C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 324 (2001) 559–567 © 2001 Académie des sciences/Éditions scientifiques et médicales Elsevier SAS. Tous droits réservés S0764446901013269/REV

Revue / Review Developmental and evolutionary hypotheses for the origin of and endosperm

William E. Friedman*

Department of Environmental, Population and Organismic , University of Colorado, Boulder, Colorado 80309, USA Received 13 October 2000; accepted 4 December 2000 Communicated by Christian Dumas

Abstract – The discovery of a second fertilization event that initiates endosperm in flowering , just over a century ago, stimulated intense interest in the evolutionary history and homology of endosperm, the genetically biparental -nourishing that is found only in angiosperms. Two alternative hypotheses for the origin of double fertilization and endosperm have been invoked to explain the origin of the angiosperm reproductive syndrome from a typical non-flowering reproduc- tive syndrome. Endosperm may have arisen from a developmental transformation of a supernumerary embryo derived from a rudimentary second fertilization event that first evolved in the ancestors of angiosperms (endosperm homologous with an embryo). Conversely, endosperm may represent the developmental transformation of the cellular phase of non-flowering seed plant female ontogeny that was later sexual- ized by the addition of a second fertilization event in a strongly progenetic female gametophyte (endosperm homologous with a female gametophyte). For the first time, explicit developmental and evolutionary transitions for both of these hypotheses are examined and compared. In addition, current data that may be congruent with either of these hypotheses are discussed. It is clear that much remains to be accomplished if the evolutionary significance of the process of double fertilization and the formation of endosperm is to be fully understood. © 2001 Académie des sciences/Éditions scienti- fiques et médicales Elsevier SAS double fertilization / endosperm development / endosperm evolution / heterochrony / homology

Résumé – Origine évolutive de la double fécondation et de l’albumen. Il y a plus d’un siècle, la découverte d’une seconde fécondation à l’origine du développement de l’albumen chez les angiospermes a suscité un grand intérêt quant à son origine évolutive. L’albumen est le tissu nutritif de l’embryon ; il a une origine génétique bi-parentale et se trouve seulement chez les plantes à fleurs. Deux hypothèses alternatives concernant l’origine de la double fécondation et de l’albumen ont été proposées pour tenter d’expliquer l’origine du système de reproduction des angiosper- mes à partir d’un système typique des phanérogames sans fleur. La première considère que l’albumen a évolué grâce au développement d’un embryon surnuméraire issu d’une seconde fécondation chez les espèces à l’origine des angiospermes : dans ce cas, l’albumen peut être considéré comme homologue à un embryon. La seconde considère qu’il représente une modification du gamétophyte femelle d’une phanérogame sans fleur qui, après sexualisation, aboutit à l’addition d’une seconde fécondation : dans ce cas, l’albumen est homologue à un gamétophyte femelle. Pour la première fois et de

*Correspondence and reprints. E-mail address: [email protected] (W.E. Friedman).

559 W.E. Friedman / C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 324 (2001) 559–567 façon explicite, les transitions observées au cours du développement du sac embryon- naire et leurs évolutions sont décrites en détail et comparées. De plus, des données récentes de nature cellulaire et moléculaire permettent de discuter ces deux hypothèses. Il reste cependant beaucoup d’investigations à entreprendre pour comprendre la signification évolutive de la double fécondation et de l’albumen. © 2001 Académie des sciences/Éditions scientifiques et médicales Elsevier SAS double fécondation / développement de l’albumen / évolution de l’albumen / hétérochronie / homologie

