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The four-celled female gametophyte of Illicium (Illiciaceae; Austrobaileyales): implications for understanding the origin and early evolution of monocots, eumagnoliids,and eudicots¹

²Department of Botany, University of Tennessee, Knoxville, Tennessee 37996 USA; ³Department of Ecology and Evolutionary Biology, University of Colorado, Boulder, Colorado, 80309 USA

Received for publication June 19, 2003. Accepted for publication October 30, 2003.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The recent consensus that Amborellaceae, Nymphaeales, and Austrobaileyales form the three earliest-diverging lineages of angiosperms has led comparative biologists to reconsider the origin and early developmental evolution of the angiosperm seven-celled/eight-nucleate (Polygonum-type) female gametophyte. Illicium mexicanum (Illiciaceae; Austrobaileyales) develops a four-celled/four-nucleate female gametophyte. The ontogenetic sequence of the Illicium female gametophyte is consistent with that of all other Austrobaileyales and also with all Nymphaeales and is likely a plesiomorphy of angiosperms. A character analysis based on more than 250 embryological studies indicates that a transition from an ancestrally four-celled/four-nucleate Illicium-like female gametophyte to a seven-celled/eight-nucleate female gametophyte occurred in the common ancestor of the sister group to Austrobaileyales (a clade that includes monocots, eumagnoliids, and eudicots). Comparative analysis of reconstructed ancestral female gametophyte ontogenies identifies specific early stages of ontogeny that were modified during this transition. These modifications generated two important angiosperm novelties—a set of three persistent antipodal cells and a binucleate central cell, which upon fertilization yields a triploid endosperm. Early angiosperms are anatomically quite diverse in these two features, although triploid endosperm, composed of one paternal genome and two maternal genomes, is a conserved feature of the overwhelming majority of angiosperms.

Key Words: ancestral character state reconstruction • evolution of development • female gametophyte • heterotopy • Illicium • modularity • origin of angiosperms • triploid endosperm

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

We have in the eight-nucleate [embryo] sac, it seems, a structure of marvelous constancy in development and arrangement of parts, evolved in the course of long ages, the exceptions to which only strengthen the view that it is a primitive type of embryo sac.

Maneval, 1914 : p. 10

The monosporic seven-celled/eight-nucleate Polygonum-type female gametophyte (embryo sac) has long been associated with the origin of flowering plants (Maneval, 1914 ; Chiarugi, 1927 ; Schnarf, 1936 ; Maheshwari, 1950 ; Johri, 1963 ; Davis, 1966 ; Bhandari, 1971 ; Foster and Gifford, 1974 ; Stebbins, 1974 ; Palser, 1975 ; Takhtajan, 1976 ; Favre-Duchartre, 1984 ; Cronquist, 1988 ; Battaglia, 1989 ; Haig, 1990 ; Donoghue and Scheiner, 1992 ; Tobe et al., 2000 ). However, its historical and developmental origin has been elusive because of the lack of close extant angiosperm relatives and the absence of a fossil record of female gametophyte anatomy. Valuable insights have been gained from broad comparisons of modern angiosperm female gametophytes with those of their distantly related seed plant relatives (Porsch, 1907 ; Battaglia, 1951 ; Takhtajan, 1976 , 1991 ; Favre-Duchartre, 1977 , 1984 ; Friedman and Carmichael, 1998 ; Friedman, 2001 ). But due to a perceived lack of variation among what were long considered the most "primitive" flowering plants, comparative biologists have never proposed a hypothesis for the specific developmental events that gave rise to the unique features of the angiosperm seven-celled/eight-nucleate female gametophyte.

Recent molecular phylogenetic analyses have identified a "basal grade" of three angiosperm lineages (Mathews and Donoghue, 1999 ; Parkinson et al., 1999 ; Qiu et al., 1999 , 2000 ; Soltis et al., 1999 , 2000 ; Graham and Olmstead, 2000 ; Graham et al., 2000 ; Zanis et al., 2002 ). These three clades consist of the monotypic Amborellaceae, the Nymphaeales (Nymphaeaceae and Cabombaceae), and the Austrobaileyales (Austrobaileyaceae, Trimeniaceae, Illiciaceae, and Schisandraceae). Amborellaceae and Nymphaeales are the first diverging branches of angiosperms (although their position relative to each other is unresolved; APG II, 2003 ). Austrobaileyales is sister to a monophyletic group that includes the monocots, eumagnoliids, and eudicots (over 99.9% of extant angiosperm species).

Until a few years ago, there had been few modern studies of female gametophyte development in these three ancient angiosperm lineages. Recently, the Amborella female gametophyte was found to be monosporic and seven-celled/eight-nucleate (Tobe et al., 2000 ). In contrast, the Nuphar (Nymphaeaceae) female gametophyte was found to be monosporic and four-celled/four-nucleate, as is almost certainly the case in all members of the Nymphaeales (Williams and Friedman, 2002 ; Friedman and Williams, 2003 ). In Austrobaileyales, we have recently shown that Schisandraceae possess four-celled and four-nucleate female gametophytes (Friedman et al., 2003 ). Conflicting reports for the rest of Austrobaileyales prevent recognition of ancestral female gametophyte traits for the clade.

Recent molecular analyses support the monophyly of Austrobaileyales (Qiu et al., 1999 ; Renner, 1999 ; Soltis et al., 2000 ; Zanis et al., 2002 ) and indicate that Austrobaileyaceae, with the single genus Austrobaileya, is sister to the remainder of the family (Fig. 1). Trimeniaceae also contains a single genus, Trimenia (Piptocalyx has been assigned to Trimenia; Phillipson, 1993 ), and is sister to a clade composed of Schisandraceae and Illiciaceae. Within Schisandraceae, Kadsura and Schisandra are paraphyletic with respect to each other (Liu et al., 2000 ; Hao et al., 2001 ). Illiciaceae contains the single genus Illicium (Fig. 1). In this study we examine the developmental embryology of Illicium mexicanum A. C. Smith (Illiciaceae).

