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Heterochrony and developmental innovation: evolution of female gametophyte ontogeny in Gnetum, a highly apomorphic seed plant.

Almost all of the more than 250,000 extant species of seed plants engage in a complex life cycle that alternates between two organismal generations, the sporophyte and the unisexual male and female gametophytes. Within this alternation of generations, the more familiar diploid sporophyte (e.g., the oak tree) is the dominant phase, while the haploid male gametophyte (e.g., the oak pollen tube) and female gametophyte (e.g., the oak embryo sac) are small and nutritionally dependent upon the sporophyte. Among seed plants, the overwhelming majority of studies of the developmental and evolutionary bases of morphological diversity have focused on the sporophyte phase of the life cycle.

Although male and female gametophytes are the less conspicuous components of an alternation of generations that defines each seed plant species, these organisms have defined ontogenies and are focal points for selection and the consequent evolutionary modification of development (Friedman 1993). Nevertheless, only a few studies (Coulter 1914; Eames 1961; Takhtajan 1972, 1976; Stebbins 1974; Favre-Duchartre 1978, 1979, 1984; Friedman 1987a,b, 1993; Choi and Friedman 1991; Takhtadzhian 1991; Friedman and Gifford 1997) have focused explicitly on the developmental and evolutionary bases for structural variation among male and female gametophytes of seed plants. This lack of developmental and evolutionary study of seed plant gametophytes is unfortunate, because gametophytes are sexual organisms with diverse somatic ontogenies (Coulter 1914; Favre-Duchartre 1978; Guerrant 1988). In this paper, we analyze the underlying developmental causes for evolutionary diversification of seed plant female gametophytes and specifically focus on the evolution of one of the most highly apomorphic seed plant female gametophytes.

Extant seed plants comprise five clades [ILLUSTRATION FOR FIGURE 1 OMITTED]: cycads, Ginkgo, conifers, angiosperms, and Gnetales (Ephedra, Welwitschia, Gnetum). There is compelling evidence that Gnetales are monophyletic and, along with flowering plants, are the only living members of a highly derived anthophyte clade ([ILLUSTRATION FOR FIGURE 1 OMITTED]; Crane 1985; Doyle and Donoghue 1986, 1992; Donoghue 1989; Zimmer et al. 1989; Rothwell and Serbet 1994; Doyle et al. 1994; Doyle 1996, 1998). Molecular phylogenetic analyses (Zimmer et al. 1989; Hamby and Zimmer 1992; Hasebe et al. 1992; Chase et al. 1993; Chaw et al. 1995; Goremykin et al. 1996) indicate that within Gnetales, Ephedra is basal, whereas Gnetum and Welwitschia are derived sister taxa (for review of hypotheses of phylogenetic position of Gnetales see Doyle 1998). Relationships among basal extant seed plants (cycads, Ginkgo, and conifers) are currently unresolved. While many lineages of seed plants are now extinct, it is evident based on the stratigraphic record (Rothwell and Scheckler 1988) and phylogenetic analyses (Doyle and Donoghue 1986; Rothwell and Serbet 1994) that early fossil seed plants such as Hydrasperma (a hydrasperman seed fern) and Pachytesta (a medullosan seed fern) are basal to all extant lineages.

In all seed plants, the entire ontogeny of the seed plant female gametophyte occurs within the confines of the sporophytic tissues of the ovule. Nevertheless, there is considerable diversity in overall size, mature structure, and function of tissues among female gametophytes [ILLUSTRATION FOR FIGURE 2 OMITTED]. Most seed plant female gametophytes are known to pass through a similar sequence of developmental stages (Singh 1978; Gifford and Foster 1989). These organisms are initiated from a haploid megaspore (formed by meiosis within the body of the diploid sporophyte) and enter a period of free nuclear development that may be extensive (all nonflowering seed plants) or quite limited (angiosperms). Free nuclear development is followed by a process of cellularization of the coenocytic gametophyte. In most nonflowering seed plants (cycads, Ginkgo, conifers, Ephedra), this cellularization phase is succeeded by a period of additional growth and development, during which two or more female gametangia (each with a single egg cell) are formed. At fertilization (sexual maturity), the female gametophyte consists of thousands of somatic cells that will ultimately be involved in the nourishment of embryos [ILLUSTRATION FOR FIGURE 2 OMITTED]. In contrast, among most angiosperms partial cellularization of the free nuclear female gametophyte into a seven-celled (eight-nucleate) structure (with one egg cell) terminates development [ILLUSTRATION FOR FIGURE 2 OMITTED]. No additional growth phase ensues (Gifford and Foster 1989).

The female gametophytes of all nonflowering clades of seed plants ultimately function in the nourishment of developing embryos. However, the timing and extent of development of embryo-nourishing tissues within the female gametophytes of seed plants are highly variable. Female gametophyte somatic tissues are provisioned with nutrients that will eventually be transferred to the developing embryo during the cellular growth phase in nonflowering seed plants (Maheshwari and Singh 1967). In Ginkgo and cycads, these embryo-nourishing tissues are completely provisioned (by the maternal sporophyte) prior to fertilization (Reynolds 1924; Favre-Duchartre 1958). Conifers and most members of the Gnetales (i.e., Ephedra and Welwitschia) express both prefertilization and postfertilization provisioning of embryo-nourishing tissues of the female gametophyte (Martens 1971; Singh 1978; Haig and Westoby 1989). In flowering plants, the female gametophyte does not function to nourish developing embryos. Instead, endosperm, which develops and is provisioned exclusively after fertilization, serves to nourish the developing embryo (Friedman 1998).