. Version abrégée L’albumen a été décrit soit comme homologue à un embryon, soit encore comme homologue au stage de la cellularisation du gamétophyte femelle stimulé par une seconde fécondation. La découverte de la double fécondation chez les Divers arguments expérimentaux ont permis récem- plantes àfleurs, il y a tout juste un siècle, a suscité de ment de prouver l’existence d’une double fécondation nombreuses recherches sur la signification de chez des plantes sans fleur, notamment chez des genres l’albumen, un des deux produits de la double fécon- appartenant aux Gnétales, et Gnetum. Cela dation. En effet, l’albumen, véritable nourrice pour tend à suggérer qu’un tel mécanisme aurait pu exister l’embryon plantule, a une origine génétique chez un ancêtre commun aux Gnétales et aux bi-parentale complexe (deux doses de gènes d’origine angiospermes. De même, l’analyse développementale maternelle pour une d’origine paternelle). Et, bien que qui considère les méchanismes hétérochroniques la double fécondation a été généralisée chez les apporte un argument à l’homologie entre l’albumen et angiospermes, dèsledébut du vingtième siècle, son le développement par cellularisation du gamétophyte existence n’a pas encore été démontrée avec certitude femelle. Un siècle aprèssadécouverte, la double chez des espèces primitives, en particulier Amborella, fécondation fait toujours l’objet de recherches actives espèce la plus primitive, considérée comme la sœur de en évolution et développement, notamment en ce qui toutes les plantes àfleurs actuelles. concerne l’albumen.