View larger version (13K): [in this window] [in a new window]   Fig. 1. Phylogeny of Austrobaileyales (from Renner, 1999 ; Zanis et al., 2002 )

Establishing ancestral states of female gametophytes in Austrobaileyales is critical for understanding the origin of its sister clade, which comprises the overwhelming majority of angiosperms (monocots, eumagnoliids, and eudicots). Most members of this clade possess a seven-celled/eight-nucleate female gametophyte (Maheshwari, 1950 ; Davis, 1966 ; Palser, 1975 ). Based on a comprehensive review of reports on female gametophyte ontogeny we reconstruct ancestral features of the seven-celled female gametophyte of monocots, eumagnoliids, and eudicots. In so doing, we provide insight into developmental events that occurred early in angiosperm history during the transition from an ancestral four-celled/four-nucleate female gametophyte similar to that of Illicium to the novel seven-celled/eight-nucleate female gametophyte that is a major feature of the life cycle of over 99% of flowering plants.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Illicium mexicanum, or star anise, is a small evergreen shrub on the coastal plain of northeastern Mexico. It is now considered conspecific with I. floridanum of the Florida panhandle to Louisiana, based on morphology (Vincent, 1997 ). Illicium species have hermaphroditic flowers with many carpels per flower, but only one ovule per carpel (Smith, 1947 ; Robertson and Tucker, 1979 ). Flowers and buds at various stages of development were collected in July of 2000, 2001, and 2002 from specimens in the University of California Botanical Garden in Berkeley, California (Accession #91.0030; Tamaulipas, Mexico).

Light microscopy
Flowers were either fixed for 24 h in 3 : 1 (95% ethanol : acetic acid) and stored in 70% ethanol or fixed in 4% acrolein in 50 m mol/L PIPES buffer (pH 6.8, with 5 m mol/L EGTA and 1 m mol/L MgSO₄) and stored in PIPES buffer. Ovules were dehydrated through an ethanol series, then infiltrated and embedded with glycol methacrylate (JB-4 embedding kit; Polysciences, Warrington, Pennsylvania, USA). Embedded ovules were serially sectioned into 5-µm thick ribbons and mounted on slides. Slides were stained with either 0.1% toluidine blue or 0.25 µg/mL of DAPI (4', 6-diamidino-2-phenylindole) in 0.05 mol/L TRIS (pH 7.2). Digital imaging was done on a Zeiss Axiophot microscope (Carl Zeiss, Oberkochen, Germany) equipped with a Zeiss Axiocam digital camera using both brightfield and fluorescence optics. Fluorescence was visualized with an HBO 100W burner with excitation filter (365 nm, band pass 12 nm), dichroic mirror (FT395), and barrier filter (LP397) (Zeiss). Images were processed with Adobe Photoshop 6.0 (Adobe Systems, San Jose, California, USA). Image manipulations were restricted to operations that were applied to the entire image, except as noted in specific figure legends.

Microspectrofluorometry
Sections from ovules fixed in 3 : 1 fixative were stained with DAPI for 1 h at room temperature in a light-free environment. Microspectrofluorometric measurements of relative DNA levels of DAPI-stained nuclei were performed within 2 h with a Zeiss MSP 20 microspectrophotometer with digital microprocessor coupled to a Zeiss Axioskop microscope equipped for epifluorescence (HBO 100W burner) with excitation filter (365 nm, band pass 12 nm), dichroic mirror (FT395), and barrier filter (LP397) and a Zeiss Plan Neofluar 40x objective. Prior to each recording session, the photometer was standardized by recording the fluorescence emitted from a fluorescence standard (GG 17). This reading was taken to represent 100 relative fluorescence units (RFU). At the completion of each session, an additional reading of the fluorescence standard confirmed that little or no deviation in the relative fluorescence value of the fluorescence standard had occurred during data recording. Relative DNA content for each nucleus was determined by summation of individual fluorescence values of each of the serial sections through that nucleus. A net photometric value for each section of a nucleus was obtained by recording an initial reading of the nucleus and subtracting a background value obtained from cytoplasm adjacent to the nucleus. These steps removed background fluorescence due to the glycol methacrylate from the photometric analysis of relative DNA content.

Because nuclei differed in size, it was occasionally necessary to use a larger iris to quantify fluorescence. We checked for measurement bias introduced by the use of the larger iris by performing repeated measurements on single DAPI-stained nuclei (alternating the order of the iris used). The result indicated that, for a given nucleus, the large iris (1.0 mm diameter) produced a statistically higher reading than the small iris (0.63 mm diameter) (mean for 1.0 mm iris = 102.55 RFU vs. mean for 0.63 mm iris = 90.31 RFU; paired t test: P < 0.0005; two-tailed test, N = 24 nuclei). We corrected for measurement bias by multiplying the value of any measurement made with the 1.0 mm iris by a factor of 0.8806.

Character analyses
We used MacClade 4.03 (Maddison and Maddison, 2000 ) to analyze character evolution. The terminal taxa were monophyletic families as circumscribed in APG II (2003) . Placement of genera within families was based on Mabberly (1997) and Royal Botanic Gardens, Kew (2003) (see also Stevens, 2001 ). We determined character states exclusively from primary embryological reports. Traits were coded as known if reports included at least one depiction of the trait and if the text and figures were internally consistent. Where there was variation for a trait within a family, we determined the ancestral state of the family by parsimony, after mapping the character onto a recent phylogeny for the family (traits were considered unordered and unpolarized). Molecular phylogenies were favored over morphological phylogenies. In some cases, characters were mapped onto alternative molecular phylogenies. When conclusive polarization could not be achieved, we coded the taxon as variable.