Within the context of the structurally diverse female gametophytes of seed plants, it has long been recognized (Coulter 1914) that some of the most anomalous seed plant female gametophytes are found in species of Gnetum, which with Welwitschia and Ephedra comprise the Gnetales. Although descriptions of female gametophyte structure and development in Gnetum are highly variable and often contradictory (Gifford and Foster 1989), recent work has established that at sexual maturity the female gametophyte of G. gnemon is a single large coenocytic cell containing hundreds of free nuclei [ILLUSTRATION FOR FIGURE 2 OMITTED]. Free nuclei within the single-celled coenocytic female gametophyte are fertilized by sperm released from pollen tubes (Lotsy 1899; Carmichael and Friedman 1995, 1996); egg cells do not differentiate in G. gnemon. After fertilization, the female gametophyte in G. gnemon develops into a relatively large, multicellular structure that serves to nourish developing embryos, similar to the female gametophytes of cycads, Ginkgo, conifers, and Ephedra (Carmichael and Friedman 1996). The entirely coenocytic nature of the female gametophyte at the time of fertilization and the lack of defined egg cells in G. gnemon (and perhaps other species of Gnetum) are unparalleled among land plants. In addition, the strategy of exclusively postfertilization development of embryo-nourishing tissues in the female gametophyte is unique among seed plants.

Since the beginning of this century, there has been speculation that the female gametophyte of Gnetum is evolutionarily juvenilized (paedomorphic) and that this may account for its highly reduced and divergent morphology at sexual maturity (Coulter 1909; Favre-Duchartre 1965; Takhtajan 1972, 1976; Takhtadzhian 1991). While these earlier accounts provide some insight, they fail to identify specific heterochronic modifications of an ancestral (plesiomorphic) pattern of female gametophyte development that might have occurred during the evolution of Gnetum. Preliminary evidence also suggests that novel developmental and structural patterns, unrelated to heterochrony, may be expressed during the early ontogeny of the female gametophyte of Gnetum (Martens 1971; Carmichael and Friedman 1995, 1996; Friedman and Carmichael 1996). Thus, the unique developmental and morphological aspects of the female gametophyte of Gnetum may be a reflection of the combined effects of heterochrony and the evolution of structural innovations ("novel substitutions" sensu Hufford 1995, 1996).

In this study, female gametophyte ontogeny in G. gnemon is compared explicitly to the plesiomorphic developmental patterns expressed in the female gametophytes of basal extant seed plants (cycads and Ginkgo) and to Ephedra, which is basal within Gnetales [ILLUSTRATION FOR FIGURE 1 OMITTED]. Timing of sexual maturation relative to rate of somatic development in the female gametophyte of Gnetum is described, and novel developmental patterns associated with specific phases of somatic ontogeny are identified. We then analyze the complex interplay of heterochrony, developmental arrest, and structural innovation that account for the highly modified ontogeny of the female gametophyte in Gnetum.

Finally, given that most paradigms for the evolution of development have been derived from diverse studies of metazoans, we examine whether basic aspects of developmental evolution in the female gametophytes of seed plants conform to the standard models of heterochrony (Gould 1977; Alberch et al. 1979; Fink 1982; McNamara 1986; McKinney 1988; Raft and Wray 1989; Hall 1992; Hall and Miyake 1995; Zelditch and Fink 1996; Reilly et al. 1997). As will be shown, specifically with respect to the evolution of paedomorphic organisms, current concepts for the evolution of juvenilization need to be reexamined in light of evidence derived from the female gametophytes of Gnetum for a previously undescribed pattern of progenesis.


Plant Materials

Under greenhouse conditions, several male and female plants of G. gnemon were fertile throughout the year and produced pollen and ovules, respectively. Over the course of two years, ovules were collected at various time intervals before and after hand-pollination.

Light Microscopy

Ovules collected for observation were immediately trimmed prior to chemical fixation. Integuments as well as extraneous nucellar tissue surrounding the female gametophyte were carefully removed from each ovule. Trimmed ovules were fixed in 4% acrolein dissolved in 100 mM PIPES buffer, pH 6.8, for 24 h at room temperature. The ovules were then rinsed three times in PIPES buffer, dehydrated through an ethanol series (10%, 20%, 30%, 50%, 75%, 95%, 100%, 2 h per step), and infiltrated with glycol methacrylate (JB-4 Embedding Kit, Polysciences, Inc.). The samples were infiltrated over a seven-day period to ensure the complete displacement of ethanol with glycol methacrylate. Ovules were then embedded and the embedding medium was polymerized in an oxygen-free environment by flushing nitrogen gas through a closed chamber. Embedded ovules were serially sectioned on a Sorvall JB-4A microtome at thicknesses of 3-5 [[micro]meter] with a glass knife made from a microscope slide. Sections were mounted on microscope slides, stained with toluidine blue (O'Brien and McCulley 1981), and preserved with mounting medium.


Relative DNA levels of 4[prime],6-diamidino-2-phenylindole (DAPI) stained female gametophyte nuclei were measured according to modified methods of Coleman et al. (1981) and Friedman (1991). Pollinated ovules were fixed at room temperature for 24 h in 3:1 ethanol:acetic acid and transferred to 75% ethanol for storage at 4 [degrees] C. Later, ovules were dehydrated through an ethanol series, embedded in glycol methacrylate, and serially sectioned as described above. The resulting slides were flooded with a solution of 0.5 [[micro]gram]/ml DAPI and 0.1 mg/ml p-phenylenediamine (added to reduce fading; Florijn et al. 1995) in phosphate buffered-saline, pH 7.2, for 15 min at room temperature in the dark. Cover slips were then mounted and the slides were placed in a dark, humid environment prior to observation.

