Developmental Evolution of the Sexual Process in Ancient Flowering Plant Lineages

Recent investigations of ancient angiosperm lineages are yielding data critical to a fundamental reassessment of the origin and early evolution of flowering plants. To “reconstruct” the reproductive features of the earliest flowering plants, biological characters must be examined in an appropriate phylogenetic sampling of extant angiosperms, and their history must then be traced back through 130 million years of time. This task requires, first, a clear formulation of the phylogenetic interrelationships among basal angiosperms. Once robust phylogenetic hypotheses are available, comparative analyses can be used to infer and reconstruct the evolutionary history of a broad range of biological characters. Thus, the study of the origin and early evolution of flowering plants, like that of any group of organisms, relies on the interplay of phylogenetic and comparative biology. Incorrect (or vague) assessments of phylogenetic relationships, as well as misconstrued biological character data, will result in flawed evolutionary historical hypotheses. Both of these pitfalls have plagued the reconstruction of early angiosperm reproductive history for more than a century. After a long period of empirical and intellectual stagnation, critical new reproductive data coupled with more robust phylogenetic hypotheses are radically altering the conceptual landscape. Many of the century-old paradigms about the origin and early evolution of flowering plant reproductive features are in the midst of being substantially overthrown. For more than a century, the “defining” features of flowering plant reproductive biology have been thought to be well circumscribed. Typically, in angiosperms, the pollen grain (male gametophyte) is bicellular when it is shed from the anther and produces two sperm during the growth of the pollen tube within the tissues of the gynoecium. The female gametophyte is almost always monosporic in origin and usually develops into a seven-celled, eight-nucleate organism before fertilization. The fertilization process in flowering plants is marked by two separate gametic fusion events that produce a diploid embryo and a triploid embryo-nourishing endosperm. Finally, in most flowering plants, the embryo initiates a cellular pattern of development and the endosperm proceeds through a free nuclear (syncytial) phase of development (but eventually becomes cellularized) (Table 1 Developmental Features of the Angiosperm Reproductive Syndrome With subsequent proliferation of antipodals (Diboll and Larson, 1966). Floyd and Friedman (2000). Developmental Features of the Angiosperm Reproductive Syndrome With subsequent proliferation of antipodals (Diboll and Larson, 1966). Floyd and Friedman (2000). In this review, we examine the reproductive features of flowering plants associated with the fertilization process from an evolutionary developmental perspective. We address three basic questions. First, did the earliest flowering plants possess a process of double fertilization? The definitive answer (as of now) is maybe. As we show, after a century and a half of study of the plant fertilization process, a key taxon central to reconstructing the ancestral characteristics of extant flowering plants has yet to be examined for this process. Indeed, until two years ago (Williams and Friedman, 2002), there were no definitive studies demonstrating a process of double fertilization in any of the most ancient lineages of angiosperms (for a review of earlier studies, see Friedman and Floyd, 2001). Thus, the long-standing assumption that the first flowering plants expressed a second fertilization event that initiated a genetically biparental endosperm has yet to be proven. Second, what was the developmental pattern and mature structure of the ancestral angiosperm female gametophyte? We present an analysis of the very recent and surprising discovery that the female gametophyte in the earliest flowering plants probably was not of the Polygonum type (monosporic, seven celled, and eight nucleate) but rather was four celled and four nucleate at maturity (Friedman et al., 2003; Friedman and Williams, 2003; Williams and Friedman, 2004). This finding has led to a fundamental reassessment of female gametophyte developmental evolution. Most importantly, this discovery has provided strong evidence of the fundamentally modular nature of the angiosperm female gametophyte. The female gametophyte developmental module is characterized by the positioning of a single nucleus within a cytoplasmic domain (early in female gametophyte ontogeny), followed by two free nuclear mitoses to yield four nuclei within the domain, and lastly, by the formation of three uninucleate cells accompanied by the sequestration of the fourth nucleus in the central region of the female gametophyte (central cell). Here, we present evidence in support of the hypothesis that the evolutionary diversification of the angiosperm female gametophyte has been accomplished largely through the ectopic expression of this basic developmental module. Third, what were the ploidy and genetic constitution of early angiosperm endosperm? Because endosperm is initiated by the fertilization of the central cell, the ploidy and genetic constitution of endosperm are tied directly to the development and mature structure of the female gametophyte. Thus, any