	RESULTS

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Megasporogenesis
In Illicium mexicanum, each ovule initiates from one to eight megasporocytes (most produce at least three). Megasporocytes form in a small area about four cells deep within the nucellus. By the time meiosis I occurs, at least 10 nucellar cells lie between the megasporocyte(s) and the micropyle, and the megasporocyte(s) is often at the far chalazal end of the ovule. As each megasporocyte enlarges, it acquires a micropylar-chalazal polarity such that the nucleus is situated in the micropylar region, and a dense zone of DNA-staining material (data not shown) occurs in the chalazal region (Fig. 2A). Meiosis I produces two uninucleate cells (Fig. 2B). The chalazal-most cell retains the cytoplasmically dense zone. After meiosis II and cytokinesis, four uninucleate megaspore cells occur in either a linear (Fig. 2C) or T-shaped (Fig. 2D) arrangement. The chalazal-most cell contains a single large nucleus and the cytoplasmically dense zone (Fig. 2C, D; the cytoplasmically dense zone appears in adjacent sections, not shown). Formation of the cytoplasmically dense zone and its partitioning to the chalazal-most megaspore is a feature of several other Austrobaileyales (Kapil and Jalan, 1964 ; Swamy, 1964 ; Endress, 1980a ; Friedman et al., 2003 ), as well as of Nymphaeales and some derived angiosperms (reviewed in Friedman and Williams, 2003 ). At the next stage, the chalazal cell becomes vacuolate and begins to grow, whereas the other megaspores have signs of degeneration (Fig. 2E). Thus, the Illicium female gametophyte is monosporic and develops from the chalazal-most megaspore cell.

View larger version (160K): [in this window] [in a new window]   Fig. 2. Megasporogenesis in Illicium. (A) Megasporocyte in prophase of meiosis I. Note the cytoplasmically dense zone in the chalazal region. (B) Dyad stage, following meiosis I (cell wall between two daughter cells indicated by arrow). The cytoplasmically dense zone occurs in the chalazal cell. (C) linear tetrad of megaspores. (D) T-shaped tetrad of megaspore cells. The chalazal-most megaspore becomes the functional megaspore. (E) Functional megaspore, or one-nucleate female gametophyte. The cytoplasmically dense zone is no longer apparent. Two of three degenerate megaspores are visible in this section. All sections stained with toluidine blue. Micropylar pole is at top and chalazal pole at bottom. c, cytoplasmically dense zone; d, degenerating megaspore; fm, functional megaspore. (C), (D), and (E) are composite images from adjacent sections. Scale bar = 10 µm

View larger version (217K): [in this window] [in a new window]   Fig. 3. Female gametophyte development in Illicium. (A) One-nucleate female gametophyte with nucleus positioned in the micropylar domain. (B) Initiation of free-nuclear development. The first mitosis is typically perpendicular to the longitudinal axis of the female gametophyte (arrow). (C) Two-nucleate stage with both nuclei in close proximity within the micropylar domain. (D) Four nucleate stage. All nuclei are situated within the micropylar domain (composite image of two serial sections). A second subordinate female gametophyte (two-nucleate stage) can be seen at bottom right (and on the left in [E]). (E) Mature four-celled female gametophyte with two synergids, an egg cell (inset, from adjacent section), and a long, vacuolate central cell containing a single nucleus near the center or the chalazal third of the mature female gametophyte. All sections stained with toluidine blue. Micropylar pole is at top and chalazal pole at bottom. Arrows indicate nuclei. cc, central cell; ccn, central cell nucleus; e, egg cell; s, synergid cell; sf, subordinate female gametophyte. Scale bar = 20 µm

Although multiple megasporocytes usually form simultaneously in Illicium ovules, differences in their subsequent development soon become apparent. Of 45 randomly sampled mature ovules, three contained two mature four-celled female gametophytes, 25 contained one mature four-celled female gametophyte plus one or more smaller female gametophytes at the two-nucleate stage or earlier (Fig. 3D, E), and the remaining 17 possessed only a single mature four-celled female gametophyte.

Following cellularization, several aspects of differentiation could be distinguished. The three small cells at the micropylar pole form the egg apparatus, consisting of the egg and two synergids. When the egg and synergids possessed conspicuous vacuoles, the egg was relatively more vacuolate than the synergids. In synergids the vacuole was chalazal to the nucleus. In the egg the nucleus was variable in position, but it was never seen at the extreme chalazal end of the cell (as is typical of many angiosperms; Huang and Russell, 1992 ). The remainder of the mature female gametophyte consists of a long vacuolate central cell, which possessed only a single nucleus in the central region. In many angiosperms the central cell nucleus (or "secondary nucleus," if it is the product of fusion of two or more polar nuclei) migrates to a position close to the egg apparatus prior to fertilization (Russell, 1993 ), but in Illicium we never observed the central cell nucleus in this position at maturity.

In summary, development of the Illicium female gametophyte is initiated from a single megaspore and yields a four-celled/four-nucleate structure at the time of fertilization. The central cell contains a single polar nucleus that apparently migrates from the micropylar pole to the central region soon after cellularization. The chalazal domain of the female gametophyte remains "unfilled" throughout ontogeny and thus does not to contribute a second polar nucleus to the central cell.

DNA content of female gametophyte nuclei
Because the central cell of the Illicium female gametophyte only receives a single nucleus during its ontogeny, the central cell is apparently haploid and not the product of an undetected fusion of two polar nuclei (as can occur in seven-celled and eight-nucleate female gametophytes). Illicium endosperm should initially be diploid, if a second fertilization occurs. We determined the ploidy level of central cell nuclei before and after fertilization to test the hypothesis that Illicium possesses a haploid central cell nucleus (which then yields a diploid endosperm upon fertilization).