Microspectrofluorometric measurements were made with a Zeiss MSP 20 microspectrophotometer with digital microprocessor coupled to a Zeiss Axioskop microscope equipped with epifluorescence (HBO 50 W burner; Carl Zeiss, Oberkochen/Wuertt., West Germany). A UV filter set (model number 48702) with excitation filter (365 nm, band pass 12 nm), dichroic mirror (FT395), and barrier filter (LP397) were used with a Zeiss Plan Neofluar 20X objective.

Prior to measurement of nuclear DNA, the photometer was standardized by measuring a fluorescence standard (Zeiss, GG17) that was assigned 100 relative fluorescence units (RFU). Relative nuclear DNA content was determined by summation of individual fluorescence values of serial sections through each nucleus. A net photometric value for each section of a nucleus was determined by taking an initial reading of the nucleus and then subtracting background fluorescence of cytoplasm and embedding medium near the nucleus. The average RFU of sperm nuclei during telophase of the mitotic division to form two sperm represents the basic haploid, 1C quantity of DNA and was used as the reference to compare relative DNA levels of female gametophyte nuclei.


Early Female Gametophyte Development and Fertilization

General aspects of early female gametophyte development in G. gnemon have been previously reported (Lotsy 1899; Carmichael and Friedman 1996). In G. gnemon two to eight megaspore mother cells (megasporocytes) differentiate within each ovule. Megasporocytes are distinguished from surrounding nucellar cells by their large size and prominent nucleus [ILLUSTRATION FOR FIGURE 3A OMITTED]. While some of the megasporocytes may degenerate, most are capable of undergoing a free nuclear meiosis that results in the formation of multiple tetrasporic megaspores [ILLUSTRATION FOR FIGURE 3B OMITTED]. Each megaspore initiates a pattern of free nuclear development, but only the gametophyte located in the most chalazal (basal) position will continue to develop and eventually participate in fertilization [ILLUSTRATION FOR FIGURE 3C OMITTED].

Coenocytic development of the functional female gametophyte proceeds for approximately 14 days after megasporogenesis. Shortly after the initiation of free nuclear development, a single prominent central vacuole becomes apparent [ILLUSTRATION FOR FIGURES 3C-E OMITTED]. For most of the free nuclear phase of development, all gametophyte nuclei are positioned in a parietal band of cytoplasm and undergo synchronous free nuclear divisions. Near the end of free nuclear development of the functional female gametophyte, a small and distinctive chalazal region differentiates [ILLUSTRATION FOR FIGURES 3F-H OMITTED]. This chalazal zone of the single-celled female gametophyte lacks a central vacuole and is densely cytoplasmic, with nuclei distributed throughout [ILLUSTRATION FOR FIGURE 3H OMITTED].

At the conclusion of the free nuclear phase of development in G. gnemon, the functional female gametophyte consists of an enlarged, vacuolate micropylar region and a constricted, densely cytoplasmic chalazal region [ILLUSTRATION FOR FIGURE 3H OMITTED]. Free nuclei in the micropylar zone are distributed in a thin parietal band of cytoplasm, whereas nuclei in the chalazal zone are found evenly distributed throughout the cytoplasm. The functional female gametophyte contains approximately 1000 nuclei (1070 [+ or -] 24, n = 4), which indicates that the four original gametophyte nuclei pass through eight rounds of synchronous free nuclear mitotic divisions (eight rounds theoretically yield 1024 nuclei).

Ovules are receptive to pollination approximately 10 days following initiation of the free nuclear phase of female gametophyte development. After growing through nucellar tissue for three days, from one to several pollen tubes approach the micropylar end of the female gametophyte [ILLUSTRATION FOR FIGURE 4A OMITTED]. At the time of pollen tube entry into the female gametophyte, free nuclear development has ceased. The entire female gametophyte is coenocytic and there is no evidence of archegonial structure or differentiated egg cells [ILLUSTRATION FOR FIGURE 4B OMITTED].

Although egg cells do not form in G. gnemon, all nuclei in the micropylar region of the female gametophyte appear to be fecundable (Carmichael and Friedman 1995). Double fertilization events ensue (for details of fertilization see Carmichael and Friedman 1995, 1996) when each pollen tube discharges two sperm nuclei. Each sperm nucleus fuses with a separate female nucleus in the micropylar region of the female gametophyte (Carmichael and Friedman 1996). The products of double fertilization in G. gnemon are two distinct, diploid zygote nuclei. Each zygote nucleus, along with a small amount of proximal cytoplasm, is rapidly surrounded by a wall and separated from adjoining female gametophyte tissue [ILLUSTRATION FOR FIGURE 4C OMITTED].

Postfertilization Female Gametophyte Development

Immediately after fertilization, the densely cytoplasmic chalazal region of the female gametophyte is partitioned by cell walls [ILLUSTRATION FOR FIGURES 5A-F OMITTED]. Several nuclei (between two and eight) are enclosed within each cellular chamber. Cellularization of the entire chalazal end of the female gametophyte is synchronous, and this yields a multicellular tissue composed of many multinucleate cells [ILLUSTRATION FOR FIGURES 5B,E OMITTED].