1. Introduction tion of the phenomenon [3]. The second question that emerged from the discovery of the initiation of endosperm from a second fertilization event focused on the evolution- Just over a century ago, the developmental origin of the ary origin of the endosperm tissue of flowering plants. This embryo-nourishing tissue of flowering plants (endosperm) line of inquiry, one of fundamental homology assessment, was independently discovered by Nawaschin [1] of Russia was widely debated during the first decade of the twenti- and Guignard [2] of France. Until 1898, the assumption eth century, but remained unresolved [4]. As will be seen, had been that the embryo-nourishing tissue of the flower- analysis of the homology of endosperm has reemerged at ing plant seed was a developmental product of the fusion the start of the twenty-first century, as a complex and of the two polar nuclei of the angiosperm female gameto- vexing set of issues that may well define important research phyte. Working with Lilium and , Nawaschin and directions for the field of plant reproductive biology during Guignard were able to document the participation of the the coming years. second of a tube in a fusion event with the two polar nuclei of the female gametophyte. This seminal discovery, of a second fertilization event in angiosperms 2. The phylogenetic distribution that gives rise to a biparental embryo-nourishing tissue, of double fertilization represented the culmination of a century of research activ- ity in which the field of plant reproductive biology was Immediately after the announced discoveries of double essentially born and all of the diverse life cycles of major fertilization in two members of the (Lilium and lineages of plants were circumscribed. Fritillaria), workers around the world (France, Russia, The unexpected discovery of the double fertilization Germany, Japan, United States, Great Britain) began to process generated widespread interest in the solution to closely examine the developmental events surrounding two immediately evident and fundamental questions in the fertilization process in diverse angiosperms. Guignard plant reproductive biology. The first question dealt specifi- [5–7] proceeded to document a process of double fertili- cally with the phylogenetic distribution of double fertili- zation in additional taxa within the Liliaceae, as well as in zation in angiosperms and would seemingly be ‘solved’ the closely related Amaryllidaceae. Additional reports of a (but see below) within two years of the initial documenta- second fertilization event among monocots were pub 560 W.E. Friedman / C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 324 (2001) 559–567 lished by Strasburger [8] in 1900. At the same time, a race are all basal to the common ancestor of eudicots (a large to discover whether double fertilization could be found monophyletic group of dicotyledonous flowering plants among ‘dicots’ culminated, in 1900, in the nearly simul- that comprise the overwhelming majority of dicotyledon- taneous reports of double fertilization events in Ranuncu- ous angiosperms) and monocots. laceae [9, 10], [10, 11] and Monotropaceae A century after double fertilization was raised to the [8]. status of a defining feature of flowering plants, it has In 1901 and 1902, double fertilization events were become evident that the synapomorphic status of a sexu- reported in [12], Najadaceae [13], additional ally formed endosperm has yet to be fully confirmed [3]. members of the Ranunculaceae [14], Solanaceae [15], There have been just three reports ever of a putative fusion Gentianaceae [15], [16], Asclepiadaceae of a second sperm with the two polar nuclei (or their fusion [17], Juglandaceae [18], and Ceratophyllaceae [19]. By product) in the most basal angiosperm clades: for Brasenia 1903, sixteen families of flowering plants were known to (Cabombaceae) [45], Nymphaea () [46], have a second fertilization event that initiated a biparental and (Illiciaceae) [47]. None of these studies yielded endosperm [20]. any light micrographs or transmission electron micro- The rapid accumulation of evidence of the potentially scope images of a second fertilization event. Rather, a widespread distribution of double fertilization in both single small drawing indicating proximity of a putative monocots and ‘dicots’ (dicotyledonous flowering plants sperm nucleus and the fused polar nuclei