosperm rela- tionships. Several multigene analyses have converged on three angiosperm clades, the ``ANITA'' grade, that are basal to the common ancestor of the monocots and eudicots (Ma- thews and Donoghue 1999; Parkinson et al. 1999; Qiu et al. 1999, 2000; Soltis et al. 1999; Graham and Olmstead 2000; Graham et al. 2000; Zanis et al. 2002). These three clades, consisting of Amborellaceae, Nymphaeales, and the Illi- ciales/Trimeniaceae/Austrobaileyaceae (ITA) clade, are now considered to represent the earliest diverging branches of the angiosperm tree. Many embryological studies of taxa within the newly rec- ognized basal ANITA grade exist. Remarkably, these works often ®nd the mature female gametophyte to be four-celled and four-nucleate, and not seven-celled and eight-nucleate as in the putative plesiomorphic Polygonum type. Within the ANITA grade, four-celled female gametophytes have been reported in four of six genera studied in Nymphaeaceae, one of two genera in Cabombaceae, and two of three genera in Illiciales (Yoshida 1962; Swamy 1964; Solntseva 1981; Ba- tygina et al. 1982; Galati 1985; Winter 1987, 1990, 1993; Titova 1990; Winter and Shamrov 1991a,b; Van Miegroet and Dujardin 1992; Orban and Bouharmont 1998; Shamrov 1998). Unfortunately, many of these studies con¯ict with others in which seven-celled/eight-nucleate female gameto- phytes have been reported, often for the same taxa (Cook 1902, 1906; Conard 1905; Seaton 1908; Kapil 1962; Hayashi 1963a,b; Kapil and Jalan 1964; Khanna 1964, 1965, 1967; Ramji and Padmanabhan 1965; Schneider 1978; Batygina et al. 1980; Schneider and Jeter 1982). To compound the un- FIG. 1. Convergence in form of female gametophytes of early certainty, many of these embryological works lack even a angiosperm lineages. (A) A seven-celled/eight-nucleate female ga- single drawing as documentation, while others offer inter- metophyte undergoes degeneration of three sterile cells (antipodals) and fusion of the two nuclei of the central cell to yield a four-celled pretations inconsistent with the ®gures. Moreover, Mahesh- female gametophyte with a diploid central cell. (B) A ``true'' four- wari (1947, 1950) judged all reports of four-celled female celled female gametophyte contains a single haploid nucleus in the gametophytes in taxa outside of the Onagraceae to be erro- central cell, as in Nuphar. As shown to the right of the dashed line, neous or unsubstantiated, since their development had not the ploidy of the central cell of the female gametophyte ultimately determines the ploidy and genomic composition of the primary been adequately or precisely studied. endosperm nucleus. There is a signi®cant complication to documenting four- celled/four-nucleate female gametophytes. In many angio- sperms, an incipient seven-celled/eight-nucleate female ga- Bouharmont 1998). To resolve differences in interpretation metophyte is developmentally transformed into a four-celled/ due to convergence in mature form, it is critical to document four-nucleate structure at maturity: the three chalazal antipodal the entire ontogeny of the female gametophyte and additionally cells undergo cell death and the two polar nuclei within the to quantitate the ploidy of the mature central cell. central cell fuse rapidly to form a single diploid nucleus (the In 2002, we discovered diploid endosperm in Nuphar,a secondary nucleus; Fig. 1). As a result, reports of four-celled member of one of the earliest divergent angiosperm lineages, female gametophytes are especially subject to scrutiny, be- the Nymphaeales (Williams and Friedman 2002). In that cause of the dif®culty in differentiating a ``true'' four-celled study we determined endosperm ploidy directly using quan- female gametophyte from a ``cryptic'' seven-celled female titative microspectro¯uorometry to assess the DNA content gametophyte (in which antipodals have degenerated and polar of the primary endosperm nucleus. Identi®cation of the dip- nuclei have fused). Indeed, major comparative embryological loid and sexual origin of endosperm in Nuphar provided the reviews characterize female gametophytes in all seven ANITA ®rst independent documentation of the nature of the second grade families as possessing ephemeral antipodals and under- fertilization event in any ANITA grade angiosperm. Although going rapid fusion of the two polar nuclei within the central female gametophyte ontogeny was not described, the ®nding cell (Table 1). Thus, female gametophytes in basal angio- that diploid endosperm in Nuphar resulted from fertilization sperms should all appear as four-celled and four-nucleate of a haploid uninucleate central cell con®rmed that its mature structures at maturity. However, a few reviews claim female female gametophyte was truly four-celled and four-nucleate gametophytes in some ANITA grade taxa are truly four-celled (see Fig. 1). structures in which two sets of mitotic divisions (from the The present study seeks to understand the developmental functional megaspore) yield four nuclei that are partitioned constraints and pathways associated with endosperm evolu- into four cells (Battaglia 1986; Batygina 1994; Orban and tion in basal angiosperms. This understanding can only be 218 W. E. FRIEDMAN AND J. H. WILLIAMS

TABLE 1. Characteristics of late female gametophyte ontogeny of ANITA-grade angiosperms, based on major embryological reviews. Anti- podals are reported to degenerate soon after their formation; within the central cell, polar nuclei are said to fuse before fertilization.