Approximately three days after fertilization, nuclei within each multinucleate cell of the chalazal region fuse. The result is a multicellular tissue composed of highly polyploid uninucleate cells [ILLUSTRATION FOR FIGURES 5C,F OMITTED]. The polyploid nature of this chalazal female gametophyte tissue was confirmed through microspectrofluorometric analysis. Prior to the initial cellularization of the chalazal portion of the female gametophyte [ILLUSTRATION FOR FIGURES 5A,D OMITTED], free nuclei contain the 2C quantity of DNA (basic haploid, 1C quantity of DNA = 10 RFU; free nuclei = 18.95 [+ or -] 1.54 RFU; n = 5) indicating a position in [G.sub.2] of the cell cycle (these are haploid nuclei). Just after cellularization, nuclei in the chalazal multinucleate cells [ILLUSTRATION FOR FIGURES 5B,E OMITTED] still contain the 2C quantity of DNA (21.14 [+ or -] 2.22 RFU; n = 7). Nuclear DNA levels within the chalazal uninucleate cellular female gametophyte tissue [ILLUSTRATION FOR FIGURES 5C,F OMITTED] ranged between 7C and 10C (range = 70.52-102.69 RFU; n = 13) as would be expected from the fusion of several haploid nuclei within each cell.

Although considerable activity is evident in the small chalazal region of the female gametophyte after fertilization, development of the larger micropylar free nuclear portion of the female gametophyte largely terminates at this point [ILLUSTRATION FOR FIGURE 4C OMITTED]. Most free nuclei remain in situ within the parietal band of cytoplasm in the vacuolate micropylar portion of the female gametophyte and cellularization of this coenocytic tissue was not observed, except in the immediate vicinity of the zygotes. The developmentally arrested micropylar portion of the female gametophyte is rapidly obliterated by a proliferation of tissues derived from the chalazal region [ILLUSTRATION FOR FIGURES 6A-D OMITTED].

When first formed, the relatively small chalazal cellular tissue is separated by a considerable distance (hundreds of micrometers) from zygotes that reside within the vacuolate micropylar portion of the female gametophyte. Once polyploid uninucleate cells are established, the chalazal cellular tissue becomes mitotically and cytokinetically active and enters a period of extensive growth. Within two weeks, the chalazal portion of the female gametophyte enlarges to fill the developmentally arrested vacuolate micropylar region of the female gametophyte where zygotes are situated [ILLUSTRATION FOR FIGURES 6B-D OMITTED]. After the micropylar end of the female gametophyte fills with tissue derived from the chalazal end of the female gametophyte, general cell division and growth continue for an additional 10 weeks. During this time, meristematic activity is located primarily in the outermost layer of cells, with the majority of growth occurring at the chalazal end of the female gametophyte [ILLUSTRATION FOR FIGURE 6 OMITTED]. Through the first six to seven weeks of the cellular growth phase, the cells of the entire female gametophyte remain vacuolate and devoid of any significant starch reserves.

Somatic growth and development of the female gametophyte is complete within three months of fertilization [ILLUSTRATION FOR FIGURE 6F OMITTED]. Cell division ceases and proembryos can be found growing within the tissues of the female gametophyte. Large numbers of starch grains are found throughout the female gametophyte and these starch reserves nourish developing embryos. The maximum size attained by the female gametophyte is roughly 1.6 cm long and 0.9 cm wide. At this point, female gametophytes of G. gnemon are mature and ready for dispersal (within seeds).


To examine evolutionary modifications of development that are manifest in the female gametophyte of Gnetum, it is first necessary to determine the pattern of female gametophyte development that characterized the ancestors of Gnetum. This requires clear estimation of the phylogenetic position of Gnetum within the Gnetales and more global hypotheses of the relationships of Gnetales to other groups of seed plants [ILLUSTRATION FOR FIGURE 1 OMITTED]. Within this context, phylogenetically grounded comparisons of female gametophyte development in Gnetum with more plesiomorphic seed plants allow for explicit determination of the direction (polarity) and nature of evolutionary transformations of developmental patterns and processes.

Plesiomorphic Seed Plant Female Gametophyte Developmental Pattern

Although some ambiguity remains about the precise interrelationships of seed plants, comparisons with early, basal fossil seed plant taxa such as Hydrasperma and Pachytesta (see below) indicate that female gametophytes of extant cycads and Ginkgo likely retain a plesiomorphic pattern of development. Among conifers, female gametophyte development is highly variable with respect to duration of somatic growth and timing of resource allocation to embryo-nourishing tissues (Singh 1978); and determination of character polarity for female gametophytes of conifers has not been attempted. Therefore, conifers will not play any further significant role in this analysis of seed plant female gametophyte evolution. Finally, it is evident that Ephedra, which is basal among Gnetales, has also retained a pattern of female gametophyte development that is highly similar to that of basal seed plants (Martens 1971; Moussel 1983). Thus, precise circumscription of female gametophyte ontogenies in basal seed plants and Ephedra represents the starting point for analysis of the evolution of the female gametophyte in Gnetum.

In cycads and Ginkgo, female gametophytes are monosporic in origin and pass through an initial coenocytic phase in which free nuclei are positioned in a parietal band of cytoplasm that surrounds a single central vacuole [ILLUSTRATION FOR FIGURE 7A OMITTED]. Free nuclei pass through several rounds of relatively synchronous free nuclear divisions (Maheshwari and Singh 1967). The duration and number of nuclei formed during this free nuclear phase of development varies among taxa. In Ginkgo, free nuclei proceed through 13 rounds of mitosis over a period of approximately 60 days (Favre-Duchartre 1958). In cycads, from eight to 10 rounds of free nuclear divisions take place over a 30- to 75-day period (Chamberlain 1906; Reynolds 1924; Baird 1939; Brough and Taylor 1940).