accompanied are now known to be paraphyletic – see below) led each publication [45–47]. In Amborella, a taxon whose Sargant [21] to conclude that this unique reproductive biology is now seen to be central to the reconstruction of process was likely to be a general feature of all flowering ancestral angiosperm features, nothing whatsoever is plants. In an early (1904) retrospective, Guérin [22] con- known of its fertilization biology (it has never been stud- curred: “Par les nombreux résultats obtenus en moins de ied). The fertilization process in Austrobaileyaceae, Trime- deux années chez les Monocotylédones et les Dicotylé- niaceae and Schisandraceae also remains unexamined. dones, l’existence dans les Angiospermes d’une double Ironically, a century after double fertilization was fécondation, l’une donnant naissance à l’embryon, l’autre elevated to the status of a defining (synapomorphic) and à l’albumen, pouvait être considéréedésormais comme general feature of angiosperms [21, 22], evidence of a un fait définitivement acquis à la science”. Within just two double fertilization process in the most basal angiosperms years of the initial discoveries of double fertilization in two is, at best, scant. It is an unfortunate reality that at the members of the Liliaceae, double fertilization was viewed outset of the twenty-first century, virtually nothing is known asadefining feature of all flowering plants. of the fertilization process in the most basal, and poten- Much progress has been made in the study of the tially plesiomorphic, angiosperms. If a second fertilization fertilization biology of angiosperms since the initial burst event is to be conclusively demonstrated in basal of activity associated with the reports of Nawaschin and angiosperms, micrographs (light, fluorescence, transmis- Guignard. With the advent of transmission electron micros- sion electron microscopy) of developmental events asso- copy, the participation of a sperm in a second fertilization ciated with a triploid fusion, as well as DNA quantitation event in angiosperms was conclusively documented in of the putative fertilization product, will be essential. both monocots (three members of the Poaceae: Hordeum, Triticum, and Triticale) and eudicots (seven taxa: Gos- sypium, Linum, Spinacia, Plumbago, Populus, Glycine, 3. The question of the homology and Nicotiana) [23–35]. The presence of double fertiliza- of endosperm – early debate tion in both monocots and eudicots indicates that this feature of reproductive biology was a characteristic of the Prior to the discovery of a second fertilization event in common ancestor of these two large angiosperm clades, flowering plants, endosperm had been widely viewed as a and of most, but not necessarily all, angiosperms [3]. developmental phase within the ontogeny of the female Interestingly, the condition for basal angiosperm lineages gametophyte, whose initiation was marked by the fusion is far less certain. of the two polar nuclei. As such, the endosperm of flow- Current phylogenetic hypotheses have, for the first time, ering plants was widely accepted to be evolutionarily conclusively identified the most basal angiosperm lin- homologous with the female gametophyte of the non- eages. These analyses [36–44] support the hypothesis that flowering seed plant life cycle, as first proposed by monotypic Amborella (an endemic of New Caledonia) or Hofmeister [21] and subsequently advanced by Stras- Nymphaeales (Nymphaeaceae plus Cabombaceae) or a burger [48]. Almost immediately after the discovery of the clade that includes Amborella plus Nymphaeales is sister sexual (biparental) origin of endosperm from a second to all other angiosperms exclusive of Amborella; and that fertilization event [1, 2], fundamental issues associated a clade which includes Illiciales (Illiciaceae plus Schisan- with the evolutionary homology of endosperm were rede- draceae) plus Trimeniaceae plus Austrobaileyaceae is sis- fined and a vigorous debate ensued. ter to the remaining angiosperms (figure 1). This phyloge- In discussions of their discoveries of double fertilization, netic hypothesis also reveals that Amborella, Nymphaeales Nawaschin referred to the phenomenon as a form of and the Illiciales–Trimeniaceae–Austrobaileyaceae clade polyembryony [5], while Guignard [2] concluded that the 561 W.E. Friedman / C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 324 (2001) 559–567