Measuring the ploidy level of individual nuclei requires prior knowledge of their position within the cell cycle. During free-nuclear development of the angiosperm female gametophyte (i.e., before cellularization and differentiation), individual haploid nuclei pass through several rounds of the cell cycle (Fig. 4A–C). Thus, during development, the genome (haploid set of chromosomes) may possess the 1C DNA content (during anaphase, telophase, and G₁ of the cell cycle); an intermediate DNA content (during DNA synthesis, or S-phase); or the 2C DNA content (during G₂ of the cell cycle, prophase, and metaphase). Nuclei at mitotic prophase possess the 2C DNA content by definition, and we measured two that had values of 176.58 RFU and 185.69 RFU (Fig. 4B). Their average, 181.1 RFU, can be taken as a standard 2C value, indicating the 1C value was approximately 90 RFU.

View larger version (102K): [in this window] [in a new window]   Fig. 4. DNA quantitation and cell cycle analysis of Illicium female gametophyte development. (A) One-nucleate female gametophyte (nucleus identified with arrow). Photometry indicates that this nucleus contains the 2C DNA content and is in G2 of the cell cycle. (B) Late two-nucleate stage, with both nuclei (arrows) close to each other within the micropylar domain of the female gametophyte. The two nuclei are in prophase of mitosis; thus their DNA content represents a known 2C standard against which to measure the relative fluorescence of other nuclei. (C) Late telophase of the second mitosis that will yield four free nuclei (pairs of arrows indicate mitotic sisters). (D) Zygote with multicellular endosperm (five endosperm nuclei, indicated by asterisks, are visible in this section). The zygote and several of the endosperm nuclei in this section possessed the 2C DNA content, consistent with diploid nuclei in G1 of the cell cycle. (B), (C), and (D) are composites of several different focal planes within a single section. All sections stained with DAPI. Micropylar pole is at top and chalazal pole is at bottom. d, degenerate megaspore; zn, zygote nucleus. Scale bar = 20 µm

To determine the ploidy of the central cell nucleus of Illicium, we examined 19 unfertilized ovules (no pollen tubes present in the micropyle; Fig. 4D). The mean fluorescence of egg nuclei was 89.1 RFU ± 20.0 SD. Because the egg is by definition haploid and the egg mean was similar to the predicted 1C value of about 90 RFU measured during free-nuclear development, the egg appears to be in G₁ of the cell cycle and possesses the 1C DNA quantity prior to fertililization. We next tested the relative fluorescence of central cell nuclei against those of accompanying egg nuclei within the same female gametophyte. The mean RFU value for Illicium central cell nuclei was 92.2 ± 18.4 SD, which is not statistically different from the mean value of egg nuclei (paired two-tailed t test: P = 0.26; N = 19 pairs). Thus, central cell nuclei, like egg nuclei, are in G₁ of the cell cycle and possess the 1C DNA content prior to fertilization.

We measured early endosperm nuclei in two ovules (approximately 80–100 endosperm cells present in each). Their average value was 166.6 ± 48.2 RFU (N = 18), which was not statistically different from a predicted diploid value based on doubling the value of haploid egg nuclei in G₁ of the cell cycle (mean predicted diploid value = 178.1 ± 40.0 RFU, N = 19; t test: P = 0.45). The mean RFU value of Illicium endosperm that we measured is consistent with the predicted RFU value of diploid nuclei in G₁ of the cell cycle. Thus the microspectrofluorometric data complement the developmental data, confirming that the central cell of the Illicium female gametophyte contains a single haploid nucleus, which after fertilization yields a diploid endosperm.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Austrobaileyales occupy a critical phylogenetic position with respect to understanding the origin of their sister clade, the angiosperms above the basal grade. Both seven-celled/eight-nucleate and four-celled/four-nucleate female gametophytes have been reported in Austrobaileyales. In this study, we documented a four-celled/four-nucleate female gametophyte in Illicium mexicanum. Within Schisandraceae, the same pattern was first discovered by Yoshida in 1962. Battaglia (1986) reviewed subsequent studies of Schisandra and confirmed Yoshida's findings, and these were extended to Kadsura by Friedman et al. (2003) . Next, we resolve conflicting interpretations of Illicium female gametophyte ontogeny. We then compare the highly concordant patterns of Illiciaceae and Schisandraceae with those found in Trimeniaceae and Austrobaileyaceae in order to reconstruct ancestral female gametophyte features of Austrobaileyales. Finally, we explore the critical evolutionary transition that must have occurred early in angiosperm history from an ancestral Illicium-like four-celled/four-nucleate female gametophyte typical of basal angiosperms to the seven-celled/eight-nucleate female gametophytes that are predominant in angiosperms above the basal grade.

Female gametophyte development in Illicium and other Austrobaileyales
In Illicium mexicanum, the female gametophyte is monosporic, with a free-nuclear phase of ontogeny within the micropylar domain that yields a four-celled and four-nucleate female gametophyte at maturity. The central cell is uninucleate and haploid, and the endosperm is diploid.

There have been reports of both monosporic (Hayashi, 1963a ; Robertson, 1973 ) and bisporic (Solntseva, 1981 ) initiation of female gametophyte development in Illicium. Solntseva (1981) concluded I. floridanum was bisporic based on her depiction of the regular occurrence of one, not three, degenerate megaspores during early female gametophyte development. However, early stages of megasporogenesis were not shown, and these are critical to distinguishing monospory from bispory (Maheshwari, 1955 ). Our study and that of Robertson (1973) provide the only photomicrographic data of megasporogenesis in Illicium, and both clearly document a pattern of monosporic development.

Within Austrobaileyales, monosporic development from the chalazal megaspore has also been found in Austrobaileya (Endress, 1980a ), Trimenia (Prakash, 1998 ), Kadsura (Hayashi, 1963b ; Friedman et al., 2003 ), and Schisandra (Yoshida, 1962 ; Hayashi, 1963b ; Kapil and Jalan, 1964 ; Swamy, 1964 ). There are no convincing reports of bispory in Austrobaileyales.

For female gametophyte development in Illicium, Solntseva (1981) reports a four-celled female gametophyte, whereas Hayashi (1963a) and Robertson (1973) report a seven-celled female gametophyte. In the earliest study, Hayashi (1963a) claims to depict fusion of two polar nuclei within the central cell as well as three persistent antipodal cells within the chalazal region.