Basal seed plant female gametophytes cellularize through a highly distinctive process of alveolar formation [ILLUSTRATION FOR FIGURE 7A OMITTED]. During this process, parietally arranged nuclei are isolated by hexagonal arrays of anticlinal cell walls that grow centripetally, thereby reducing the size of the central vacuole. The entire female gametophyte is ultimately cleaved into radially aligned uninucleate cells termed alveoli (Singh 1978). Following the formation of uninucleate alveolar cells, the female gametophyte becomes cytokinetically active and begins a period of sustained growth. Most cell division activity is confined to the outermost portion of the female gametophyte (Singh 1978). Egg cells (within archegonia) are formed during this cellular growth phase. Arhegonia, however, do not mature (are not receptive to sperm) until just prior to fertilization many weeks later (Norstog 1972).

During the cellular phase of development in basal seed plants, female gametophyte cells are initially vacuolate and devoid of any significant nutrient reserves. Near the end of somatic development, when growth is nearly complete, female gametophytes of cycads and Ginkgo accumulate maternal resources in the form of numerous starch grains and other metabolic substances (Emberger 1949; Singh 1978). In cycads (Smith 1910; Reynolds 1924) and Ginkgo (Favre-Duchartre 1956, 1958) fertilization occurs after complete provisioning of the female gametophyte, that is, completion of the entire somatic ontogenic trajectory [ILLUSTRATION FOR FIGURE 6G OMITTED]. Elapsed time from megaspore stage to somatically mature female gametophyte (Table 1) is approximately 150 days in Ginkgo (Favre-Duchartre 1958) and ranges from 120 days (Zamia; D. Stevenson and K. Norstog, pets. comm.) to almost a year (Macrozamia; Brough and Taylor 1940) in cycads. Nutrient reserves accumulated by the female gametophyte are metabolized by developing embryos after fertilization (Maheshwari and Singh 1967).

Comparisons with the exceptionally well-preserved ovules of upper Devonian Hydrasperma tenuis (Matten et al. 1984) and Carboniferous Pachytesta hexangulata (Stewart 1951) demonstrate that the earliest seed plants had female gametopbytes with ontogenies similar to extant cycads and Ginkgo. Female gametophytes of H. tenuis appear to pass through an initial free nuclear phase (Matten et al. 1980), cellularize centripetally (form alveoli), and at sexual maturity consist of thousands of cells with several archegonia (Matten et al. 1984). Ovules of P. hexangulata and H. tenuis both display a distinct sclerotesta around the female gametophyte at the time of sexual maturity (i.e., when eggs are present within the female gametophyte but embryos are absent; Stewart 1951; Matten et al. 1984). Because differentiation of a sclerotesta isolates the female gametophyte from surrounding sporophytic tissues of the ovule, ovules of extant seed plants do not differentiate a sclerotesta until after maternal nutrients are provisioned and somatic development of the female gametophyte is complete (Gifford and Foster 1989). Thus, it is likely that somatic growth and provisioning of nutrients within the female gametophytes of early seed plants such as Hydrasperma and Pachytesta also were completed prior to fertilization (Emberger 1949).

Developmental patterns of extant basal seed plant female gametophytes (cycads, Ginkgo) and fossil evidence from some of the earliest known seed plants indicate that the plesiomorphic pattern of somatic female gametophyte ontogeny [TABULAR DATA FOR TABLE 1 OMITTED] among seed plants includes three critical phases: (1) free nuclear development; (2) centripetal cellularization (formation of alveoli); and (3) cellular growth and development accompanied by nutrient provisioning. In the plesiomorphic state, sexual maturation (here defined as fertilization) occurs at the ultimate conclusion of this somatic ontogenetic trajectory. This sequence of events [ILLUSTRATION FOR FIGURE 7A OMITTED] serves as a baseline with which to compare gametophyte development in more derived seed plants.

Because Ephedra is basal in the Gnetales and appears to have retained many plesiomorphic features of reproduction, it is likely that female gametophyte development in Ephedra is a good approximation of the ancestral condition within Gnetales. In Ephedra, duration of the free nuclear phase (time to cellularization) varies among taxa, but lasts from approximately 11 days with 256 nuclei in E. distachya (Moussel 1980, 1983) to 20 days with 256 free nuclei (eight rounds of mitosis) in E. trifurca (Table 1; Land 1904). Cellularization occurs through formation of centripetal alveoli (Lehmann-Baerts 1967; Moussel 1980), as in cycads and Ginkgo. Although duration of somatic growth has not been reported in any species of Ephedra, it is known that fertilization occurs prior to the completion of the cellular growth phase of female gametophyte development (Land 1907; Lehmann-Baerts 1967; Moussel 1974; Haig and Westoby 1989). In Ephedra, the expression of each of the three phases of somatic female gametophyte development is basically similar to cycads and Ginkgo. Nothing is known of the timing of fertilization and duration of female gametophyte development in Welwitschia (Martens and Waterkeyn 1973).