Family Antipodals Polar nuclei Reference Amborellaceae ephemeral fuse before fertilization Tobe et al. 2000 Nymphaeaceae very ephemeral fuse before fertilization Davis 1966 ephemeral Ð Johri et al. 1992 Cabombaceae ephemeral fuse before fertilization Davis 1966 ephemeral Ð Johri et al. 1992 Austrobaileyaceae ephemeral Ð Johri et al. 1992 Trimeniaceae antipodals probably present Ð Johri et al. 1992 Illiciaceae ephemeral Ð Davis 1996 ephemeral Ð Johri et al. 1992 Schisandraceae very ephemeral fuse before fertilization Davis 1996 ephemeral or missing Ð Johri et al. 1992

achieved through knowledge of female gametophyte ontog- whereas several peduncles possessing open ¯owers with ma- eny. Thus, we ®rst describe the complete ontogenetic se- ture anthers served as pollen donors. These were cut, placed quence of the four-celled/four-nucleate Nuphar female ga- in water and transported to the lab. Three hours later ``re- metophyte that yields a diploid endosperm upon fertilization. cipients'' were hand-pollinated with pollen from at least two We next ask how this pattern of female gametophyte ontog- ``donors.'' Pollinations were performed by rubbing open an- eny differs from that of seven-celled/eight-nucleate female thers across the stigmatic ridges. Ovules were then chemi- gametophytes (which produce triploid endosperm) in various cally ®xed at 1, 5, 13, 22, 38, or 53 h after pollination. other early lineages of angiosperms (Amborella, basal eudi- Flowers were ®xed for 24 h either in 3:1 (95% ethanol: cots, eumagnoliids, and basal monocots). Finally we seek to acetic acid) and stored in 70% ethanol, or in a solution of understand the developmental basis for evolutionary transi- 50-mM piperozine-N, N-bis (2-ethanesulfonic acid) 1.5 so- tions between four-celled and seven-celled female gameto- dium (PIPES) buffer (also 5-mM EGTA and 1-mM MgSO4) phyte ontogenies found among ancient angiosperm lineages. with acrolein at 4% concentration (ph 6.8) and stored in As we will demonstrate, subtle yet de®nable modi®cations PIPES buffer. Specimens were dehydrated through an ethanol in the development of the female gametophyte are the motive series, and in®ltrated and embedded with glycol methacrylate force behind key evolutionary transitions in the genetics, (JB-4 embedding kit, Polysciences, Inc., Warrington, PA). ploidy, and maternal:paternal genomic ratios of endosperm. Embedded ¯owers were serially sectioned into 5-␮m thick We conclude that four-celled female gametophytes can be ribbons. Sectioned ¯owers were stained with either 0.1% to- viewed as a basic and modular unit of developmental evo- luidine blue, or with 0.25 ␮g/ml of DAPI (4Ј,6-diamidino- lution for angiosperm female gametophytes. According to our 2-phenylindole) in 0.05 M tris (ph 7.2). Digital imaging was hypothesis, the seven-celled female gametophyte is a devel- on a Zeiss axiophot microscope equipped with a Zeiss axi- opmental by-product of ectopic duplication of this basic mod- ocam digital camera using both bright ®eld and ¯uorescence ule. Thus we posit that early angiosperms possessed a diploid optics. Fluorescence was visualized with an HBO 100-W endosperm associated with a four-celled and four-nucleate burner (Carl Zeiss, Oberkochen, Germany), using a UV ®lter female gametophyte, and that a triploid endosperm and the set (model no. 48702) with excitation ®lter (365 nm, band seven-celled and eight-nucleate female gametophyte are de- pass 12 nm), dichroic mirror (FT395), and barrier ®lter rived features of angiosperms. Finally, we show that our hy- (LP397). Images were processed with Adobe Photoshop 5.0 pothesis is congruent with theoretical expectations (Haig and (Adobe Systems, San Jose, CA). Image manipulations were Westoby 1989; Queller 1989; Friedman 1995) that posit that restricted to operations that were applied to the entire image. triploid endosperm evolved after a diploid fertilization prod- uct acquired embryo-nourishing behavior prior to the origin RESULTS of extant angiosperms. Megasporogenesis and Megagametogenesis MATERIALS AND METHODS The Nuphar ovule is anatropous and bitegmic (Fig. 2A). Nuphar polysepala (Engelm.) is an aquatic perennial found The micropyle is formed by the inner integument. A single in ponds and lakes throughout western North America (orig- (rarely two) megasporocyte forms about four or ®ve cells inally known as N. polysepalum; Wiersma and Hellquist deep within the nucellus. The megasporocyte acquires a mi- 1997). Flowers are borne above water on stout peduncles. cropylar-chalazal polarity, as evidenced by the micropylar Buds and ¯owers at various stages of development were col- positioning of the nucleus at prophase/metaphase of meiosis lected in June/July of 2000 and 2001 at Red Rocks Lake I and presence of a dense zone of DNA-staining material (Brainard Lake Recreation area), Colorado. Supplementary (suggesting the presence of mitochondria and plastids; data pollinations were also conducted to study male gametophyte not shown) in the chalazal region (Fig. 2A±C). After meiosis development and fertilization. Flowers are protogynous and I, the micropylar cell of the dyad is smaller than the chalazal become receptive before buds open. Twelve peduncles with cell, and the contents of its nucleus often appear disorganized unopened mature ¯ower buds served as pollen recipients, (Fig. 2D). The chalazal cell of the dyad retains the cyto- DEVELOPMENTAL EVOLUTION OF ANGIOSPERMS 219