Although Hayashi (1963a) showed persistent antipodals in I. anisatum, Robertson (1973) assumed antipodals had degenerated early in I. floridanum. He never saw antipodals cells or the fusion of polar nuclei in I. floridanum female gametophytes. Consequently, the first photomicrographs of the Illicium female gametophyte (Robertson, 1973 ) depict only four cells and four nuclei in the mature female gametophyte: a single centrally positioned nucleus in the central cell and three uninucleate cells that comprise the egg apparatus.

The next study of female gametophyte development in Illicium (Solntseva, 1981 ) depicted free-nuclear development exclusively within the micropylar domain, and no nuclei or cells were ever seen in the chalazal region. In our study of I. mexicanum, we observed the same ontogenetic sequence depicted in Solntseva (1981) , and we used DNA quantitation to rule out the possibility that a cryptic developmental stage was missed (fusion of two polar nuclei, as assumed by Robertson, 1973 ). In Illicium, the only photomicrographs of the mature female gametophyte show a four-celled/four-nucleate structure (Robertson, 1973 ; this study), and thus it appears that Hayashi's report is in error. We conclude that Illicium possesses a four-celled/four-nucleate female gametophyte composed of an egg, two synergids, and a uninucleate, haploid central cell.

In Schisandraceae a four-celled/four-nucleate ontogenetic sequence virtually identical to that of Illicium has been described in Kadsura (Friedman et al., 2003 ) and in Schisandra (reviewed in Battaglia, 1986 , 1989 ). Some details of female gametophyte development are known in other Austrobaileyales, and these are consistent with those of Illiciaceae and Schisandraceae. In Austrobaileya (Austrobaileyaceae), as well as Trimenia (Trimeniaceae), cells resembling an egg apparatus have been reported in the micropylar region, and in all published figures, the central cell of mature female gametophytes contains a single nucleus (Endress, 1980a ; Endress and Sampson, 1983 ; Prakash, 1998 ). A figure putatively depicting two polar nuclei fusing (Prakash, 1998 ) is apparently mislabeled. Rather, a two-nucleate female gametophyte is shown, based on its 10-fold smaller size than the mature female gametophytes depicted in adjoining figures. Importantly, no antipodal cells or a second polar nucleus have ever been reported within the chalazal region of the female gametophyte of Trimeniaceae or Austrobaileyaceae.

Taxa within Austrobaileyales are remarkably invariant in the ontogeny and mature structure of their female gametophytes. All are monosporic, initiating ontogeny from the chalazal megaspore, and all have a mature female gametophyte composed of an egg apparatus and a central cell with a single, centrally positioned nucleus. In Trimeniaceae, Illiciaceae, and Schisandraceae, published figures indicate that free-nuclear stages of ontogeny are restricted to the micropylar domain. There is absolutely no evidence in any taxon for the presence of traits associated with the seven-celled/eight-nucleate female gametophyte (nuclear migration at the two-nucleate stage, a second polar nucleus, or antipodal cells). The reconstructed ancestral female gametophyte of the Austrobaileyales clade is monosporic with a four-celled/four-nucleate ontogeny similar to that of Illicium.

The ancestral female gametophyte of extant angiosperms
Modern embryological studies focused on female gametophyte development have now been completed in each of the three earliest diverging branches of angiosperms. In Amborellaceae, a single study of female gametophyte development found a monosporic, seven-celled/eight-nucleate female gametophyte (Tobe et al., 2000 ). In Nymphaeales, recent work shows that the entire clade can reasonably be concluded to possess a monosporic, four-celled/four-nucleate female gametophyte with double fertilization and diploid endosperm, similar to what has recently been shown in Nuphar (Williams and Friedman, 2002 ; Friedman and Williams, 2003 ). Based on comparative analysis of these two patterns of ontogeny, we concluded that the seven-celled/eight-nucleate female gametophyte represents a derived condition within angiosperms and that the four-celled/four-nucleate female gametophyte is the ancestral state (Friedman and Williams, 2003 ).

The finding of a four-celled/four-nucleate ontogeny in Illicium and all other Austrobaileyales adds weight to our earlier conclusions (Friedman and Williams, 2003 ). The four-celled female gametophyte of Illicium (Austrobaileyales) and the four-celled female gametophyte of Nuphar (Nymphaeales) possess a nearly identical ontogeny and mature structure. In both, the chalazal megaspore cell is functional, and its single nucleus undergoes two rounds of mitosis within the micropylar domain to form four free nuclei. Cellularization occurs to form three uninucleate cells (egg and two synergids) at the micropylar pole, and a fourth nucleus is left within the central cell. The central cell nucleus migrates to the central region of the central cell before fertilization. In both taxa, as well as in Kadsura, nuclei are positioned at G₁ of the cell cycle prior to fertilization (Williams and Friedman, 2002 ; Friedman and Williams, 2003 ; Friedman et al., 2003 ). Other taxa within Austrobaileyales (Yoshida, 1962 ; Swamy, 1964 ; Robertson, 1973 ; Endress, 1980a ; Solntseva, 1981 ; Endress and Sampson, 1983 ; Prakash, 1998 ; Friedman et al., 2003 ) and Nymphaeales (Batygina et al., 1982 ; Galati, 1985 ; Winter and Shamrov, 1991a , b ; Van Miegroet and Dujardin, 1992 ; Winter, 1993 ; Orban and Bouharmont, 1998 ) have a remarkably similar ontogeny.

The similarity of ontogeny and structure of female gametophytes in Nymphaeales and Austrobaileyales suggests they possess a common ancestor with an Illicium-like female gametophyte. Thus the common ancestor of Austrobaileyales and its sister clade, containing monocots, eumagnoliids, and eudicots, is likely to have produced a four-celled/four-nucleate female gametophyte similar to that of Illicium and other Austrobaileyales. The seven-celled/eight-nucleate female gametophyte typical of the majority of angiosperms must have originated within angiosperms above the basal grade.

