Female Gamete Competition in an Ancient Angiosperm Lineage

Female gamete competition in an ancient angiosperm lineage

Julien B. Bacheliera,b,c and William E. Friedmana,b,c,1

aDepartment of Ecology and Evolutionary Biology, University of Colorado, Boulder, CO 80309; bDepartment of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA 02138; and cArnold Arboretum, Harvard University, Boston, MA 02131

Edited* by Peter H. Raven, Missouri Botanical Garden, St. Louis, MO, and approved May 19, 2011 (received for review March 23, 2011) In Trimenia moorei, an extant member of the ancient angiosperm ly diversification of floral morphology and anatomy are widely clade Austrobaileyales, we found a remarkable pattern of female viewed to have led to increased levels of pollen reception and gametophyte (egg-producing structure) development that strik- hence, male–male competition (4). Collectively, the evolution of ingly resembles that of pollen tubes and their intrasexual compe- the carpel, extragynoecial compitum, transmitting tissue, callose tition within the maternal pollen tube transmitting tissues of most plugs, and insect pollination seems to have resulted in signifi- flowers. In contrast with most other flowering plants, in Trimenia, cantly enhanced levels of prefertilization male competition and multiple female gametophytes are initiated at the base (chalazal maternal choice, and thus may have played a major role in the end) of each ovule. Female gametophytes grow from their tips and early diversification of angiosperms (4–9). compete over hundreds of micrometers to reach the apex of the Despite all of the attention paid to prefertilization mecha- nucellus and the site of fertilization. Here, the successful female nisms of male competition and female choice, there has never gametophyte will mate with a pollen tube to produce an embryo been any discussion of the possibility that female gametophytes and an endosperm. Moreover, the central tissue within the ovules (haploid egg-producing structures within ovules) might engage in of Trimenia, through which the embryo sacs grow, contains starch a process of competition. Interestingly, recent embryological and other carbohydrates similar to the pollen tube transmitting studies of several members of the Nymphaeales and Austro- tissues in the styles of most flowers. The pattern of female game- baileyales have revealed that, in contrast with most angiosperms, tophyte development found in Trimenia is rare but by no means more than one female gametophyte is occasionally or frequently EVOLUTION unique in angiosperms. Importantly, it seems that multiple female initiated in each ovule (10–14). For us, this suggests the potential gametophytes are occasionally or frequently initiated in members for competition between female gametophyte genomes during fi of other ancient angiosperm lineages. The intensi cation of pollen the developmental period leading up to the time of fertilization. tube (male gametophyte) competition and enhanced maternal se- To gain insight into whether female gametophytes, in addition lection among competing pollen tubes are considered to have to male gametophytes, also engage in intrasexual competition been major contributors to the rise of angiosperms. Based on fl Trimenia before fertilization among early lineages of owering plants, we insights from , we posit that prefertilization female ga- examined ovule and gametophyte development in Trimenia (Tri- metophyte (egg) competition within individual ovules in addition meniaceae). Previous studies of Trimenia reported that as many as to male gametophyte (sperm) competition and maternal mate 20 megasporocytes differentiate in each ovule and that several choice may have been key features of the earliest angiosperms. female gametophytes will proceed to develop (11, 15). These brief reports provide circumstantial evidence for potentially high levels evolution of flowering plants | reproduction | Darwin of intragametophytic competition. However, none of these earlier studies examined the development of female gametophytes from ver since Charles Darwin concluded that the early diversi- their initiation to the time of fertilization. As will be seen, because fi “ ” E cation of angiosperms was an abominable mystery, numer- Trimenia occupies a relatively basal position within the Austro- ous hypotheses have been advanced to explain their extraor- baileyales, it has the potential to help resolve the question of dinary evolutionary radiation (1, 2). These hypotheses suggest that whether female gametophyte competition before fertilization was fl anumberofkey owering plant synapomorphies were intimately a critical feature of the earliest phases of angiosperm evolution. associated with