Figure 1. Current hypothesis of relationships of extant basal angiosperms based on recent molecular phylogenetic analyses [36–43]. Three clades (Amborellaceae, Nymphaeales, Illiciales–Trimeniaceae–Austrobaileyaceae) have been identified as the earliest (extant) divergent angiosperms and are basal to the common ancestor of monocots and eudicots. endosperm was a transitory organism (‘organisme transi- hypothesis of homology with the female gametophyte, toire’). In 1900, Sargant [21] proposed that the endosperm Strasburger [8] referred to endosperm as a ‘secondary of flowering plants might be homologous with a supernu- prothallium’. merary embryo. Sargant hypothesized that the ancestors of angiosperms had a double fertilization process that origi- nally yielded two [49] and that one of these 4. The question of the homology embryos had been developmentally transformed into an of endosperm – recent analysis and new embryo-nourishing structure. Interestingly, the hypothesis that endosperm might be homologous with an embryo hypotheses predates the discovery of double fertilization. In 1887, LeMonnier [50] proposed that the fusion of the two polar The developmental and evolutionary underpinnings of nuclei (then believed to be the sole contributors to the the hypothesis that endosperm is homologous with an initiation of endosperm) could itself be viewed as a sexual embryo have been explicitly analyzed during the last event and that the endosperm derived from this fusion twenty years [52–61] and have recently been reviewed [4, could be considered a distinct and separate organism or 60]. Essentially, the endosperm–embryo homology hypoth- embryo. esis posits the following evolutionary events in the ances- In contra-distinction to the hypothesis that endosperm is tors of angiosperms: homologous with an embryo, Strasburger [8], and later – Origin of a second fertilization event that produces a Coulter [51], argued that the formation of endosperm supernumerary embryo. tissue should be viewed as a second phase in the devel- – Acquisition of embryo-nourishing function by the super- opment of the female gametophyte. In essence, Stras- numerary embryo. burger viewed the endosperm within the female gameto- – Reduction of the embryo-nourishing role and size of the phyte not as a separate entity (as would be the case with female gametophyte (eventually to a seven-celled mature the embryo-origin hypothesis), but rather as a continua- structure with no embryo-nourishing function) [60]. tion of female gametophyte development that is stimu- – Loss of individual fitness by the supernumerary embryo lated by the second fertilization event. In keeping with his (associated with the acquisition of embryo-nourishing 562 W.E. Friedman / C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 324 (2001) 559–567 function) compensated for by gains in the inclusive fitness angiosperms, if endosperm is hypothesized to be derived of the compatriot embryo [60]. from (homologous with) a developmental component of – Transformation of development of the second fertiliza- the female gametophyte, can be viewed as a post- tion product from an indeterminate embryo– fertilization cellular growth phase. pattern to a determinate pattern characteristic of When the general and putatively plesiomorphic endosperm. angiosperm female gametophyte ontogeny (including the – Addition of a second female nucleus to the second endosperm phase) is compared with the general seed plant fertilization event. female gametophyte ontogeny (figure 2), both ontogenies Even though the original ideas for the endosperm– reveal the same sequence of events: free nuclear develop- female gametophyte homology hypothesis are over a cen- ment, cellularization of the , and a final phase of tury old, there has been little explicit articulation of the cellular growth. However, several aspects of the ontoge- specific evolutionary and developmental transitions that netic trajectory of flowering plant female might have been required to transform the ontogeny of a differ from non-flowering seed plant female gametophytes. non-flowering seed plant female gametophyte into that of The first stage of the ontogeny of the female gametophyte, an angiosperm, in which the endosperm comprises the the proliferation of free nuclei, has been significantly terminal phase of its development. What follows is an reduced (from many rounds of mitosis) to only three attempt to define specific developmental and evolutionary successive divisions in angiosperms to yield eight nuclei transitions in the reproductive biology of the ancestors of [63, 64, 66, 67]. Correlated with the small number of free angiosperms that would be congruent with the origin of nuclei in the angiosperm female gametophyte, the cellu- endosperm from a female gametophyte. larization phase is much abbreviated in duration com- If endosperm is homologous with a phase of female pared with the female gametophytes of non-flowering gametophyte development, it is almost certain that hetero- seed plants. However, the final growth phase of the chronic alterations to the reproductive process of the angiosperm female gametophyte (assuming endosperm ancestors of angiosperms played a central role in the proliferation is a phase of female gametophyte develop- evolution of this syndrome. All extant non-flowering seed ment) can be considered roughly similar, in duration and plants form large embryo-nourishing female gametophytes. extent, to the proliferative phase in non-flowering seed Most non-flowering seed plant female gametophytes go plants. through a free nuclear phase (mitosis without cytokinesis), The most profound alteration in the ontogeny of the followed by cellularization of the single celled syncytium angiosperm female gametophyte is the acceleration of the (cytokinesis without mitosis) and a third phase of cellular point of fertilization from late in the somatic ontogeny (as growth (mitosis coupled with cytokinesis) to produce a in non-flowering seed plants) to a point just after cellular- large (many thousands of cells) female gametophyte. These ization of the eight nucleate syncytium. Thus, the three generalized phases of female gametophyte develop- angiosperm female gametophyte is strongly progenetic ment (figure 2) comprise the somatic ontogeny of the (figure 2), compared with its ancestors [62, 64, 67, 68]. female haploid organism [62]. During the third and final Within the context of comparisons of angiosperm female phase of female gametophyte ontogeny, gametangia gametophytes to those of non-flowering seed plants, the (archegonia containing eggs) are initiated and fertilization hypothesis that endosperm is homologous with a female occurs at some point during (conifers) or at the end of gametophyte appears to require the following evolution- (cycads, Ginkgo) the cellular growth phase [63, 64]. This ary events: general and widespread pattern of female gametophyte – A strong trend towards earlier reproductive maturity development among non-flowering seed plants can be (progenesis) in the female gametophyte of the ancestors of considered the starting point for an analysis of the evolu- angiosperms. tion of the angiosperm reproductive syndrome. – Significant abbreviation of the first two phases of the The female gametophyte in plesiomorphic angiosperms ontogeny of the female gametophyte resulting in a trun- initiates a set of three successive free nuclear divisions to cated free nuclear phase (ultimately only three successive yield a syncytium that contains eight free nuclei. Partial mitotic divisions) and associated brief cellularization cellularization of the syncytial angiosperm female game- phase. tophyte produces six uninucleate cells (three antipodals, – Introduction of a nuclear fusion event that initiates the two synergids, one egg) and a central chamber (termed the cellular phase of female gametophyte ontogeny and pro- central cell) which contains the two remaining nuclei duces a diploid and strictly maternal embryo-nourishing (polar nuclei) from the syncytial stage. Prior to, or at the tissue that develops after the time of fertilization. time of, the second fertilization event, the polar nuclei – Addition of a second sperm to the fusion event between fuse. Recent phylogenetically-based analysis of endosperm the polar nuclei to sexualize the ‘endosperm’ and render it developmental patterns in angiosperms clearly demon- genetically and developmentally biparental. strates that the cellular pattern of endosperm proliferation If the endosperm tissue of angiosperms is homologous (as contrasted with free nuclear or helobial patterns) is with the female gametophyte of non-flowering seed plants plesiomorphic for flowering plants [61, 65]. Thus, the third (i.e., derived from the final cellular phase of somatic phase of female gametophyte development in development), it is likely that a second fertilization event