Explicit Criteria for Identification of Heterochrony in Plants

One of the more common mechanisms of evolutionary change involves alterations in relative rate or timing of specific developmental events, that is, heterochrony. Heterochrony has the potential to create drastic phenotypic effects from relatively simple changes in development (Gould 1977). Although discussion of precise definitions of heterochronic phenomena continues (Reilly et al. 1997), there is general consensus about the method of analysis required to detect heterochronic modification of development (Gould 1977; Alberch et al. 1979; Fink 1982; McNamara 1986; McKinney 1988; Raff and Wray 1989; Diggle 1992; Hall 1992; Hall and Miyake 1995; Alberch and Blanco 1996; Zelditch and Fink 1996).

To determine whether heterochrony is responsible for morphological variation among related organisms, two developmental landmarks are defined: [Alpha] (onset of a developmental event) and [Beta] (offset of a developmental event; Alberch et al. 1979). These developmental landmarks represent hypotheses of homologous events or processes in ancestral and derived ontogenies. Although developmental landmarks may be any feature of ontogeny, birth and sexual maturity are often chosen to represent onset ([Alpha]) and offset ([Beta]) events when heterochrony at the level of whole organisms is being studied ("global heterochrony"; Raft and Wray 1989; Hall 1992; Raff 1996). Rate of somatic development (k) must also be determined in ancestral and derived ontogenies (Alberch et al. 1979; Fink 1982; Reilly et al. 1997). With this information in hand, absolute timing of developmental landmarks and rate of somatic development are compared in ancestral (plesiomorphic) and derived (apomorphic) organisms to ascertain the mode of heterochrony responsible for the evolution of the derived ontogeny (Gould 1977; McNamara 1986; Raff and Wray 1989; Mabee 1993).

The ontogenies of all seed plant female gametophytes are determinate, nonmetameric (the sporophytes of seed plants have an indeterminate and metameric architecture), and as a consequence of being the sexual generation of the life cycle, involve the differentiation of gametes. As such, seed plant female gametophytes pass through clearly identifiable juvenile and adult ontogenetic phases and are ideal for the analysis of the potential effects of heterochrony. In the present study, megasporogenesis marks the initiation (onset or birth) of female gametophyte development ([Alpha]), and fertilization serves to denote the offset point at which sexual maturity is reached ([Beta]). The absolute timing of these developmental landmarks during the ontogeny of the female gametophyte of G. gnemon can be compared with equivalent (homologous) events in plesiomorphic extant seed plants (cycads, Ginkgo) as well as Ephedra (which is basal within the Gnetales). Because seed plant female gametophytes are unitary (nonmetameric) organisms with extremely simple morphologies, rate of somatic development (k) is measured in terms of increase in length of the whole organism per unit time.

Heterochrony and the Developmental Evolution of the Female Gametophyte in Gnetum

Publications on development of seed plant female gametophytes that include specific data about absolute size, rate of growth, and timing of fertilization are exceedingly rare (as was noted by deSloover 1963; our survey of the literature from the past century yielded only a few taxa for which requisite data are available). However, the data that are available (Table 1) indicate that rates of somatic development of the female gametophyte in G. gnemon ([k.sub.Gnetum] [approximately equal to] 0.18mm/day) and Ephedra ([k.sub.Ephedra] [approximately equal to] 0.14 mm/day) are similar, and that both are moderately higher than that expressed in basal seed plants (the cycads Macrozamia and Zamia, and Ginkgo; [k.sub.plesiomorphic] [approximately equal to] 0.10mm/day). Moreover, it is evident that duration of coenocytic development (time to cellularization from megaspore stage) in Ephedra and Gnetum is considerably shorter than in cycads and Ginkgo (Table 1). These data are congruent with the hypothesis that female gametophytes in the common ancestor of Gnetales underwent an increase in the rate of somatic development compared with plesiomorphic seed plants.

What differs significantly between the development of female gametophytes of Gnetum and other seed plants is the timing of sexual maturation (fertilization) with respect to somatic development (Table 1, Figs. 7B, 8). Among plesiomorphic seed plants, fertilization of eggs occurs near or at the end of the cellular phase of development of the female gametophyte, months after the onset event of megaspore formation (Table 1). In the only report for Ephedra, fertilization has been advanced considerably (to a point roughly 35 days after megaspore stage; Land 1904), but still within the final phase of cellular development. In G. gnemon, pollen tubes enter the female gametophyte at an extremely early and free nuclear stage of development, and fertilization occurs approximately 14 days after the initiation of somatic ontogeny.

Heterochronic increases in the rate of somatic development cannot produce juvenilization in descendent ontogenies. Indeed, increased somatic rates ([Delta]k positive; "acceleration") typically produce peramorphic organisms. In the absence of decreased rates of somatic development ([Delta]k negative; "neoteny"), only early offset can result in a juvenilized morphology at sexual maturity. Thus, while the rate of somatic development in Gnetum has increased relative to that of its ancestors [ILLUSTRATION FOR FIGURE 8 OMITTED], precocious fertilization in G. gnemon at a juvenile stage of development (relative to the ancestral condition) is the clear result of progenesis ([Delta][Beta] is negative and results in early offset time; [ILLUSTRATION FOR FIGURE 7B OMITTED]). As a consequence of the precocious sexual maturation (fertilization) of the female gametophyte of Gnetum, prior to the transition to cellular organization, archegonial initiation is precluded, and this results in the loss of egg cells, a phenomenon unparalleled among land plants (except for Welwitschia; see Martens and Waterkeyn 1973).