their early evolutionary history and subsequent diversification (3). Foremost among the defining features of an- Results giosperms is the carpel, which encloses the ovule/seed and increases Initiation of Multiple Female Gametophytes Occurs Far from the Site the distance of pollen tube growth between the site of pollen re- of Fertilization. In T. moorei, numerous diploid megaspore mother ception and point of fertilization. This key innovation fundamen- cells (megasporocytes) within a single ovule initiate meiosis to tally altered the basic interactions between the male and female create haploid functional megaspores (a megaspore is the founding components of the reproductive process, and it resulted in higher cell of a female gametophyte). Female gametophytes are initiated levels of male gametophyte competition and greater opportunities deep within the ovule (proximate to the chalaza) in a position re- for maternal selection among potential sperm donors (4). mote from the future site of fertilization (Fig. 1). This phenomenon Recent character state reconstructions show that the common stands in marked contrast to most flowering plants, where the fe- ancestor of angiosperms likely had an extragynoecial compitum, male gametophyte is initiated and develops in a region of the ovule a construction of the carpels that enhances competition among that is close to the location where the pollen tube will arrive to pollen tubes and their selection by the maternal plant before deposit its sperm (Discussion)(16). fertilization (5, 6). In addition, the differentiation of a transmitting tissue within the carpel that supports pollen tube growth to the ovule has long been viewed as promoting male competi- Author contributions: W.E.F. designed research; J.B.B. performed research; J.B.B. and tion and increasing maternal choice of sperm donors (7, 8). The W.E.F. analyzed data; and J.B.B. and W.E.F. wrote the paper. formation of callose plugs within pollen tubes recently has been The authors declare no conflict of interest. hypothesized to have enabled male gametophytes to grow at *This Direct Submission article had a prearranged editor. higher rates and compete over greater distances than was the 1To whom correspondence should be addressed. E-mail: [email protected]. case for the immediate ancestors of angiosperms (8, 9). Finally, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the evolution of insect pollination in conjunction with the ear- 1073/pnas.1104697108/-/DCSupplemental.

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ch ch Fig. 2. Young ovule with multiple female gametophytes. (A)3Dre- Fig. 1. Young ovules showing the zone of megasporogenesis at the cha- construction (from transverse serial sections) of ovule nucellus (gray) con- lazal pole of the nucellus. (A) Very young ovule with one of the megaspore taining tube-like female gametophytes (each a different color) initiated at mother cells (mmc) in the zone of megasporogenesis (zm) at the chalazal the base of the nucellus and growing to the site of fertilization at the apex of pole (ch) of the nucellus (nuc). The asterisk marks the future site of fertil- the nucellus (micropylar pole of ovule at the top). One female gametophyte ization at the micropylar end of the nucellus. (B) Ovule showing several (green) has become dominant. (Scale bar: 200 μm.) (B–D) Transverse sections degenerated megaspores within the zone of megasporogenesis. The arrow from the series used for reconstruction in A and stained with periodic acid points to the tip of a female gametophyte (fg) that has grown out of the Schiff’s (PAS) reagent to reveal starch and other carbohydrates in pink. Each zone of megasporogenesis and is beginning to ascend through the nucellus star marks a female gametophyte of the corresponding color in the computer central tissue (indicated by arrowheads) to the future site of fertilization reconstruction; dashed lines in A indicate position of each transverse section. (asterisk). ii, inner integument; fun, funiculus. (Scale bars: 100 μm.) (B) Central tissue of the nucellus ﬁlled with starch grains (black arrows). (C) Central tissue of the nucellus with fewer and smaller starch grains and one dominant two-nucleate female gametophyte (green in A; one of two nuclei Female Gametophytes Undergo Tip Growth and Appear to Compete. shown by the green arrowhead). (D) Base of nucellus, where additional fe- Although some megaspores abort, as many as 10 megaspores male gametophytes (black stars) were initiated. (Scale bars: 25 μm.) within an individual ovule initiate female gametophytes that seem to crush and displace the diploid maternal tissue (nucellus) and Movie S3). The two free nuclei of a dominant female ga- situated above them along