563 W.E. Friedman / C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 324 (2001) 559–567

Figure 2. Comparative ontogenetic trajectories of the generalized female gametophyte of non-flowering seed plants and the (hypothetical) plesiomorphic female gametophyte of angiosperms (Polygonum type). As can be seen, the timing of fertilization in the ontogeny of the angiosperm female gametophyte has been accelerated to an early point and is a reflection of strong progenesis. If it is assumed that endosperm is a developmental component of (and is homologous with) the female gametophyte, both the female gametophytes of most non-flowering seed plants and flowering seed plants would be seen to pass through the same three developmental stages: free nuclear proliferation, cellularization of the syncytial stage, and a cellular growth phase in which mitosis and cytokinesis are coupled. According to the endosperm–female gametophyte homology hypothesis, a second fertilization event to yield a genetically biparental cellular phase of gametophyte development would have originated after the evolution of a strongly progenetic female gametophyte. Green arrowheads indicate the time of fertilization within the ontogeny of the female gametophyte in cycads and Ginkgo (terminal), conifers (within the cellular growth phase), and angiosperms (prior to the cellular growth phase of the endosperm). was not a primary factor in the origin of the angiosperm homozygous and strictly maternal embryo-nourishing tis- reproductive syndrome. Rather, acceleration of the timing sue. This chain of events, if endosperm is homologous of fertilization (progenesis) within the ontogeny of the with a component of female gametophyte ontogeny, stands female gametophyte must have been a central factor. in marked contrast with the ‘endosperm–embryo homol- Within the context of this hypothesis, origin of a post- ogy hypothesis’ where a second fertilization event that fertilization embryo-nourishing tissue (as found in produces a supernumerary embryo is the starting point for angiosperms) would be entirely unassociated with the the origin of endosperm. evolution of a second fertilization event. A subsequent sexualization of endosperm might well have produced If endosperm of flowering plants is homologous with the genetic [54, 55, 69] and/or -related [67, 70] benefits cellular growth phase of the female gametophyte of non- to development that improved upon an originally diploid flowering seed plants, the modern concept of the