transition from a four-celled, four-nucleate female gametophyte with a haploid central cell to a seven-celled, eight-nucleate female gametophyte with a binucleate (diploid) central cell (Polygonum type) likely marks a transition from the diploid endosperms characteristic of the earliest angiosperms to the triploid endosperms found among most extant flowering plants (Friedman and Williams, 2003). The current data are congruent with the hypothesis that the earliest flowering plants produced diploid biparental endosperms (Friedman and Williams, 2003) and that this condition was followed by a rapid (in geological terms) transition to triploid biparental endosperms in the common ancestor of 99% of extant angiosperms ∼10 million years after the origin of flowering plants (Williams and Friedman, 2004). Importantly, the linkage between female gametophyte development and endosperm ploidy and genetics (Friedman and Williams, 2003) indicates that the evolutionary history of endosperm is tied directly to upstream events associated with the development of the female gametophyte. Throughout the 20th century, the broad consensus on the reproductive characteristics of the earliest angiosperms derived from a general phylogenetic hypothesis that “Magnolia-like” plants and their relatives (magnoliids) constitute a plexus of ancient lineages (in essence, the paraphyletic wellspring) from which all other angiosperms evolved (Bessey, 1897, 1915; Arber and Parkin, 1907; Takhtajan, 1969; Dahlgren, 1980; Cronquist, 1981; Walker and Walker, 1984; Donoghue and Doyle, 1989; Thorne, 1992). As a consequence, members of the Magnoliales and other groups, such as the Laurales and Winterales, frequently were studied to reconstruct the embryological features of the earliest flowering plants (Maneval, 1914; Earle, 1938; Hayashi, 1965; Sastri, 1969; Bhandari, 1971a, 1971b). Embryological studies throughout the 20th century affirmed that a monosporic seven-celled, eight-nucleate female gametophyte (Polygonum type) is prevalent among magnoliids (Hayashi, 1965; Bhandari, 1971a, 1971b; Tobe et al., 2000) and among angiosperms as a whole (it is estimated to be present in >70% of extant angiosperm species [Maheshwari, 1950; Johri et al., 1992]). The development of the Polygonum type of female gametophyte begins with a single functional megaspore that initiates a series of three free nuclear divisions to yield eight nuclei within a syncytium. The syncytium then cellularizes to produce an egg apparatus (two synergids and an egg cell) at the micropylar pole and three antipodal cells at the chalazal pole. The remaining two nuclei of the female gametophyte are partitioned into the remainder of the syncytium, or central cell. Given the near ubiquity of Polygonum-type female gametophytes among magnoliid lineages, the conclusion was reached early and often in the 20th century that the first flowering plants had a monosporic seven-celled, eight-nucleate female gametophyte (Porsch, 1907; Schnarf, 1931; Johri, 1963; Davis, 1966; Foster and Gifford, 1974; Stebbins, 1974; Palser, 1975; Takhtajan, 1976; Favre-DuChartre, 1984; Cronquist, 1988; Battaglia, 1989; Haig, 1990; Donoghue and Scheiner, 1992; Johri et al., 1992). Furthermore, all variants of female gametophyte structure and developmental patterns in angiosperms were thought to be derived in some manner from this starting point. Twentieth century embryological studies also affirmed that a second fertilization event that initiates an endosperm was ubiquitous among flowering plants, including magnoliids (Sargant, 1900; for a review of this topic, see Friedman, 2001a). Thus, soon after its discovery, a double fertilization process was viewed as a defining and ancestral feature of angiosperms. Because the central cell of a Polygonum-type female gametophyte is initially binucleate (the two haploid polar nuclei may fuse before the second fertilization event to yield a diploid “secondary nucleus”), the product of the fertilization of the central cell is a triploid endosperm with a biparental genetic constitution and a 2:1 ratio of maternal to paternal genomes; this was universally concluded to be the ancestral condition for flowering plants. The twentieth century views of what characterized the “primitive” suite of angiosperm reproductive features were accepted with little dissent. Indeed, based on all of the generally accepted paradigms of flowering plant reproductive biology, evolutionary biologists could confidently report that “[d]ouble fertilization and the development of copious endosperm from a triple fusion nucleus [in a Polygonum-type female gametophyte] are clearly primitive characters within the angiosperms” (Cronquist, 1988). There was little reason to question these views—that is, until 1999. Flowers of Basal Angiosperm Lineages. (A) Carpellate flower of the dioecious taxon Amborella trichopoda. (B) Hermaphrodite flower of Nuphar polysepala. (C) Staminate flower of the monoecious taxon Kadsura japonica. Phylogeny of Angiosperms Based on Recent Molecular Analyses. Amborella, Nymphaeales, and Austrobaileyales constitute a set of lineages that are more ancient (in terms of time of origin) than monocots, eumagnoliids, or eudicots. is that the common ancestor of and is not the common ancestor of extant angiosperms. Thus, comparative studies of and as and be used to infer directly the ancestral characteristics of flowering plants