The ancestral female gametophyte of angiosperms above the basal grade
Austrobaileyales is well supported as sister to all angiosperms above the basal grade by recent molecular analyses (Soltis et al., 2000 ; Zanis et al., 2002 ) and by the possession of a shared deletion in the PISTILLATA gene (Stellari et al., in press). Angiosperms above the basal grade include monocots, eumagnoliids, and eudicots (as well as Chloranthaceae and Ceratophyllaceae) and form a clade that is strongly supported by molecular analyses (Zanis et al., 2002 ) and by several morphological synapomorphies (Doyle and Endress, 2000 ; Endress and Igersheim, 2000 ).

The female gametophytes of early-diverging lineages within angiosperms above the basal grade appear to be mostly seven-celled and eight-nucleate (Johri et al., 1992 ), but their ontogeny and mature structure are highly variable. Below we map female gametophyte traits onto a recent molecular phylogeny with the primary goal of reconstructing the ancestral ontogeny and mature structure of the female gametophyte of angiosperms above the basal grade. A secondary interest was to determine the nature and phylogenetic position of early modifications of ancestral states. Thus, we restrict the analysis to the earliest diverging lineages within monocots and eudicots, to all families within the relatively smaller eumagnoliid clade, and to several other families of uncertain but early placement, Ceratophyllaceae and Chloranthaceae (Table 1). To determine ancestral and derived conditions of angiosperms above the basal grade, we incorporated all families listed in Table 1 into the ordinal-level phylogeny in Fig. 1 of Soltis et al. (2003) , which is based on the results of Qiu et al. (1999) , Soltis et al. (1999 , 2000 ), and Zanis et al. (2002) . The topology for families within orders was based on Soltis et al. (2000) , except for Alismatales (Les and Cleland, 1997 ), Piperales (Nickrent et al., 2002 ), and Laurales (Renner and Chanderbali, 2000 ).

A critical invariant feature of all seven-celled/eight-nucleate female gametophytes, not present in four-celled Illicium-like female gametophytes, is the migration of nuclei to opposite poles at the onset of female gametophyte development. In monosporic, seven-celled/eight-nucleate female gametophytes, nuclear migration occurs at the two-nucleate stage of ontogeny resulting in the placement of one nucleus at each pole of the female gametophyte (Fig. 5). In Zea mays, nuclear migration at the two-nucleate stage has been shown to be a genetically based trait associated with the acquisition of a polarized cytoskeletal array (Huang and Sheridan, 1994 , 1996 ; Vollbrecht and Hake, 1995 ). This key early developmental step is a major synapomorphy uniting monocots, Ceratophyllaceae, Chloranthaceae, eumagnoliids, and eudicots.

View larger version (17K): [in this window] [in a new window]   Fig. 5. Reconstructed female gametophyte ontogeny of the common ancestor of monocots, eudicots, and eumagnoliids. In angiosperms with seven-celled female gametophytes, migration of one nucleus to the chalazal pole at the two-nucleate stage establishes a free-nuclear ontogeny within the chalazal domain that ultimately forms three antipodal cells and a polar nucleus. In the micropylar domain, an egg, two synergids, and a polar nucleus are formed by the same process. At maturity, the two polar nuclei migrate to the center of the central cell and fuse. A second fertilization event within the central cell forms a triploid primary endosperm nucleus. Antipodal cells do not undergo cell death, but persist during early endosperm development. Micropylar pole is at top and chalazal pole at bottom. a, antipodal cell; c, central cell nucleus (or polar nucleus); e, egg cell; pen, primary endosperm nucleus; s, synergid cell; sn, secondary nucleus; z, zygote

The establishment of the chalazal domain generates two novel female gametophyte traits—a binucleate central cell and an antipodal cell complex. All seven-celled female gametophytes possess two polar nuclei in the central cell just after cellularization occurs. In most of these, the two polar nuclei migrate to the center of the central cell where they fuse before fertilization; this is the ancestral state (Fig. 5; Table 1).

A second innovation associated with the origin of the novel chalazal developmental domain is a group of three cells, or antipodals. The chalazal position of antipodal cells places them directly in between the endosperm and the flow of nutrients from the maternal sporophyte to the embryo. Antipodal cells often display morphology indicative of nutritive, secretory, and/or transfer function, even when their post-fertilization development is short (Kapil and Bhatnagar, 1981 ; Willemse and Van Went, 1984 ). Alternatively, antipodal cells may undergo a process of cell death in which they degenerate soon after their formation.

Rapid cell death of antipodals has been thought to be the plesiomorphic condition of angiosperms (Bhandari, 1971 ; Tobe et al., 2000 ), but it has also been suggested that the ancestral state was for enlargement or multiplication of antipodals (Sargant, 1900 ; Battaglia, 1951 ; Wyatt, 1955 ). Parsimony analysis indicates that the common ancestor of angiosperms above the basal grade possessed three antipodal cells that persist after fertilization. In contrast to the above views, our analysis is consistent with the insight of McLean and Ivimey-Cook (1956) that variation in antipodal cell behavior should be seen as elaboration of an ancestrally simple antipodal cell complex. Monocots, Ceratophyllaceae, some Chloranthaceae, and many basal eudicots possess three simple antipodals that persist after fertilization without further development. Developmental modifications of antipodal cells occur in phylogenetically derived positions within basal monocots and eumagnoliids and are common in many basal eudicots and Chloranthaceae. These include multiplication of antipodal cells, multiple nuclei within cells, various forms of endopolyploidy, extreme cell growth, and cell morphology suggesting haustorial function (Table 1) (reviewed in Huss, 1906 ; Schnarf, 1931 ; Kapil and Bhatagnar, 1981 ; D'Amato, 1984 ).