FIG. 2. Megasporogenesis in Nuphar. (A) The ovule has two integuments and assumes an anatropous orientation during the megasporocyte stage. (B) Major features of the megasporocyte are a micropylar-positioned nucleus (here in early prophase with nucleolus still visible), and a cytoplasmically dense zone within the chalazal region. (C) At metaphase of meiosis I the megasporocyte has completed growth (composite image from two sections). (D) At the dyad stage, after meiosis I, the megasporocyte has been subdivided by a cell wall. The chalazal cell is larger and retains the cytoplasmically dense zone. (E) Meiosis II often fails or is delayed in the micropylar cell of the dyad (D), whereas the chalazal cell of the dyad has undergone meiosis II to form two cells. The chalazalmost cell of these two cells becomes the functional megaspore (indicated by a bracket) and contains the cytoplasmically dense zone. (F) The functional megaspore becomes vacuolate and enlarges. The cytoplasmically dense zone is redistributed from the chalazal zone to a region close to the nucleus. The two nonfunctional megaspores appear degenerate and become crushed by growth of the functional megaspore. All ®gures are oriented with the micropylar pole at top, chalazal pole at bottom. Scale bar, 15 ␮m (50 ␮ms in A). c, cytoplasmically dense zone; fm, functional megaspore; o, ovule. plasmically dense zone (Fig. 2D). Meiosis II usually fails or gametophyte, but instead remain in close proximity to each is severely delayed in the micropylar cell of the dyad, whereas other in the micropylar domain (Fig. 3D). A second round the chalazal cell of the dyad undergoes meiosis II to produce of mitosis in the female gametophyte is synchronous. After two daughter cells (Fig. 2E). The result of megasporogenesis the second mitosis, each pair of nuclei initially remains in is the formation of a linear triad (rarely a tetrad) of cells, of close proximity within the micropylar domain (Fig. 3E). At which the chalazalmost cell, containing the cytoplasmically the four-nucleate stage of development, the chalazalmost nu- dense zone, becomes the functional megaspore (Fig. 2E, F). cleus migrates to a position near the center of the female Thus, in Nuphar, the female gametophyte is monosporic and gametophyte (Fig. 3F). develops from the chalazal megaspore. Cellularization occurs at the four-nucleate stage, yielding As the functional megaspore enlarges, its nucleus becomes three small cells at the micropylar pole that will comprise situated in the micropylar portion of the cell (Figs. 3A±C). the egg apparatus (egg and two synergids; Fig. 3F, G). The Growth of the one-nucleate female gametophyte produces a remainder of the female gametophyte (the ``central cell'') is wide micropylar region and a narrow elongate chalazal re- occupied by a large vacuole and a single nucleus positioned gion. Because these regions of the female gametophyte differ at the point where the large micropylar chamber becomes in shape and possess distinct and presumably autonomous constricted and elongate (Fig. 3F). Migration of the central patterns of free-nuclear development at all later stages of cell nucleus to a position close to the egg apparatus does not ontogeny, we hereafter refer to them as domains (Boisnard- occur (as it does in many derived groups of angiosperms; Lorig et al. 2001). The club shape of the female gametophyte Russell 1993), but a thin strand of cytoplasm is seen leading is retained up to and beyond fertilization, and is also char- from the egg apparatus to the central cell nucleus. Subsequent acteristic of other members of the Nymphaeaceae, Cabomba, development of the female gametophyte is characterized by Trimeniaceae, and some eumagnoliids (Earle 1938; Batygina enlargement of the central cell nucleus and vacuolation of et al. 1982; Endress and Sampson 1983; Heo et al. 1998), the egg nucleus (synergids are relatively less vacuolate). but not of other basal angiosperms such as Amborella, Acorus, Thus, structural development results in a four-celled/four- Chloranthaceae, or Ceratophyllaceae (Buell 1938; Vijayar- nucleate structure within the micropylar domain of the Nu- aghavan 1964; Batygina et al. 1982; Tobe et al. 2000). phar female gametophyte. The chalazal domain of the female At the one-nucleate stage the entire chalazal domain is gametophyte remains ``un®lled'' throughout ontogeny. vacuolate and most of the cytoplasm is closely invested around the nucleus in the micropylar domain, and the cy- Fertilization toplasmically dense zone is no longer seen (Fig. 3C). The ®rst mitosis occurs within the micropylar domain. The two In hand-pollinated ¯owers, pollen germinated within one daughter nuclei do not migrate to opposite ends of the female hour and penetrated the stigmatic tissues within ®ve hours. 220 W. E. FRIEDMAN AND J. H. WILLIAMS

FIG. 3. Stages of growth and development of the Nuphar female gametophyte. (A) The functional megaspore becomes vacuolate and (B) begins growth. (C) At the one-nucleate stage, the nucleus is positioned within the micropylar domain (seen here in early prophase; note nucleolus is still visible). (D) At the two-nucleate stage (slightly oblique view), just after the ®rst mitosis, both nuclei remain within the micropylar domain (no migration of a nucleus to the chalazal domain occurs). (E) At the four-nucleate stage (slightly oblique view), just after the second round of mitosis, both pairs of nuclei remain within the micropylar domain. (F) At the four-celled/four-nucleate stage, the single polar nucleus has migrated to the constriction between the micropylar and chalazal domains and the egg apparatus has differentiated. (G) The egg apparatus of the mature female gametophyte consists of a vacuolate egg cell (seen in bottom panel, which is an enlargement of F) and two synergids (top and bottom panels; from separate sections). All ®gures are oriented with the micropylar pole at top, chalazal pole at bottom. Arrows indicate the structural transition from the broad micropylar domain to the elongate chalazal domain. C±F are composites taken from several serial sections or planes of focus. Scale bar, 25 ␮m. A±F are to same scale. e, egg; s, synergid.