Although fertilization in G. gnemon occurs at a juvenilized stage of gametophyte development, the overall somatic ontogeny of the organism does not truncate at the point of sexual maturity. After fertilization, the chalazal end of the female gametophyte cellularizes and enters a period of growth and development that continues for nearly three months (Figs. 6, 7B; Table 1). The extent of cellular growth in G. gnemon is similar to that of plesiomorphic nonflowering seed plant female gametophytes and is probably greater than in Ephedra. Toward the end of somatic growth in Gnetum, maternal resources are allocated to the embryo-nourishing female gametophyte tissue, as is the case in all nonflowering seed plants. At somatic maturity, the embryo-nourishing female gametophyte of G. gnemon is similar in structural organization to the mature female gametophytes of cycads, Ginkgo, conifers, and Ephedra [ILLUSTRATION FOR FIGURES 6F,G, 8 OMITTED].

Acceleration of sexual maturation and completion of the ancestral somatic ontogeny in the derived progenetic female gametophyte of Gnetum stands in marked contrast with the many described occurrences and standard models of progenesis among metazoans (Gould 1977; Alberch et al. 1979; McNamara 1986; Diggle 1992; Hanken and Wake 1993; Reilly et al. 1997), where truncation of the ancestral somatic ontogeny is always a correlate of accelerated reproductive maturation. For most evolutionary biologists who study animal development, progenesis is synonymous with truncation and miniaturization in the derived organisms (Hanken and Wake 1993). However, modifications of somatic development need not accompany the evolution of precocious sexual maturation (Reilly et al. 1997). In the female gametophyte of Gnetum, although change in offset time (acceleration of fertilization) has a dramatic effect on morphology at fertilization, it has had no correlated effect on the overall duration of somatic development or the size and morphology of the derived organism at somatic maturity.

The entirely postfertilization development and provisioning of an embryo-nourishing tissue in the female gametophyte of G. gnemon is unique among nonflowering seed plants and is analogous to postfertilization endosperm development in flowering plants [ILLUSTRATION FOR FIGURE 9 OMITTED]. Moreover, as a consequence of nuclear fusions during cellularization, the female gametophyte of Gnetum is composed of highly polyploid cells. Endosperm, a synapomorphy of angiosperms, has been considered a key adaptation integrally associated with the evolutionary radiation of flowering plants (Stebbins 1974; Tiffney 1981). Among the many reasons cited for the importance of endosperm in the angiosperm life cycle are the increased efficiency of postfertilization provisioning of the embryo-nourishing endosperm by the maternal sporophyte and the polyploid nature of endosperm (for discussion of adaptationist perspectives on endosperm evolution see Donoghue and Scheiner 1992). Gnetum has independently adopted a strategy of postfertilization provisioning of a polyploid embryo-nourishing tissue similar to that of angiosperms through novel modifications of female gametophyte ontogeny [ILLUSTRATION FOR FIGURE 9 OMITTED]. Because Gnetum comprises roughly 35 species (Price 1996) and angiosperms number more than 250,000 species, the adaptive significance (i.e., status as a key innovation, sensu Sanderson and Donoghue 1996) of a polyploid, postfertilization embryo-nourishing tissue, and its role in the evolutionary radiation of flowering plants, is questionable.

Developmental Arrest and Structural Innovation in the Female Gametophyte of Gnetum

Heterochrony has been viewed by many biologists as the dominant or even sole developmental phenomenon responsible for morphological evolution (e.g., Gould 1977; McNamara 1988; Hall 1992; Reilly et al. 1997). Based on comparisons with the plesiomorphic ontogeny of basal seed plants (cycads, Ginkgo) and Ephedra, the female gametophyte of Gnetum shows definitive evidence of progenetic modification. However, heterochrony alone does not account for all of the unusual developmental patterns expressed in Gnetum. Examination of the sequence of developmental stages (for discussion of ontogenetic sequence models see Alberch 1985; O'Grady 1985; Kluge 1988; Langille and Hall 1989; Mabee 1993; Hufford 1995, 1996; Alberch and Blanco 1996) expressed by the female gametophyte of Gnetum indicates that phase-specific, nonheterochronic developmental innovations have played important roles in the evolution of the female gametophyte of Gnetum.

The pattern of early free nuclear development in G. gnemon is identical to that expressed in the female gametophytes of cycads, Ginkgo, and Ephedra, and hence is plesiomorphic. In all of these female gametophytes, a single central vacuole forms after megasporogenesis and nuclei are positioned within a parietal band of cytoplasm. However, prior to completion of free nuclear development in G. gnemon, a divergent cytoplasmic organization appears in the chalazal region of the female gametophyte. At the completion of free nuclear development in G. gnemon, the female gametophyte consists of a plesiomorphic vacuolate micropylar zone with parietal placement of nuclei and a structurally apomorphic densely cytoplasmic chalazal zone. No equivalent of the free nuclear chalazal tissue in the female gametophyte of Gnetum occurs in Ephedra or more basal seed plants. As such, the free nuclear chalazal zone in Gnetum represents a phase-specific developmental innovation or novel substitution (sensu Hufford 1995).

After fertilization, the plesiomorphic micropylar and apomorphic chalazal regions of the female gametophyte of G. gnemon differ significantly in developmental fate. The micropylar (fertile) region does not cellularize (to any significant extent) or undergo further development [ILLUSTRATION FOR FIGURE 7B OMITTED]. Developmental arrest in a free nuclear condition precludes the plesiomorphic pattern of centripetal cellularization that is associated with the transition from free nuclear to cellular organization among the female gametophytes of all basal seed plants. Concurrent with developmental arrest of the micropylar portion of the female gametophyte, the structurally divergent free nuclear chalazal region in G. gnemon cellularizes.