The nested phylogenetic positions of taxa with early cell death (Table 1) suggest that this character can also be seen as a developmental modification of an ancestrally simple and "persistent" state. Early antipodal cell death is rare in monocots and eudicots but is shared by virtually all eumagnoliids that have been studied (Table 1). It may be one of the strongest morphological synapormorphies for this recently defined clade.

In summary, the reconstructed female gametophyte of the common ancestor of angiosperms above the basal grade is monosporic, seven-celled and eight-nucleate, and has a clearly defined ontogenetic sequence (Fig. 5). After the first free-nuclear division, the two newly formed nuclei migrate to opposite poles. Two more mitoses occur simultaneously to yield four nuclei at each pole. Cellularization occurs to produce three cells at each pole, and the fourth nucleus from each quartet is partitioned to the central cell. In the micropylar domain, the three cells differentiate to become an egg and two synergids. In the chalazal domain, three antipodal cells form, produce vacuoles and persist after the time of fertilization. In the central cell, the two haploid polar nuclei migrate to the center and fuse before fertilization to produce a diploid secondary nucleus. Double fertilization occurs and the fusion of sperm cell and central cell is inferred to yield a triploid endosperm (Fig. 5; Table 1).

The evolutionary developmental origin of the seven-celled and eight-nucleate female gametophyte
The reconstructed ancestral female gametophyte of the clade comprising monocots, eumagnoliids, and eudicots exhibits a unique seven-celled/eight-nucleate ontogeny. In contrast, the two lineages basal to this clade, Austrobaileyales and Nymphaeales, share a nearly identical four-celled/four-nucleate female gametophyte ontogeny. This particular phylogenetic arrangement of characters suggests for the first time a specific hypothesis for the developmental events that led to the origin of the seven-celled female gametophyte in monocots, eumagnoliids, and eudicots.

As was earlier recognized, the angiosperm female gametophyte is composed of essentially modular structures defined by quartets of nuclei positioned within one, two, or even four developmental domains (Porsch, 1907 ; Schnarf, 1936 ; Maheshwari, 1950 ; Cocucci, 1973 ; Swamy and Krishnamurthy, 1975 ; Favre-Duchartre, 1977 , 1984 ; Battaglia, 1989 ; Haig, 1990 ). Each quartet defines a developmental module (sensu Wagner, 1989 , 1996 ; Raff, 1996 ). A developmental module possesses an ontogeny that occurs within one spatial domain of the female gametophyte as follows: (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 confined to the central cell of the female gametophyte (Friedman and Williams, 2003 ).

In Fig. 6, we compare the ontogenetic sequences of the reconstructed ancestral Illicium-like four-celled female gametophyte with that of its presumed descendant, the reconstructed ancestral seven-celled female gametophyte of monocots, eumagnoliids, and eudicots. In the Illicium-like four-celled female gametophyte, free-nuclear development takes place only within the micropylar domain, and the ontogenetic sequence comprises a single developmental module (Fig. 6). In the reconstructed ancestral seven-celled female gametophyte, free-nuclear development occurs within both the micropylar and the chalazal domains, and these parallel ontogenetic sequences comprise two developmental modules (Fig. 6). It is immediately apparent that the micropylar developmental module is a conserved feature of both ontogenies (and of virtually all angiosperm female gametophytes), whereas the chalazal developmental module of seven-celled/eight-nucleate female gametophytes is a novelty (Fig. 6). The chalazal module of the seven-celled female gametophyte is similar in structure and ontogeny to the micropylar developmental module, suggesting an origin by duplication (Friedman and Williams, 2003 ).

View larger version (32K): [in this window] [in a new window]   Fig. 6. Comparative development of female gametophytes of early angiosperms. The Illicium-like four-celled/four-nucleate female gametophyte of Austrobaileyales is at the top, and the reconstructed seven-celled/eight-nucleate female gametophyte of the common ancestor of the clade that includes monocots, eumagnoliids, and eudicots is below. Female gametophytes of angiosperms are fundamentally modular structures in which individual developmental modules consist of "quartets" of nuclei. Each module (outlined in a grey box) is initiated from a single nucleus that undergoes two free-nuclear mitoses to yield four nuclei. Cellularization of a four-nucleate module involves partitioning of three uninucleate cells, while the fourth nucleus is contributed to the central cell of the female gametophyte. The confinement of both nuclei to the chalazal domain at the two-nucleate stage of development in I. mexicanum results in the establishment of a single modular quartet. Conversely, in angiosperms with seven-celled female gametophytes, one of the nuclei migrates to the chalazal pole at the two-nucleate stage (two-headed arrow) initiating a second (chalazal) developmental module (in addition to the micropylar module) that ultimately forms three antipodal cells and a polar nucleus. Newly-formed polar nuclei migrate to the center of the central cell during maturation of the female gametophyte. Antipodal cells do not undergo cell death. Micropylar pole is at top and chalazal pole at bottom. a, antipodal cell; c, central cell nucleus (or polar nucleus); e, egg cell; s, synergid cell; sn, secondary nucleus

Given our current understanding of flowering plant phylogeny, the seven-celled/eight-nucleate female gametophyte of monocots, eumagnoliids, and eudicots originated in their common ancestor at the time of, or soon after, Austrobaileyales and the main line of angiosperms diverged from each other (Fig. 7). The novel chalazal module was composed of two features early in its history: an antipodal complex of three relatively simple persistent cells, and a single polar nucleus partitioned to the central cell (where it migrates to the central region and fuses with the polar nucleus donated by the micropylar module) (Fig. 6). These two features are both predicted from knowledge of the ontogeny of the Illicium-like four-celled female gametophyte. Antipodal cells and the binucleate central cell (yielding a triploid endosperm upon fertilization) originated in the common ancestor of angiosperms above the basal grade (Fig. 7). They have no homologue in nonflowering seed plants.