With the discovery that Amborella, Nymphaeales, and Austrobaileyales are the most ancient extant angiosperm lineages and (or some of the biological features of the earliest angiosperms (Friedman et al., it was that to of the origin of flowering plants have to be through study of these the remaining through of Amborella, Nymphaeales, and Austrobaileyales with early lineages of monocots, eumagnoliids, and can robust hypotheses be the nature of the common ancestor of extant angiosperms. members of the Winterales, and other Amborella and within the Austrobaileyales were examined during the 20th century (Friedman et al., 2003). In the of the Nymphaeales, embryological studies were the often were Friedman and Williams, 2003). After the recent of the most ancient lineages of angiosperms, it that there was little evidence of a second fertilization event in any of the members of the Amborella, Nymphaeales, and Austrobaileyales (Friedman and Floyd, 2001). years there were four of a fusion of a second sperm nucleus with the two polar nuclei (or their fusion the of the female gametophyte in the most basal angiosperm and (Hayashi, In to these there were that some early angiosperm lineages not have a second fertilization event and the ancient of these studies evidence in support of their in the Basal Angiosperm Nuphar polysepala. (A) nucleus with before the first fertilization event the of (B) nucleus before the of (C) nucleus in the of cell nucleus before the second fertilization event the of endosperm nucleus before the of The sperm can be the central cell endosperm nucleus in the of The pattern in basal angiosperms is fundamentally to that of all that have been studied by Friedman, et al., but from the cell of and other derived angiosperms et al., plants, found in and to be among the phase of the cell is expressed in before fertilization a second fertilization event has been in the and the of this process was a feature of the common ancestor of extant flowering plants on an of the condition in To the fertilization event in Amborella has not been Amborella is eventually to an endosperm from a process, it can be confidently concluded that double fertilization was present in the common ancestor of all extant flowering plants. Amborella a second fertilization the that the first flowering plants a endosperm and that double fertilization evolved in the common ancestor of all extant flowering plants other than Amborella or that a second fertilization process was present in the first angiosperms but was in the that produced double fertilization is eventually to be a feature of the common ancestor of flowering plants, this will not this process from and it fertilization as two fertilization events in a single female gametophyte by two sperm from a single pollen also in plants, among members of the and some and Friedman, the second fertilization event in plants a diploid not an endosperm. through and 1992; and et al., 1989; and 1990; and 1992; et al., et al., and Doyle, et al., phylogenetic analyses to that was the most extant plant to angiosperms. Thus, it was most to double fertilization in and angiosperms as been derived from a common plant ancestor 1990; and Friedman, Recent plant phylogenetic analyses have a between and angiosperms, or have produced or have been to any clear phylogenetic of among extant lineages et al., et al., et al., et al., and et al., et al., and and et al., and et al., For the time the phylogenetic of a Thus, the origin of double as in angiosperms, and the of endosperm an in the of evolutionary time. to the of the basal phylogenetic of Amborella, Nymphaeales, and Tobe and that the female gametophyte of Amborella produces seven cells and eight This finding initially to that there had been a in hypothesis what the most ancient flowering plant lineages, the century-old that the first angiosperms had a Polygonum-type female gametophyte Based on a review of the Johri et al., Tobe et that members of the and Austrobaileyales had Polygonum-type female the long-standing of the embryological characters of these studied In to Amborella and what was to be the in and there were that the female gametophytes in and Austrobaileyales not be of the Polygonum with by and by et 1981; Battaglia, and and 1992; and that the female gametophytes in Austrobaileyales and four cells at an egg cell, two and a uninucleate central cell. these to be by other studies that seven-celled, eight-nucleate female gametophytes in and Austrobaileyales Hayashi, and and 1965; of the embryological studies of and Austrobaileyales key data that could be to between hypotheses of female gametophyte in the of Angiosperm at seven-celled, eight-nucleate female gametophyte of the antipodal cells and fusion of the two nuclei of the central cell to yield a female gametophyte with a diploid central cell female gametophyte a single haploid nucleus in the central cell egg cell. Many that the magnoliid taxon study had a Polygonum-type female gametophyte (in ancient lineages of flowering plants must have this type of female concluded that had a seven-celled, eight-nucleate in their developmental Thus, female gametophytes in and usually were characterized as and rapid fusion of the two polar nuclei within the central cell 1966; Johri et al., 1992; Tobe et al., in a Basal Angiosperm (A) in (B) with nuclei to the micropylar pole of the female gametophyte. (C) and views of the transition from