The synchronous mode of cellularization to form multinucleate cells and subsequent nuclear fusions to yield polyploid uninucleate cells in the chalazal end of the female gametophyte of Gnetum differ fundamentally from the plesiomorphic centripetal formation of uninucleate alveoli in cycads, Ginkgo, and Ephedra [ILLUSTRATION FOR FIGURE 7 OMITTED]. This apomorphic pattern of cell formation in the chalazal end of the female gametophyte in Gnetum represents an additional stage-specific developmental innovation in its somatic ontogeny. The novel pattern of free nuclear development in the chalazal zone (apomorphic cytoplasmic organization) and subsequent novel pattern of cellularization of this chalazal tissue may represent correlated innovations of development in the female gametophyte of Gnetum. If this is so, then mode of cellularization (centripetal vs. synchronous) among seed plant female gametophytes may well be linked to the preceding pattern (vacuolate vs. densely cytoplasmic) of free nuclear development.

Once cellularization of the chalazal zone is complete, the female gametophyte engages in a normal (plesiomorphic) cellular growth phase to yield a somatic embryo-nourishing tissue similar to the female gametophytes of all other nonflowering seed plants [ILLUSTRATION FOR FIGURE 7 OMITTED]. Expression of meristematic activity in the periphery of the cellular female gametophyte and duration of cellular growth are roughly similar to that of Ephedra, cycads, and Ginkgo. Thus, developmental innovations associated with the early free nuclear and cellularization phases of female gametophyte ontogeny in Gnetum do not affect the expression of the subsequent plesiomorphic cellular growth phase or the morphology of the gametophyte at somatic maturity. Although developmental perturbations in early stages of ontogeny may result in more global "ontogenetic repatterning" (Roth and Wake 1985), the early expression of structural innovations in the female gametophyte of G. gnemon are clearly phase specific and isolated in their effects on subsequent development and morphology. The dissociation (sensu Raff et al. 1991) of effects of innovations early in development in Gnetum from mature morphology are similar to patterns described in various clades of direct developing frogs and sea urchins (Raff 1992, 1996) where major modifications of early development have not had significant effects on the adult somatic morphology.


Evolutionary modification of development lies at the heart of all explanations of biological diversity. As a consequence of their sexual and organismic status, seed plant gametophytes are focal points for selection and the consequent evolutionary modification of development (Friedman 1993). Although the female gametophyte of Gnetum is highly divergent among seed plants, coenocytic organization at sexual maturity, absence of egg cells, lack of centripetal alveolar cellularization, and postfertilization development of embryo-nourishing tissues in Gnetum can be understood to have resulted from a complex array of modifications of development.

Comparison of female gametophyte ontogeny in Gnetum with basal and plesiomorphic seed plants reveals that the female gametophyte of G. gnemon continues to express the three phases of seed plant female gametophyte somatic development (free nuclear development, cellularization, cellular growth) common to more plesiomorphic seed plants. Within this basic somatic ontogeny, heterochrony, developmental arrest, and stage-specific developmental innovations have each played critical roles in the ontogenetic evolution of this highly apomorphic female gametophyte.

Heterochronic changes have produced an extraordinary acceleration of the timing of fertilization relative to somatic development in the female gametophyte of Gnetum. As a consequence of extreme progenesis, the female gametophyte matures sexually (i.e., is fertilized) at a free nuclear stage of development. A major effect of progenesis is to preclude the differentiation of egg cells, with the result that free nuclei within the female gametophyte function as gametes. Progenesis, without truncation of the entire organismal ontogeny, results in the evolution of postfertilization development of embryo-nourishing female gametophyte tissues in Gnetum.

Developmental arrest and stage-specific developmental innovations have also played key roles in the evolution of female gametophyte ontogeny in Gnetum. Developmental arrest of the plesiomorphic micropylar portion of the free nuclear female gametophyte following fertilization precludes centripetal cellularization, which is normally associated with the phase change from free nuclear organization to cellular organization among plesiomorphic seed plant female gametophytes. The chalazal portion of the female gametophyte expresses an apomorphic free nuclear organization and subsequent pattern of cellularization. Centripetal cellularization (expressed in plesiomorphic seed plants) does not occur in the chalazal tissue of the female gametophyte of Gnetum. Instead, simultaneous cellularization yields a multinucleate cellular tissue and subsequent nuclear fusions produce polyploid uninucleate cells. This novel chalazal tissue in the female gametophyte of Gnetum develops into a large multicellular embryo-nourishing tissue much like the mature female gametophytes of basal seed plants.

The beginning and end points of the somatic ontogeny of the female gametophyte of Gnetum are essentially identical to those of other nonflowering seed plants [ILLUSTRATION FOR FIGURE 7 OMITTED]. However, the ontogenetic trajectory that connects the megaspore with the mature female gametophyte in Gnetum is highly divergent from the ontogenetic paths of more plesiomorphic seed plants. In essence, the ontogeny of the female gametophyte of Gnetum can be viewed as the "road not taken" by other seed plants and, from a developmental perspective, "that has made all of the difference."


We thank P. Diggle, J. Hanken, and A. deQueiroz for suggestions for the improvement of the manuscript. This research was supported by grants from the National Science Foundation (IBN-9696013) and North Dakota EPSCoR, and equipment grants-in-aid of research from Apple Computer, Lasergraphics, Carl Zeiss, Research and Manufacturing Company, Leica Instruments, Fisher Scientific, and Olympus America.


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Author:Friedman, William E.; Carmichael, Jeffrey S.
Date:Aug 1, 1998
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