the chalazal to micropylar axis (Fig. 1). Shortly thereafter, free nuclear mitosis within several of the metophyte undergo a second mitotic division and form two pairs one-nucleate female gametophytes yields a set of two-nucleate of nuclei arranged transversely to each other. Cellularization of gametophytes that begin to grow to the distant apex of the nu- a four-nucleate female gametophyte is followed by differentiation cellus, where fertilization will ultimately occur (Fig. 2 and Movie of the four cells into two synergids, an egg cell, and a uninucleate S1). The nuclei pairs migrate to the growing tips of the female central cell (Fig. 4). Thus, in T. moorei, the female gametophyte is gametophytes. At this stage of development, the base of the four-celled and four-nucleate at sexual maturity (Fig. 4), which is nucellus contains several two-nucleate female gametophytes the case with all other extant members of the ancient angiosperm intertwined with each other as well as degenerated megaspores clades Austrobaileyales and Nymphaeales (14, 17). (Fig. 2). More than one tube-like female gametophyte commonly In 15 of 200 ovules of Trimenia that were examined, two or develops beyond this zone and continues to grow between the three female gametophytes ultimately reached the site of fertil- cells of the central tissue of the nucellus (Fig. 3 and Movie S2). ization at the apex of the nucellus. However, not all of the female Typically, between two and ﬁve female gametophytes extend into gametophytes that emerged from the nucellar apex underwent the distal half of the nucellus to the micropyle of the ovule (Fig. sexual maturation; some remained at the two-nucleate stage of 3). Computer reconstructed images of multiple female game- development. In several cases, we observed two female gametophytes within an ovule show that the path of growth may be tophytes within an individual ovule that had cellularized and dif- highly convoluted and that the individual tubes are often entan- ferentiated into four-nucleate and four-celled structures (Fig. 4). gled with each other. The digital reconstructions also show that, although many female gametophyte tubes are initiated at the base Transmitting Tissue for Growth of Female Gametophytes Differen- of the ovule, there is attrition, such that only one to three female tiates Within the Nucellus. The concurrent growth of the multiple gametophytes ultimately reach the site of fertilization (Figs. 1–4). tube-like female gametophytes in Trimenia occurs in the central tissue of the nucellus. Strikingly, this region of maternal tissue Maturation Produces Four-Celled, Four-Nucleate Female Gameto- contains conspicuous starch grains and other carbohydrates phytes. When a dominant tubular female gametophyte reaches (Figs. 1B and 2). Both starch grains and carbohydrates accumu- the apex of the nucellus (Fig. 3), it enlarges circumferentially at its late early in the development of the ovule at the time when distal end and crushes the surrounding tissue of the nucellus, megasporocytes start to differentiate. The nutrient reserves dis- thereby exposing it directly to a pollen tube with its sperm (Fig. 4 appear progressively as the tube-like two-nucleate female game-

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Fig. 3. Nearly mature ovule with multiple female gametophytes. (A)3D reconstruction of nucellus (gray) and tube-like female gametophytes (each

a different color) initiated at the base of the nucellus and growing to the site EVOLUTION Fig. 4. Mature ovule with three dominant female gametophytes. (A)3D of fertilization at the apex of the nucellus (micropylar pole of ovule at the reconstruction of ovule nucellus (gray) and tube-like female gametophytes top). (Scale bar: 200 μm.) (B–D) Transverse sections from series used for the (each a different color) protruding through the depression in the nucellar reconstruction in A and stained with PAS and toluidine blue. Each star marks apex (micropylar pole of ovule at the top). (Scale bar: 200 μm.) (B–E) a female gametophyte of the corresponding color in the computer re- Transverse sections taken from the series used for the reconstruction in A construction; dashed lines in A indicate the positions of each transverse and stained with PAS and toluidine blue. Each star marks a female game- section. (B) Dominant two-nucleate (green arrowheads) female gameto- tophyte of the corresponding color in the computer reconstruction; dashed phyte (green in A) has enlarged circumferentially after reaching depression lines in A indicate position of each transverse section. (B) Enlarged apices of in nucellus apex. (C) Five additional female gametophytes have grown be- the three dominant female gametophytes; a pair of