two nuclei in to four nuclei in of the female gametophyte before to an egg The central cell nucleus is with an of the of the Angiosperm single nucleus is in a domain of the female gametophyte. This nucleus then initiates two free nuclear mitoses to yield four of these nuclei are partitioned into uninucleate the fourth nucleus is to the common of the central cell of the female gametophyte. There are two female gametophyte present in the most ancient of extant the monosporic four-celled, four-nucleate characteristic of and Austrobaileyales (Friedman et al., 2003; Friedman and Williams, 2003; Williams and Friedman, and the monosporic seven-celled, eight-nucleate of Amborella et al., the of evolutionary transition is, on of we have that four-celled, four-nucleate female gametophytes may be among angiosperms (Friedman and Williams, 2003). this hypothesis is the first angiosperm female gametophytes were of a single four-celled, four-nucleate module (Friedman and Williams, 2003). in angiosperm ectopic expression of this basic developmental module in the of a second developmental module within the female gametophyte. of the of the of Basal Angiosperm with of the of Amborella and and nuclear event at the in the Polygonum-type female gametophyte to the of a second developmental module. We have that the of a module in the angiosperm female gametophyte was accomplished initially at the through the developmental of a apparatus that the nuclei into cytoplasmic at of the female gametophyte The of this nuclear process was the of two in the female gametophyte and the of two modular The result was the formation of a seven-celled, eight-nucleate Polygonum-type female gametophyte (Friedman and Williams, 2003). of the Angiosperm The of the female gametophyte of to a angiosperm female gametophyte was accomplished through a process of in which the of is within the and to the of at a of the in the female gametophyte of the common ancestor of monocots, eumagnoliids, and also in the Amborella in the origin of triploid endosperm. endosperms in flowering plants were million years the modular hypothesis is the chalazal module (or of monocots, and a developmental that during the earliest of the diversification of angiosperms. Thus, the second polar nucleus and the three antipodal cells of the seven-celled, eight-nucleate female gametophyte are angiosperm and are not with any of the female gametophyte of plants. This conclusion in marked to the long-standing hypothesis that the antipodal cells of the seven-celled, eight-nucleate female gametophyte are with a the of plant female gametophytes (Sargant, 1900; Battaglia, Favre-DuChartre, The developmental and functional of antipodals and the chalazal polar nucleus in basal and basal that the chalazal module of the Polygonum-type female gametophyte of two three antipodal cells that development (Williams and Friedman, and a polar nucleus that was partitioned into the central cell, it in a second fertilization In more derived angiosperm lineages, antipodal cells have in these the proliferation of nuclei to of cell and cell associated with Schnarf, 1931; and 1981; 1984; and The phylogenetic of with more antipodals is in with the hypothesis of origin by modular of gametic of the egg apparatus into and subsequent for (Williams and Friedman, 2004). for the of Angiosperm (A) module found in and Austrobaileyales as well as in and The central cell nucleus is in egg cell. (B) found in Polygonum-type female The central cell nuclei are in (C) found in the female gametophyte. The central cell nuclei are in found in the female gametophyte. module a single before the In this female there are no The central cell nuclei are in The key to between angiosperm female gametophytes with of in the of early development to nuclei within or four cytoplasmic and (Friedman and Williams, 2003). The of nuclear to at the early can for the transition from a four-nucleate to an eight-nucleate female gametophyte in the common ancestor of monocots, eumagnoliids, and eudicots. the of nuclear at the can for the origin of the four-celled, four-nucleate female gametophyte from an eight-nucleate as is the in analysis indicates that the of had a Polygonum-type female gametophyte and et al., is not the group in which the chalazal module has been In and the chalazal module to have been and in this with a from eight-nucleate female gametophyte) to four-nucleate female gametophyte) and Battaglia, The monosporic seven-celled, eight-nucleate Polygonum-type female gametophyte has long as the for analysis of the origin and subsequent evolution of the angiosperm female gametophyte 1931; 1950; Johri, 1963; Davis, 1966; Foster and Gifford, 1974; Stebbins, 1974; Palser, 1975; Takhtajan, 1976; Favre-DuChartre, 1984; Cronquist, 1988; Battaglia, 1989; Haig, 1990; Donoghue and Scheiner, 1992; Johri et al., 1992; Tobe et al., is that this type of female gametophyte is among basal angiosperms in a single extant Amborella the four-celled, four-nucleate type of female gametophyte the ancient angiosperm lineages and Austrobaileyales and may be for flowering plants as a Given that the genetic constitution of endosperm is directly to the genetic constitution of the central cell of the female in female gametophyte development have the to directly the ploidy and of endosperm. We that in the modular of female gametophytes may have to with the functional biology of the gametophyte has to