Pollen tubes underwent mitosis of the generative cell between proached or penetrated by a pollen tube at 13 and 22 hours ®ve and 13 hours after pollination. Pollen tubes were ®rst after pollination. At 38 and 53 hours, unfertilized female seen entering the micropyle 13 hours after pollination. One, gametophytes were rarely seen. or occasionally both, synergids degenerated before the ovule Double fertilization events in Nuphar were observed at 13 was penetrated by a pollen tube. Most ovules had been ap- and 22 hours after pollination. In some ovules both sperm DEVELOPMENTAL EVOLUTION OF ANGIOSPERMS 221 cells and the tube cell nucleus were observed just after entry construct the ontogeny typical of early lineages of angio- into the female gametophyte, and in others the two sperm sperms with monosporic seven-celled/eight-nucleate female nuclei were found in the region between the egg cell and gametophytes based on recent studies of Amborella and basal central cell. Figure 4 shows an even later stage of fertilization, lineages of eumagnoliids, eudicots, and monocots (Batygina in which both egg cell and central cell have been penetrated et al. 1982; Palser et al. 1989; Grayum 1991; Johri et al. by sperms. In this ®gure (and in other observations), fusion 1992; Tobe et al. 1993; Floyd et al. 1999; Tobe et al. 2000). of the sperm nucleus and central cell nucleus is completed In angiosperms with seven-celled/eight nucleate female ga- before fusion of the sperm nucleus with the egg nucleus. The metophytes, the chalazal megaspore cell enlarges and vac- second fertilization event, involving fusion of a sperm nu- uolation typically occurs on either side of the single centrally cleus and central cell nucleus, was observed many times, and positioned nucleus (Maheshwari 1950). However, early an- always occurred far from the egg apparatus, where the central giosperm lineages with Polygonum-type development appear cell nucleus is positioned in mature female gametophytes to be variable in their pattern of vacuolation and nuclear (Fig. 3F, 4A). positioning. The ®rst nucleus may be positioned in the mi- cropylar, chalazal, or central region, and this trait may even DISCUSSION vary within species (Palser et al. 1989; Floyd et al. 1999; Tobe et al. 2000). Irrespective of initial nuclear positioning, A second fertilization event that yields a triploid endo- in all Polygonum-type female gametophytes, the ®rst free- sperm has been viewed as a synapomorphy of angiosperms nuclear mitosis produces two daughter nuclei that migrate to for over a hundred years (Sargant 1900; GueÂrin 1904). In opposite poles. At the end of this migration, a central vacuole light of recent molecular phylogenies, de®nitive data on dou- separates the two nuclei and their associated cytoplasm. ble fertilization and the nature of the second fertilization At the two-nucleate stage in Polygonum-type female ga- product, in what are now recognized as the earliest lineages metophytes, the micropylar nucleus and the chalazal nucleus of angiosperms, were found to be nonexistent (Friedman and undergo two synchronized rounds of mitosis. After the ®rst Floyd 2001). The ®rst unambiguous documentation of double round, each polar domain of the female gametophyte contains fertilization in an angiosperm lineage basal to the monocot/ two nuclei; and after the second round, each domain contains eudicot common ancestor was published for Nuphar in 2002 four nuclei (for a total of eight free nuclei). At the eight- (Williams and Friedman 2002). The process of double fer- nucleate stage, cell walls partition three nuclei at each pole, tilization in Nuphar, however, does not yield a triploid en- while the remaining free nucleus from each group of four dosperm, supposedly one of the most de®ning features of migrates toward the center of the female gametophyte. As a ¯owering plants. Instead, the second fertilization event in result, two nuclei are left within the central portion of the Nuphar yields a diploid endosperm. female gametophyte (the central cell). Thus, at the time of The discovery of diploid endosperm in an ancient lineage cellularization, the Polygonum-type female gametophyte is of angiosperms has the potential to offer new insights into seven-celled and eight-nucleate (Johri et al. 1992). Early an- the early evolution of endosperm. Ultimately, important en- giosperm lineages with Polygonum-type female gameto- dosperm characteristics such as ploidy and maternal:paternal phytes are characterized by rapid degeneration of antipodals genome ratios are directly tied to the ontogeny and mature (the three cells at the chalazal end of the female gametophyte) structure of the female gametophyte. Below we focus our and fusion of the two polar nuclei (within the central cell) discussion on: (1) unique aspects of female gametophyte de- before fertilization (Johri et al. 1992; Tobe et al. 2000). In velopment in Nuphar that produce a four-celled/four-nucleate more derived angiosperm taxa, there is a high degree of var- structure, and comparison of the ontogenetic sequences for iation in both the timing of nuclear fusion within the central female gametophytes of Nuphar and the Polygonum type, cell, and the fate of the three antipodals, which may degen- particularly among basal angiosperms; (2) evaluation of pre- erate, fuse with each other, undergo endoreduplication, en- viously published reports to determine the distribution of large, and/or proliferate (McLean and Ivimey-Cook 1956; patterns of female gametophyte development in basal angio- Dahlgren 1991; Johri et al. 1992). sperms; (3) potential avenues for evolutionary transitions be- Both Nuphar and Polygonum-type female gametophytes tween four-celled and seven-celled female gametophytes; and initiate ontogeny from the chalazalmost megaspore. But Nu- (4) developmental and genetic arguments that favor polari- phar and Polygonum-type female gametophytes differ con- zation of the female gametophyte as ancestrally four-celled/ spicuously at early and intermediate stages of development four-nucleate and endosperm as ancestrally diploid in ¯ow- (Fig. 5). In Polygonum-type female gametophytes, after the ering plants. ®rst mitosis (during the two-nucleate stage), nuclei separate and migrate to opposite poles. At the two-nucleate stage in Comparison of the Nuphar Ontogenetic Sequence to the Nuphar, migration of nuclei to opposite poles does not take Polygonum-Type Sequence place; both nuclei remain within the micropylar domain of Among angiosperms, monosporic seven-celled/eight-nu- the female gametophyte and undergo one additional mitotic cleate female gametophytes have been almost universally division to yield four free nuclei within the micropylar do- viewed as ancestral (Palser 1975; Haig 1990; see Rajan 1976 main (Fig. 5). for an exception). Thus, the common, and presumably Po- The immediate consequence of the presence or absence of lygonum-type female gametophyte has served as the standard nuclear migration at the two-nucleate stage is that in seven- against which to compare variant female gametophyte types. celled/eight-nucleate female gametophytes (Polygonum- Because the Polygonum type is itself variable, we here re- type), subsequent free-nuclear divisions occur simultaneous- 222 W. E. FRIEDMAN AND J. H. WILLIAMS