View larger version (22K): [in this window] [in a new window]   Fig. 7. Early evolution of the angiosperm female gametophyte. Before the origin of extant flowering plants, the female gametophyte underwent extreme progenesis from a gymnosperm-like female gametophyte with thousands of cells to one with only four cells. The plesiomorphic angiosperm female gametophyte underwent free-nuclear ontogeny exclusively within the micropylar domain to form an egg cell, two synergids, and single nucleus partitioned into the central cell. The ontogeny of the four-celled female gametophyte represents a single developmental module. The micropylar developmental module was then duplicated and ectopically expressed in the chalazal domain. The novel chalazal developmental module yielded a binucleate central cell, as well as three antipodal cells. Double fertilization originated before the origin of Nymphaeales (and possibly before the origin of all extant angiosperms). Thus, triploid endosperm with a 2 : 1 maternal-to-paternal genome ratio originated in the common ancestor of monocots, eumagnoliids, and eudicots, as a consequence of double fertilization and the origin of the chalazal module. Amborellaceae also possesses a chalazal developmental module, but the occurrence of a second fertilization event and the ploidy of its endosperm have not yet been documented

The origin and early evolution of endosperm genetics and ploidy
The four-celled female gametophytes of Illicium and other Austrobaileyales and Nymphaeales yield, or can be inferred to yield, a diploid and sexually derived endosperm composed of one maternal and one paternal genome. Diploid endosperm in Austrobaileyales and in Nymphaeales undergoes a vigorous precocious development (relative to the zygote) and clearly functions in nutrient storage and embryo nourishment (Endress, 1980a ; Johri et al., 1992 ; Floyd et al., 1999 ; Floyd and Friedman, 2000 , 2001 ; Williams and Friedman, 2002 ; this study).

It now appears that double fertilization in early angiosperms originally yielded a fully functional diploid endosperm similar to that of Illicium and Nuphar. Triploid endosperm with its increased ploidy level and characteristic 2 : 1 maternal-to-paternal genome ratio evolved later in flowering plant history. The transition from a diploid endosperm typical of Austrobaileyales to a triploid endosperm in the common ancestor of monocots, eumagnoliids, and eudicots must have occurred at least 120 million years ago, based on the oldest fossil records of taxa in either clade (Friis et al., 1999 ; Doyle, 2000 ).

Triploid endosperm and the 2 : 1 maternal-to-paternal genome ratio have been viewed as selectively advantageous by virtually all who have considered endosperm evolution (see reviews of Sargant, 1900 ; Brink and Cooper, 1947 ; Stebbins, 1976 ; Queller, 1989 ; Donoghue and Scheiner, 1992 ). Our study seems to support this position in that the phylogenetic distribution of taxa possessing diploid and triploid endosperm suggests a "key innovation" hypothesis (sensu Simpson, 1953). The clade in which triploid endosperm originated (angiosperms above the basal grade) has an exceedingly high species diversity (approximately 250 000 species; Mabberly, 1997 ) relative to its sister, Austrobaileyales, which possesses the plesiomorphic trait (diploid endosperm) and has approximately 100 living species. The transition from a diploid to a triploid endosperm appears to be associated with a dramatic increase in diversification rate. However, most early angiosperm lineages, whether they possess diploid endosperm (e.g., Nymphaeales, Austrobaileyales) or triploid endosperm (e.g., Ceratophyllaceae, Chloranthaceae, Winteraceae, Calycanthales, Nelumbonaceae, Platanaceae, Eupteleaceae, Buxales, and Trochodendrales), have extremely low absolute diversification rates (Magallón and Sanderson, 2001 ). Endosperm triploidy and the 2 : 1 maternal-to-paternal genome ratio may have been advantageous, but any causal relationship with angiosperm diversification is likely to have a more complex explanation.

We still know little of the reproductive biology of many relict early angiosperm lineages (Friedman and Floyd, 2001 ). We do know that after the origin of triploid endosperm and antipodal cells many different evolutionary experiments in embryo nourishment have occurred. Parent-of-origin genetic effects and the endosperm balance number documented in many phylogenetically derived flowering plants with triploid endosperm (Johnston et al., 1980 ; Lin, 1984 ; Haig and Westoby, 1991 ; Grossniklaus et al., 1998 ; Scott et al., 1998 ; Luo et al., 2000 ; Carputo et al., 2003 ) are premier examples of such experimentation and may explain why there are few cases of reversions to functional diploid endosperms in angiosperms above the basal grade.

Conclusions
The Austrobaileyales occupy a critical phylogenetic position for understanding the early evolutionary history of monocots, eumagnoliids, and eudicots. The four-celled/four-nucleate ontogeny and mature structure of the Illicium female gametophyte are now established as plesiomorphic features of angiosperms, shared by Austrobaileyales, as well as by an even earlier branching lineage, the Nymphaeales. The evolutionary developmental origin of the seven-celled/eight-nucleate female gametophyte in the common ancestor of monocots, eumagnoliids, and eudicots can be traced by comparison to that of its reconstructed immediate predecessor, the four-celled/four-nucleate, Illicium-like female gametophyte.

The ontogenetic sequence of the Illicium female gametophyte can be described as a developmental module in which a single nucleus undergoes two mitotic divisions within the micropylar domain of the female gametophyte to give rise to a four-celled/four-nucleate structure at maturity. The reconstructed ancestral seven-celled/eight-nucleate female gametophyte of angiosperms above the basal grade consists of a micropylar developmental module similar to that of Illicium and a second developmental module (closely similar to the micropylar module) expressed within the chalazal domain. The novel chalazal developmental module of angiosperms above the basal grade is a duplicated structure that originated by the insertion of a nuclear migration event at the two-nucleate stage of ontogeny and ectopic expression of the micropylar developmental module. A major consequence of this unique angiosperm innovation is the formation of a binucleate central cell, which upon fertilization yields a triploid endosperm with a 2 : 1 maternal-to-paternal genome ratio. These reproductive features, long thought to characterize flowering plants as a whole, originated in the common ancestor of the sister clade to Austrobaileyales, the lineage leading to over 99% of living angiosperms.