female gametophyte developmental that have in the genetics and ploidy of endosperm. the critical of associated with evolutionary in endosperm genetics and ploidy will be that the of free nuclei during the early of female gametophyte development and the of developmental that are this the key of the chalazal module was the chalazal polar nucleus that in a second fertilization event and the genetic constitution and ploidy of endosperm. Thus, the set of three in their in the earliest Polygonum-type female can be viewed as an evolutionary and in essence, as a developmental associated with the evolution of an maternal nuclear to the central cell, accomplished through modular (Friedman and Williams, 2003). The (or expression of the modular of the angiosperm female gametophyte has provided a for the of of endosperm ploidy and of maternal to paternal to endosperm. endosperm ploidy and in the ratio of maternal to paternal is with and by analyses of the of and during the early evolution of endosperm and 1989; and and 1988; Friedman, to these between and the of in the embryo-nourishing tissues of of a maternal and based between for from the maternal in the maternal to endosperm the ploidy of endosperm. also is that the structure that to endosperm is an analysis has that the embryo-nourishing of endosperm must have evolved the second fertilization product was a diploid Finally, ploidy of endosperm has been viewed as an to of through of Thus, the hypothesis that the first angiosperms had diploid endosperms (as a result of a female gametophyte) and that this condition (in geological terms) to a triploid endosperm with (as a result of a seven-celled, eight-nucleate female gametophyte) is with a range of earlier based analyses of the evolution of in essence, had this For more than a century, the question of what endosperm is, from an of has plant and evolutionary The of the diploid condition of early angiosperm endosperms may to address this an there are three to the question of what endosperm is, all of which have been for more than a century (Sargant, 1900; 1900; 1907; endosperm is the of an embryo Friedman this the of a of the female gametophyte by or an that during the earliest of angiosperm evolution and is, in essence, not with any of the double fertilization is a feature of the common ancestor of angiosperms, the that diploid endosperms among basal angiosperms and are likely to be that before the of embryo-nourishing the ancestral second fertilization product was diploid ancestral condition is congruent with an embryo for are double fertilization events in may not be to of flowering plants, it is that the second fertilization product in and is a diploid embryo and Friedman, as to be the in that are to have a second fertilization process that the of by an embryo could have been for by in the of a embryo that is the of the embryo-nourishing of its and in the endosperms of and 1984; and et al., et al., et al., et al., et al., et al., et al., 2003) have of it is are present in all triploid endosperms with a 2:1 ratio of an question eventually will be among basal angiosperms with diploid endosperms and a ratio of comparative based on the most of angiosperms, the evolutionary developmental hypothesis (Friedman and Williams, 2003; Williams and Friedman, 2004). The earliest angiosperms a four-celled, four-nucleate female gametophyte that antipodals and a second polar The developmental of the female gametophyte of cells with to the condition of the first angiosperms the of the of the of from to female and the of the This condition is to be found among and million years after the origin of angiosperms, ectopic of the basic modular that produces the egg apparatus and a uninucleate haploid central cell a second developmental module expressed in the chalazal domain of the female gametophyte of the common ancestor of monocots, eumagnoliids, and also in an ancestor of Amborella Thus, the antipodals of the seven-celled, eight-nucleate female gametophyte (Polygonum type) a and egg Because the ploidy and genetic constitution of endosperm is always tied directly to the development and mature structure of the female the transition from a monosporic four-celled, four-nucleate female gametophyte with a haploid central cell to a monosporic seven-celled, eight-nucleate female gametophyte with a binucleate (diploid) central cell also marked the transition from the diploid genetically biparental endosperms of the earliest angiosperms to the triploid genetically biparental endosperms found among most extant flowering plants, including and Finally, the of the first angiosperms a process of double fertilization the answer will be as soon as the condition is in double fertilization is found to be a defining feature of the first angiosperms, this on the century-old question of the evolutionary origin of double fertilization most importantly, the of endosperm. than a century after the of study of the reproductive biology of flowering plants, to be about the basic features of the process in angiosperms in general more in the most ancient lineages of this of plants. a new of the evolutionary developmental for the diversification of flowering plant gametophyte fertilization process, and endosperm genetics is to from studies by recent into angiosperm and a more of plant structure We and for critical and to the and for with and This was by a from the to and by a from the of on and