FIG. 5. Comparison of ontogenetic sequences typical of early an- giosperm lineages. Nuphar ontogeny (see Table 2 for other basal angiosperm taxa that may be similar) is depicted on left; Polygo- num-type ontogeny typical of Amborella and basal monocots, basal eudicots, and basal eumagnoliids on right. Stages of ontogeny: FM, functional megaspore stage (three slashes indicate degenerate mega- spores; often there are only two in ANITA grade lineages); stages 1, 2, 4, and 8 correspond to the one- to eight-nucleate syncytial stages of development. C is the cellularization stage. D is the dif- ferentiation stage during which mature features of individual cells of the female gametophyte are acquired. mp, micropylar pole; ch, chalazal pole.

ly in both the micropylar and chalazal domains of the female gametophyte, whereas in Nuphar, subsequent free-nuclear di- visions occur only in the micropylar domain (Fig. 5). In Nu- phar, the chalazal domain remains ``un®lled'' throughout ontogeny. In both ontogenies syncytial development ends when four nuclei occupy the micropylar domain (Battaglia

FIG. 4. The double fertilization process in Nuphar polysepala.(A) Newly fertilized female gametophyte, stained with the DNA-bind- ← ing ¯uorochrome, DAPI. At the micropylar end (top), the large, vacuolate zygote cell contains a sperm nucleus and an egg nucleus largement of the sperm nucleus-egg nucleus fusion event from A. in the process of fusion. To the right of the zygote are remnants of The round sperm nucleus is still largely intact and in contact with a synergid nucleus and tube cell nucleus within the cytoplasm of the egg nucleus, which is more visible in the next section below it the burst synergid. The remainder of the female gametophyte con- (C). (D) Enlargement of the primary endosperm nucleus from A, sists of the elongate central cell, which contains a single primary containing brightly staining sperm chromatin. The central cell nu- endosperm nucleus (pen). Fusion of the sperm and central cell nuclei cleus chromatin is largely localized to the nuclear envelope. Sperm to form the primary endosperm nucleus has already occurred, as chromatin is also visible in the next section (E). Scale bar, 25 ␮m evidenced by the presence of highly condensed sperm chromatin, (A) and 5 ␮m (B±E). e, egg; pen, primary endosperm nucleus; sc, which is distinct from the central cell nucleus chromatin. (B) En- sperm chromatin; sn, sperm nucleus; z, zygote. DEVELOPMENTAL EVOLUTION OF ANGIOSPERMS 223

TABLE 2. Reports of female gametophyte ontogeny in ANITA-grade angiosperms. Reports were considered conclusive if at least two distin- guishing ontogenetic stages were depicted (uni- vs. bi-polar distribution of nuclei during ontogeny), and if text was consistent with ®gures. Studies that report on female gametophyte development, but were inconclusive (four- or seven-celled female gametophyte claimed) are: Nuphar: Hofmeister 1858 (egg apparatus only), Winter 1990 (4); Barclaya: Schneider 1978 (7), Winter 1990 (4); Nymphaea: Cook 1902, 1906 (7), Conard 1905 (7), Seaton 1908 (7), Khanna (1967) (7), Winter 1990 (4), Orban and Bouharmont 1998 (4); Victoria: Khanna 1967 (7), Winter 1990 (4), Winter and Shamrov 1991b (4); Cabomba: Cook 1906 (7), Ramji and Padmanabhan 1965 (7), Schneider and Jeter 1982 (7), Titova 1990 (7); Brasenia: Cook 1906 (7), Khanna 1965 (7); Illicium: Hayashi 1963a (7); Kadsura: Hayashi 1963b (7); Schisandra: Kapil 1962 (7), Kapil and Jalan 1964 (7), Hayashi 1963b (7). There are no reports for Ondinea, Austrobaileya, and Trimeniaceae.

Arrangement of nuclei Mature female Genus during free-nuclear stages gametophyte Reference Amborella bi-polar seven-celled Tobe et al. 2000 Nuphar micropylar pole four-celled This study micropylar pole four-celled Winter 1987 micropylar pole four-celled Winter and Shamrov 1991a micropylar pole four-celled Shamrov 1998 Barclaya micropylar pole four-celled Winter 1987; 1993 Nymphaea micropylar pole four-celled Van Miegroet and Dujardin 1992 micropylar pole four-celled Winter and Shamrov 1991b Euryale bi-polar seven-celled Khanna 1964 Cabomba micropylar pole four-celled Galati 1985 micropylar pole four-celled Titova 1988* micropylar pole four-celled Batygina et al. 1982 Illicium micropylar pole four-celled Solntseva 1981; see also Battaglia 1986, 1989 Schisandra micropylar pole four-celled Yoshida 1962 micropylar pole four-celled Swamy 1964; see also Battaglia 1986 * Entire ontogenetic sequence reproduced in Batygina 1994.