synergid cells (pink yond the lower one-half of nucellus (one of the two nuclei of a subordinate arrowheads) of one of two sexually mature female gametophytes (pink star). female gametophyte indicated by the orange arrowhead). (D) Base of nu- (C) A pair of synergid cells (green arrowheads) of the second sexually mature cellus, where additional female gametophytes (black stars) were initiated. female gametophyte (green star). (D) Polar nucleus (green arrowhead) of (Scale bars: 25 μm.) the second mature female gametophyte (green star). (E) Polar nucleus (pink arrowhead) of the other sexually mature female gametophyte (pink star). (Scale bars: 20 μm.) tophytes grow through the central nucellus. After two-nucleate female gametophytes have grown through most of the length of the central tissue of the nucellus, small starch grains and carbo- the site of fertilization before the embryo sac had become sex- hydrates can only be observed in the very distal part of the nu- ually mature or had completed its growth through the nucellus. cellus (Fig. 2). Callose appears in the cell walls of the central tissue of the nucellus. Even more striking, callose is present in the Discussion walls of growing female gametophytes (Fig. 5). However, callose Four-Nucleate Female Gametophyte in Trimenia Is Consistent with plugs, as found in the pollen tubes of most angiosperms (including Most Other Ancient Angiosperm Lineages. Although earlier studies those of Trimenia) (Fig. 6), were not observed in the tube-like indicated that the female gametophyte in Trimenia was Polygo- female gametophytes. num-type (11, 15), our results clearly show that it is monosporic in origin and four-celled and four-nucleate at maturity. This finding Pollen Tube Development and Male Gametophyte Competition. Pol- is consistent with the many recent reversals of embryological len grains were rarely observed on the stigma, and often, they reports that originally claimed that ancient lineages of extant seemed to have aborted shortly after germinating and producing flowering plants have a seven-celled, eight-nucleate (Polygonum- a short tube. This behavior is consistent with earlier reports of type) embryo sac (12–14, 19–22). Indeed, none of the members of self-incompatibility in Trimenia (18). When male gametophytes the three most deeply divergent clades of extant angiosperms did continue to develop, their pollen tubes extended beyond the (Amborella, Nymphaeales, and Austrobaileyales) display the receptive stigmatic surface and through the carpel ground tissue. common (among angiosperms) Polygonum-type female gameto- Pollen tubes entered the ovarian cavity (locule) through the open phyte (17). It now seems likely that the first flowering plants were mouth of the ascidiate carpel, which is filled with a secretion. characterized by a four-celled, four-nucleate female gametophyte More than one pollen tube rarely entered the locule and ex- (14, 17), as found in Trimenia and other members of the Aus- tended farther along the base of the funicle. As many as three trobaileyales and Nymphaeales that have been reinvestigated pollen tubes were observed in our samples to reach the tip of an during the past decade (12–14, 19, 21, 22). ovule and enter the micropyle (Fig. 6). However, even if more than one pollen tube entered the micropyle, only one male Homoplastic Evolution of Tube-Like Female Gametophytes Among gametophyte ultimately engaged in a process of double fertil- Angiosperms. In T. moorei, more than one female gametophyte is ization. In several instances, the dominant pollen tube reached regularly initiated within each individual ovule. In addition, the

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Fig. 5. Callose in female gametophyte tubes. (A) Composite image of adjacent median longitudinal sections (white frames) of a mature ovule. Cal- lose fluorescence is evident in cell walls of the mature female gametophyte (star) that has reached the site of fertilization (asterisk) and central tissue of nuc nucellus. ch, chalaza; nuc, nucellus; zm, zone of megasporogenesis. (Scale bar: 200 μm.) (B) Central tissue of nucellus above zone where multiple female gametophytes are initiated. Callose fluorescence is evident in cell walls Fig. 6. Callose in pollen tubes. Composite image of adjacent median lon- of central tissue of nucellus (n) and in the cell wall of the dominant growing gitudinal sections of a mature ovule. Callose fluorescence is evident in the female gametophyte (star). (C) Base of nucellus. Callose fluorescence can be several pollen tubes (pt) growing between the cells of the micropyle formed seen in one degenerating megaspore (d) and the dominant female game- by the inner integument (ii). One pollen tube has reached