564 W.E. Friedman / C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 324 (2001) 559–567 angiosperm female gametophyte as a seven-celled, eight- tial development of chalazal and micropylar ‘domains’ nucleate organism at somatic and sexual maturity will [61]. Thus, there is accumulating evidence of the embryo- require substantial revision: the seven-celled, eight nucle- like nature of endosperm in flowering plants. It would be ate stage would represent a sexually mature, but somati- most valuable to know whether specific patterns of gene cally immature female gametophyte and the post- expression associated with embryogenesis are also asso- fertilization development of endosperm would represent ciated with endosperm development in basal angiosperms. the terminal phase of angiosperm female gametophyte Conversely, the recently described fertilization- development, homologous with the cellular growth phase independent endosperm mutants in Arabidopsis [82–85] within the ontogenetic sequence of female gametophyte could be interpreted to support the homology of development in non-flowering seed plants (figure 2). endosperm with a phase of female gametophyte ontogeny. In the fertilization-independent endosperm mutants stud- ied to date, a strictly maternal ‘endosperm’ tissue initiates 5. Current evidence that relates to development from the fused polar nuclei, in the absence the origin of endosperm of fertilization. This suggests that a second fertilization event may not be necessary for the developmental estab- lishment of the embryo-nourishing tissue of flowering Outside of flowering plants, regular double fertilization plants. However, the interpretation of the phenotype of events have been documented in Ephedra and Gnetum fertilization-independent endosperm mutants as an [71, 72], two members of the Gnetales. The product of the ‘endosperm’ may be premature. Fertilization-independent second fertilization event in Gnetales is a diploid super- endosperm mutants are known to initiate a free nuclear numerary that initiates embryo development [58, proliferation of the fused polar nuclei of the central cell, 73]. This pattern of double fertilization is remarkably but there is no evidence that this ‘tissue’ cellularizes, similar to the condition that Sargant [21] originally hypoth- undergoes cellular growth and ultimately assumes the esized might have characterized the ancestors of basic features of a functional embryo-nourishing angiosperms. If the second fertilization event in Gnetales endosperm [82–85]. It is entirely possible that the ‘real’ and angiosperms is homologous, endosperm is likely to phenotype of known fertilization-independent endosperm represent a developmental transformation of an embryo mutants is one in which the cell cycle of the fused polar [60]. It is worth noting that the most recent seed plant nuclei of the central cell of the embryo sac is activated in phylogenetic hypotheses suggest that Gnetales may be the absence of a second fertilization event, but the suite of most closely related to conifers, and more distantly related cellular and molecular developmental programs associ- to angiosperms [74–80]. If double fertilization events are ated with the differentiation of an actual endosperm tissue homologous in Gnetales and angiosperms, a plesiomor- are not activated. phic pattern of double fertilization, to produce two embryos, must have been present in the common ancestor 6. Conclusions of Gnetales and angiosperms [3]. Irrespective of interrelationships of seed plants and The seminal discovery of the developmental origin of homology assessment of double fertilization events among endosperm in flowering plants from a second fertilization major seed plant lineages, the issue still remains that event by Nawaschin and Guignard in 1898 and 1899 endosperm must have an evolutionary antecedent: it is represented the crowning achievement of nineteenth cen- either a homologue of the female gametophyte or it is a tury comparative plant reproductive biology. During this developmentally transformed embryo [3]. The fundamen- period, beginning with the accidental discovery of the tal debate of the early twentieth century remains in 1824 [86], all of the basic sexual life cycles unchanged at the outset of the twenty-first century, and of major lineages of land plants were described, and many evaluation of the homology and evolutionary history of of the most profound questions of homology and evolu- endosperm represents a complex and formidable task. tionary history of plants were first articulated. New developmental data may be relevant to the deter- Remarkably, a century after the initial debate on the mination of the homology of endosperm. For example, in evolutionary significance of the process of double fertili- vitro endosperm of Zea (formed outside of the physical zation in flowering plants, much remains to be accom- constraints of an ) typically forms a globular region of plished if the evolutionary history of double fertilization is densely cytoplasmic cells and a filamentous region of to be completely revealed and the homology of endosperm larger, more vacuolate cells similar to the bipolar differen- is to be definitively resolved. A century after the field of tiation of embryos [81]. In addition, recent studies of comparative fertilization biology of plants reached a zenith endosperm development in diverse basal angiosperm taxa of activity, there is a clear need for a renewal of efforts in [61, 65] reveal that plesiomorphic endosperm develop- this discipline. The new phylogenetic hypotheses for basal ment in flowering plants shares many basic developmen- angiosperms indicate that virtually nothing is known of the tal properties in common with embryos. The early ontog- reproductive biology of the earliest angiosperm lineages. eny of both embryos and endosperms in basal angiosperms Studies of the fertilization process, ranging from descrip- involves unequal partitioning of the first cell and differen- tions of basic developmental events to cell biology and 565 W.E. Friedman / C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 324 (2001) 559–567 ultrastructure, are desperately needed in basal and endosperm of basal angiosperms may have much to angiosperms. And with time, comparative studies of pat- contribute to our understanding of the origin and early terns of molecular developmental events associated with evolution of the defining reproductive biology of flower- the fertilization process, the female gametophyte, embryo ing plants.

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