1989). Thus, at the time of cellularization Nuphar and Po- picted at least two critical de®ning stages of ontogeny are lygonum-type female gametophytes have four and eight nu- included in Table 2. References for inconclusive reports are clei, respectively (Fig. 5). listed in the table legend. For analysis of character evolution, each genus was coded as known if at least two independent The Distribution of Female Gametophyte Types in ANITA- reports depicted ontogenetic sequences that were structurally Grade Angiosperms consistent and could reasonably be matched to the key de- velopmental landmarks that distinguish four-celled and sev- In early angiosperm lineages seven-celled and four-celled female gametophytes are especially likely to appear similar en-celled ontogenies. For Amborella, full photographic doc- at maturity as a result of antipodal cell death and the early umentation of the entire ontogenetic sequence clearly dem- fusion of polar nuclei in female gametophytes of Polygonum onstrates a seven-celled/eight-nucleate female gametophyte type (Fig. 5). Nevertheless, two key developmental land- (Tobe et al. 2000). All other ANITA-grade taxa were coded marks can be used to distinguish true four-celled from cryptic as unknown. The common ancestor of angiosperms above the seven-celled female gametophytes during ontogeny: (1) pres- ANITA grade was coded as seven-celled, because the female ence or absence of nuclear migration at the two-nucleate gametophytes of basal lineages of eumagnoliids, eudicots, stage, and (2) presence or absence of nuclei in the chalazal and most monocots are reported to be Polygonum type (Palser zone of the female gametophyte at subsequent developmental et al. 1989; Grayum 1991; Johri et al. 1992; Floyd et al. stages through the time of cellularization (Fig. 5). 1999; Tobe et al. 2000). There are over 40 studies that have reported aspects of In ANITA-grade angiosperms, only Amborella is known female gametophyte development in ANITA-grade angio- to have a seven-celled/eight-nucleate female gametophyte, sperms. All indicate the presence of either a monosporic sev- whereas ®ve genera in two major basal clades are inferred en-celled/eight-nucleate Polygonum-type female gameto- to produce ``true'' four-celled/four-nucleate female game- phyte or a monosporic four-celled/four-nucleate Nuphar-like tophytes at maturity (Nuphar, Nymphaea, and Cabomba of female gametophyte. Only one publication to date provides Nymphaeales; and Schisandra and Illicium of the ITA clade; de®nitive micrographic and developmental data, that of Tobe Table 2). Because of the current uncertainty associated with et al. (2000) for Amborella. Of the other published studies, the phylogenetic positions of Amborella and Nymphaeales, many con¯ict with each other, and drawings or micrographs character states were mapped onto two alternative published of critical developmental stages are often lacking, or are in- topologies: (1) Amborella sister to all angiosperms and Nym- consistent with the accompanying text. Because of the im- phaeales sister to all angiosperms except Amborella, or (2) portant phylogenetic positions of ANITA-grade taxa, a crit- Amborella sister to Nymphaeales and this clade sister to all ical evaluation of these reports is essential. other angiosperms (Mathews and Donoghue 1999; Parkinson Based on the developmental landmarks that distinguish et al. 1999; Qiu et al. 1999, 2000; Soltis et al. 1999; Graham Polygonum-type and Nuphar female gametophytes, we ex- and Olmstead 2000; Graham et al. 2000; Zanis et al. 2002). amined these 40 reports in an attempt to establish which Under either of these two topologies, based solely on opti- developmental pattern is likely to characterize each taxon mization of the fewest character state changes in basal an- (genus). Reports that were internally consistent and that de- giosperms, the plesiomorphic state for the female gameto- 224 W. E. FRIEDMAN AND J. H. WILLIAMS

FIG. 6. Evolution of female gametophyte ontogeny and associated endosperm ploidy in early angiosperm lineages. Two published alternative topologies of the ANITA grade taxa were considered, because of the uncertain phylogenetic positions of Amborella and Nymphaeales: A±C, Amborella sister to all other angiosperms; D±E, Amborella plus Nymphaeales sister to each other in a monophyletic group sister to all other angiosperms (see text for citations). Endosperm ploidy is known for Nuphar and many monocots and eudicots, and is inferred (on the basis of female gametophyte structure) for Amborella, Nymphaea, Cabomba, Schisandra, Illicium, and eumagnoliids. Under either phylogenetic hypothesis the plesiomorphic state for angiosperms is equivocal.

phyte (and inferred endosperm ploidy) of angiosperms is cots, and eudicots are homoplasious with Amborella (Fig. equivocal. 6C). Figure 6 shows ®ve equally parsimonious character maps Interestingly, recent studies have shown that Amborella (on two topologies) tracing female gametophyte evolution in exhibits many traits that may be homoplasious with more basal angiosperms (all involve three state changes). Under derived angiosperms. Traits shared in a potentially homo- either phylogenetic hypothesis, if four-celled female game- plasious fashion with derived angiosperms above the ANITA tophytes are ancestral, then seven-celled female gameto- grade include a seven-celled/eight-nucleate female gameto- phytes are independently derived twice, once in Amborella phyte (Figs. 6A, C, D) and presumably triploid endosperm; and once in the common ancestor of the eumagnoliids, mono- an orthotropous ovule with cup-shaped outer integument (En- cots, and eudicots (Fig. 6A, D). Conversely, if seven-celled dress and Igersheim 2000; Yamada et al. 2001); and succes- female gametophytes are ancestral, four-celled female ga- sive microsporogenesis (Furness et al. 2002). These and other metophytes may be derived twice (Fig. 6B, E), or once, if morphological characters (Doyle and Endress 2000; Sampson seven-celled female gametophytes in eumagnoliids, mono- 2000; Endress 2001; Hesse 2001) indicate that monotypic DEVELOPMENTAL EVOLUTION OF ANGIOSPERMS 225