the tip of the tophyte (star). (Scale bars: 10 μm.) female gametophyte (star) and grown along its surface towards the synergids (arrowheads). Arrows indicate the surface of the nucellus (nuc). (Scale μ multiple female gametophytes begin their development at the bar: 50 m.) base of the nucellus in a position remote from the future site of fertilization. These two features of ovule development in Tri- that engage in a form of competition evolved independently menia stand in marked contrast to the condition in most angio- several times over the course of flowering plant history. sperms, where a single functional megaspore produces a single female gametophyte near the nucellar apex (16). As female Intrasexual Female Gametophyte Competition Is Common Among gametophytes develop in Trimenia, they grow concurrently to the Ancient Lineages of Angiosperms. Among angiosperms, the initia- apex of the nucellus and form intertwined tube-like structures tion of multiple female gametophytes is not uncommon (24, 29– with callosic walls. Strikingly, the central tissue of nucellus, 31). Even in nonflowering seed plants (gymnosperms), a number through which the female gametophytes grow, seems to function of taxa are reported to initiate more than one female gameto- as a nutritive transmitting tissue. phyte per ovule (24, 30). For years, these observations largely Female gametophyte development in Trimenia, thus, strongly have been treated as mere teratological features of reproductive resembles that of pollen tubes (male gametophytes) in many development. To our knowledge, the concept of competition ways. Female gametophytes seem to undergo tip growth to (before fertilization) between meiotically related female game- produce a tubular (rarely branched) morphology, contain callose tophytes within a seed plant ovule has only been noted a few times in their cell walls (as do all flowering plant pollen tubes), and in the last century (24, 26, 31, 32). However, Haig (31), on the develop within a maternal diploid tissue that is highly reminis- fi cent of the pollen tube transmitting tissue of the carpel. More- basis of inclusive tness analysis, predicted that competition be- over, as is typical of most angiosperm pollen tubes, a form of tween meiotic products in an angiosperm ovule ought to occur competition seems to be the rule: many female gametophytes are and might be a thoroughly underappreciated phenomenon. initiated within each ovule, but only one can successfully engage Among ancient angiosperm lineages, Trimenia clearly repre- in the fertilization process. sents an extreme case in that its female gametophytes develop in The reproductive pattern typified by Trimenia in which mul- a fashion highly similar to pollen tubes and seem to compete tiple female gametophytes are initiated far from the site of fer- over substantial distances within the nucellus of the ovule. The tilization, engage in a pattern of tip growth, and seem to compete female gametophytes of most extant members of early divergent over a substantial distance within the nucellus is rare among angiosperm lineages do not produce extensive tube-like struc- angiosperms but by no means unique. This suite of characters tures. However, this fact does not necessarily imply that female has also been reported sporadically in taxa within the Lauraceae gametophyte (egg) competition is absent in ovules of these (23), Paeoniaceae (24), Crassulaceae (25), Onagraceae (26), clades. In all but one of the extant families of Austrobaileyales Oleaceae (27), and Santalales (28). The occurrence of such a and Nymphaeales, multiple female gametophytes per ovule are remarkable mechanism in phylogenetically disparate groups commonly or occasionally formed (10–14, 21). This same phe- suggests that tube-like growth of multiple female gametophytes nomenon also has been reported in the magnoliid lineage Laur-

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1104697108 Bachelier and Friedman Downloaded by guest on September 26, 2021 ales (33) and in Ceratophyllum (34), the sister clade to eudicots. an ovule in Trimenia coupled with the prevalence of the pro- As in Trimenia, more than one female gametophyte may reach duction of multiple female gametophytes per ovule among many maturity, but only one will participate in a double fertilization other ancient angiosperm lineages strongly suggest that male event to produce an embryo and its associated endosperm. We gametophytes were not alone in the competition to parent prog- posit that the potential for direct competition among multiple eny within seeds. Rather, a combination of intrasexual competi- female gametophytes within an ovule may actually be common in tion among male gametophytes and among female