Amborellaceae may be highly apomorphic, an obvious re- unlikely explanation for developmental transitions between minder that basal taxa need not be plesiomorphic taxa. four-celled and seven-celled female gametophytes. The late modi®cation hypothesis uses the total number of Modularity of the Angiosperm Female Gametophyte and the nuclei present in the female gametophyte as landmarks to Evolution of Endosperm Genetics and Ploidy establish comparable ontogenetic states. An alternative de- velopmental landmark is the point of sexual maturation. Nu- The monosporic seven-celled/eight-nucleate Polygonum- phar and Polygonum-type female gametophytes share an type female gametophyte has long served as a focal point for identical sexual offset point: in both, cellularization and dif- discussion of the origin and subsequent evolution of the an- ferentiation of an egg apparatus and a central cell occur after giosperm female gametophyte (Schnarf 1931; Maheshwari four nuclei are formed within the micropylar domain through 1950; Johri 1963; Davis 1966; Foster and Gifford 1974; Steb- two rounds of mitosis (and irrespective of the total number bins 1974; Palser 1975; Takhtajan 1976; Favre-DuChartre of nuclei present elsewhere in the female gametophyte; Fig. 1984; Cronquist 1988; Battaglia 1989; Haig 1990; Donoghue 7). Accordingly, the eight-nucleate stage of Polygonum type and Scheiner 1992; Tobe et al. 2000). All other female ga- is the ontogenetic equivalent of the four-nucleate stage of metophyte ``types'' have been described as arising through Nuphar; and the four-nucleate stage is equivalent to the two- modi®cation of an ancestral ontogeny typical of the Polyg- nucleate stage, respectively (Fig. 7). This alignment identi®es onum type (Palser 1975; Battaglia 1989). The documentation the early Polygonum-type ontogenetic stage, consisting of of a monosporic four-celled/four-nucleate female gameto- the ®rst mitosis and migration of the two nuclei to opposite phyte in Nuphar, as well as its inferred presence in many of poles, as either: (1) a novel insertion, if the Nuphar sequence the other earliest lineages of angiosperms, suggests, for the is ancestral, or (2) a stage that has been omitted, if the Po- ®rst time, alternative interpretations of female gametophyte lygonum-type sequence is ancestral and Nuphar is derived developmental evolution. (Fig. 7). The highly reduced (relative to non¯owering seed plants) Under the ``early modi®cation'' hypothesis, the critical angiosperm female gametophyte develops in a tightly regu- ontogenetic feature that is common to Amborella and other lated developmental sequence that can be characterized by a angiosperms with seven-celled female gametophytes, and few stereotypic landmark stages (instantaneous ontogenetic that does not appear in any four-celled/four-nucleate Nuphar- states; sensu Hufford 1995). There are only two known on- like ontogenies, is a mitotic division coupled with nuclear togenetic sequences present in ANITA-grade angiosperms migration to opposite poles. Recent analyses of seven-celled (Table 2): the monosporic four-celled/four-nucleate ontoge- female gametophyte mutants indicate that nuclear migration netic sequence characteristic of Nuphar and the monosporic and positioning at the two-nucleate stage of ontogeny are a seven-celled/eight-nucleate sequence of Amborella (Tobe et direct consequence of the acquisition of a polarized cyto- al. 2000). Although the polarity of evolutionary transition is, skeletal array, which is dependent on haploid female ga- on parsimony grounds, unresolved, a comparison of the two metophyte gene expression (Huang and Sheridan 1994, 1996; ontogenetic sequences can highlight speci®c stages of de- Vollbrecht and Hake 1995). The presence or absence of this velopment at which modi®cation is likely to have occurred nuclear migration event determines whether the chalazal do- (Alberch 1985; O'Grady 1985; Kluge 1988; Langille and Hall main of the female gametophyte will undergo nuclear and 1989; Mabee 1993; Hufford 1995, 2001; Alberch and Blanco cellular development at all subsequent stages. Thus, the 1996). downstream consequence of this phase-speci®c early devel- A common form of developmental sequence evolution re- opmental modi®cation is the presence or absence of a parallel sults from modi®cation of late stages of ontogeny (Wake developmental sequence within the chalazal domain of the 1991; Mabee 1993; Hufford 1996, 1997; Raff 1996; Hall female gametophyte (Fig. 7). 1999). Within this context, transformation between seven- It has long been recognized that the angiosperm female celled/eight-nucleate and four-celled/four-nucleate ontoge- gametophyte is composed of repeated developmental motifs. nies in early angiosperms might have arisen through loss or These consist of quartets of nuclei that arise within one, two, gain of the ®nal (third) round of mitosis (as implied in Fig. or even four domains (poles) of the angiosperm female ga- 5). Under this ``late modi®cation'' hypothesis, the eight-nu- metophyte (Porsch 1907; Schnarf 1936; Maheshwari 1950; cleate stage of Polygonum type can be viewed as a late ad- Swamy and Krishnamurthy 1975; Favre-DuChartre 1976; dition to a plesiomorphic Nuphar sequence, or conversely, Battaglia 1989; Haig 1990). These quartets can be viewed as the Nuphar sequence can be derived by deletion of the eight- quasi-autonomously developing subunits of the female ga- nucleate stage of a plesiomorphic Polygonum-type sequence metophyte, or in essence, as developmental modules (sensu (Fig. 5). A clear implication of the late modi®cation hy- Wagner 1989, 1996; Raff 1996). Each ``modular quartet'' pothesis is that addition of an eight-nucleate stage (by a third develops in a characteristic location within the female ga- mitosis) to the Nuphar sequence should yield eight nuclei metophyte, and in synchrony with other modules (if there are within the micropylar zone. Conversely, deletion of the eight- other modules). Importantly, modular quartets of angiosperm nucleate stage (omission of the third mitosis) from the Po- female gametophytes are characterized by a common devel- lygonum-type sequence should leave two nuclei at each pole opmental pattern: (1) positioning of a single nucleus within of the female gametophyte at the time of cellularization (Fig. a cytoplasmic domain (pole) of the female gametophyte; (2) 5). Neither of these spatial distributions of nuclei among two free-nuclear mitoses to yield four nuclei within that do- monosporic angiosperm female gametophytes has ever been main; and (3) partitioning of three uninucleate cells adjacent seen, and thus, the late modi®cation hypothesis seems an to the pole such that the fourth nucleus is con®ned to the 226 W. E. FRIEDMAN AND J. H. WILLIAMS

ule consists of three antipodal cells and a polar nucleus po- sitioned within the central chamber (central cell), where it ultimately fuses with the polar nucleus derived from the mi- cropylar module. A few relatively derived angiosperms pos- sess four modules (e.g., Penaea-type female gametophyte), each with three uninucleate cells and a fourth nucleus donated to the common central ``cell.'' The micropylar module (en- compassing the development of an egg apparatus and asso- ciated contribution of a nucleus to the central cell) is a de- ®ning structural feature of angiosperm female gametophytes. Ectopic expression of preexisting developmental programs to generate duplicate structures is a well-established phe- nomenon in plants (Sattler 1988; Poethig 1990; Schwarz- Sommer et al. 1990; Doebley and Lukens 1998; Kellogg 2000; Frohlich and Parker 2000; Baum and Donoghue 2002) and animals (Riedl 1978; Weiss 1990; MuÈller and Wagner 1991; Raff 1996; Minelli 2000; Arthur 2002). If Nuphar-type female gametophyte ontogeny is plesiomorphic in angio- sperms, the evolution of a second (chalazal) module could have been accomplished through the developmental insertion of an early mitotic division coupled with migration of the two nuclei to opposite poles. The net effect of such an event would have been the parallel development of two modular quartets and the evolution of a seven-celled/eight-nucleate female gametophyte. Within the framework of this hypoth- esis, the iterative morphological structure (consisting of two modular quartets) of seven-celled/eight-nucleate Polygonum- type female gametophytes in early angiosperm lineages must have evolved through ectopic expression of a preexisting micropylar developmental module. An obvious prediction is that the underlying molecular developmental genetic net- works (or developmental cassettes, sensu Tabin et al. 1999; FIG. 7. The early modi®cation hypothesis for the alignment of Abouheif et al. 1999; Wray 1999; Bolker 2000; Smith 2001; ontogenetic sequences present in basal lineages of angiosperms. Arthur 2002) of the micropylar and chalazal modules in early Nuphar ontogeny (see Table 2 for other basal angiosperm taxa that Polygonum-type female gametophytes, and the micropylar may be similar) is shown on left; Polygonum-type ontogeny typical of Amborella and other basal monocots, basal eudicots, and basal module of Nuphar, will be comparable. If we are correct, this eumagnoliids, on right. Ontogeny in the micropylar region of Nu- implies that the seed plant female gametophyte was initially phar-type during the one-nucleate stage through the cellularization reduced to a minimal sexually functional size (four cells, two stage is directly comparable to that of Polygonum-type during the of which are fecundable) in the common ancestor of extant two-nucleate to cellularization stages (outlined in dashed box). The ¯owering plants, and originally produced a diploid endo- one-nucleate stage of Polygonum-type is a novelty that includes mitosis of the single nucleus and the subsequent migration of daugh- sperm. ter nuclei to opposite domains. Depending on the polarity of evo- It is worth noting that evolution of a seven-celled female lutionary transition, this stage is identi®ed as either an insertion or gametophyte from a four-celled female gametophyte via a deletion. Stages of ontogeny: FM, functional megaspore stage; module duplication is concordant with theoretical consider- stages 1, 2, 4, and 8 correspond to the one- to eight-nucleate stages; C, cellularization stage; D, differentiation stage; mp, micropylar ations that suggest a diploid embryo-nourishing tissue was pole; ch, chalazal pole. likely to be an intermediate condition between the haploid female gametophytes of gymnosperms, and the triploid ge- netically biparental endosperm of most ¯owering plants (see central region of the female gametophyte (central cell; see Queller 1989; Friedman 1995, 1998, 2001; Friedman and Fig. 7). Floyd 2001). Furthermore, inclusive ®tness, intersexual con- Well over 99% of angiosperms, including all early angio- ¯ict, and parent-offspring con¯ict theories indicate that a sperm lineages, have a micropylar module that is composed, triploid endosperm should be selectively advantageous com- at maturity, of three cells (egg plus two synergids) and a pared with a diploid endosperm (Westoby and Rice 1982; fourth nucleus positioned within the central region of the Queller 1983, 1989; Willson and Burley 1983; Haig and Wes- female gametophyte (central cell; Maheshwari 1950; Battag- toby 1989; Friedman 1995). This could be because an in- lia 1989; Haig 1990). The four-celled female gametophytes crease in ploidy level promotes endosperm vigor (Brink and of Nuphar and other ANITA-grade angiosperms contain only Cooper 1947; Stebbins 1974). Alternatively, it could be be- this single modular quartet. In contrast, the vast majority of cause detrimental effects of genetic con¯icts of interest as- angiosperms contain two modular quartets: a micropylar sociated with diploid endosperm are balanced by the addition module and an additional chalazal module. The chalazal mod- of a second maternal nucleus to produce the characteristic 2: DEVELOPMENTAL EVOLUTION OF ANGIOSPERMS 227

1 maternal:paternal genome ratio of triploid endosperm for assistance with histology. S. Harris and the University of (Johnston et al. 1980; Lin 1984; Queller 1989; Haig and Colorado at Boulder interlibrary loan deserve special thanks Westoby 1989, 1991; Moore and Haig 1991; Grossniklaus et for ®nding many obscure references. This work was sup- al. 2001). ported by a research grant from the National Science Foun- Transitions in endosperm ploidy level or maternal:paternal dation (IBN 9816107) to WEF. genome ratios, and selection upon these key characters, can only be enacted through modi®cations of female gametophyte LITERATURE CITED ontogeny that alter the number (and/or ploidy) of nuclei in Abouheif, E. and nine others. 1999. Establishing homology criteria the central cell. In almost all angiosperms, the ploidy of the for regulatory gene networks: prospects and challenges. Pp. 207± central cell is directly determined by the number of devel- 225 in G. R. Bock and G. Cardew, eds. 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