gametophytes early divergent flowering plant lineages. may have played an important role in enhancing the overall fitness Our analysis of the phylogenetic distribution of multiple female of progeny during the earliest phases of flowering plant evolution. gametophytes per ovule among ancient lineages of flowering plants indicates that this character may have arisen in the com- Materials and Methods mon ancestor of all angiosperms or have been highly homo- Material. Reproductive material of T. moorei (Oliv. ex Benth.) Philipson was plasious in its origins during the earliest phases of angiosperm collected at different stages of development in October 2009 and 2010 from evolution (Table S1). If the production of multiple competing plants growing in the subtropical rainforest of New South Wales in Australia female gametophytes is not an ancestral trait of flowering plants, (Table S2). Plant material was fixed for 24–48 h in 4% glutaraldehyde, FAA the high levels of homoplasy certainly provide circumstantial (formalin/acetic acid/alcohol), or 3:1 95% ethanol:acetic acid. evidence that the production of multiple female gametophytes per ovule was advantageous during the initial diversification of Bright-Field and Fluorescence Microscopy. Dissected floral buds, open flowers, angiosperms—and would, once again, be a strong reminder of and fruits were prepared for embedding in glycol methacrylate and were serially sectioned into 3- to 5-μm-thick ribbons (17). Subsets of serial sections the tremendous developmental lability manifest among ancient μ −1 angiosperms (3, 20). Irrespective of whether this condition is were stained with either 0.25 gmL DAPI in 0.05 M Tris buffer (pH 7.2) to detect DNA or aniline blue in 0.15 M K3PO4 buffer (pH ∼ 11) to detect callose plesiomorphic or highly homoplasious, competition between before being stained with 0.1% toluidine blue and/or periodic acid Schiff’s meiotically related but genetically unique female gametophytes reaction (17). Digital imaging and fluorescence were performed as in pre- (eggs) within an ovule seems to have been a key reproductive vious studies (17). feature of the earliest phases of flowering plant evolution. 3D Computer Reconstructions. Digital images of selected transverse serial Brief History of Intrasexual Competition. In 1794, Erasmus Darwin, sections of ovules and female gametophytes at different stages of de- Charles Darwin’s grandfather, first proposed that competition velopment were imported in a single file and manually aligned with Adobe EVOLUTION among males was an important driver of evolutionary change Photoshop CS3. Aligned images were compiled in Z-stacks and modeled in (35). Sixty-five years later, Charles Darwin devoted a section of 3D using the IMOD software package (Boulder Laboratory for 3-D Electron On the Origin of Species to the “struggle between males for Microscopy of Cells). Pictures of each model were generated using IMOD and possession of the females” (36). From its conceptual origins, compiled into a movie using the Windows Live Movie Maker software from intrasexual competition has, thus, largely but not exclusively Microsoft. Each movie features the model of an ovule presented in the text. been viewed as a highly asymmetric process in which females Each model is presented as shown in the corresponding figure and rotated mate with and may choose among the winners of battles among clockwise along the y axis through 720°. During the second full rotation, the potential male metazoan partners. nucellus is faded to show the intertwined development of the tubular female gametophytes. Concepts of male competition and female choice were first applied to the life cycles of plants in the latter half of the 20th – – ACKNOWLEDGMENTS. We thank P. K. Diggle, P. K. Endress, D. L. Marshall, century (4, 7 9, 31, 37 39). Since then, a number of develop- R. H. Robichaux, and J. H. Williams for critical reading of the manuscript; mental and genetic studies of angiosperm reproduction have J. J. Bruhl and Ian R. H. Telford for key assistance with field collections; shown that pollen tube (male–male) competition and female se- T. Eldridge, J. I. Hormaza, T. S. McGillivray, E. I. Scherbatskoy, and C.-C. Wu lection among pollen tubes (sperm donors) can result in increased for help with histological work; and E. N. Madrid and A. B. Demarest for fi assistance with 3D computer reconstructions. We also thank two anonymous levels of tness among progeny (40). The insights derived from reviewers for their comments on our manuscript. This work was supported our discovery of competing tube-like female gametophytes within by National Science Foundation Grant IOS-0919986 (to W.E.F.).

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