312 Ever Since Joseph Hooker Provided the First Scientific De

AMERICAN JOURNAL OF BOTANY RESEARCH ARTICLE

D EVELOPMENT AND EVOLUTION OF THE FEMALE GAMETOPHYTE AND FERTILIZATION PROCESS IN W ELWITSCHIA MIRABILIS (WELWITSCHIACEAE)1

W ILLIAM E. FRIEDMAN 2

Department of Organismic and Evolutionary Biology, 26 Oxford Street, Harvard University, Cambridge, Massachusetts 02138 USA; and Arnold Arboretum of Harvard University, 1300 Centre Street, Boston, Massachusetts 02131 USA

• Premise of the study: The female gametophyte of Welwitschia has long been viewed as highly divergent from other members of the Gnetales and, indeed, all other seed plants. However, the formation of female gametes and the process of fertilization have never been observed. • Methods: Standard histological techniques were applied to study gametophyte development and the fertilization process in Welwitschia . • Key results: In Welwitschia , fertilization events occur when pollen tubes with binucleate sperm cells grow down through the nucellus and encounter prothallial tubes, free nuclear tubular extensions of the micropylar end of the female gametophyte that grow up through the nucellus. Entry of a binucleate sperm cell into a vacuolate prothallial tube appears to stimulate the rapid coagulation of cytoplasm around a single female nucleus, which differentiates into an egg cell. One sperm nucleus enters the female gamete, while the second sperm nucleus remains outside and ultimately degenerates. Only a single fertilization event occurs per mating pair of pollen tube and prothallial tube. • Conclusions: Welwitschia lacks the gnetalean pattern of regular double fertilization, as found in Ephedra and Gnetum , involving sperm from a single pollen tube to yield two zygotes. Moreover, an analysis of character evolution indicates that the female gametophyte of Welwitschia is highly apomorphic both among seed plants, and speciﬁ cally within Gnetales, but also shares several key synapomorphies with its sister taxon Gnetum . Finally, the biological role of prothallial tubes in Welwitschia is examined from the perspectives of gamete competition and kin conﬂ ict.

Key words: double fertilization; embryo; evolutionary history; gametophyte; Gnetales; seed plants; Welwitschia ; Welwitschiaceae.

Ever since Joseph Hooker provided the fi rst scientifi c de- organisms. Hooker was so taken with the biological curiosities scriptions of Welwitschia mirabilis in 1863, it has been evident of Welwitschia that he engaged in an extensive correspondence that this seed plant species has the distinction of being among with Charles Darwin, Asa Gray, and T. H. Huxley on his work the most unusual and highly apomorphic of all photosynthetic with this taxon. Indeed, within this early post-Origin world, more than 30 letters alluding to and discussing Welwitschia would pass between these four closely knit colleagues and evo- 1 Manuscript received 28 October 2014; revision accepted 14 January lutionary proponents between 1861 and 1864. As Hooker wrote 2014. to Darwin (16 September 1862, Darwin Correspondence Proj- The author thanks Judy Jernstedt (University of California, Davis) for ect), “I am staggered with the intricacy of Welwitschia .” her decade of service as Editor-in-Chief of the American Journal of Botany and for her expert handling of this manuscript; Scott Russell (University of Joseph Hooker (1863) was the fi rst of three important bota- Oklahoma, handling editor), Pamela Diggle (University of Connecticut), nists to devote considerable time and effort to uncovering the Gar Rothwell (Oregon State University), and two anonymous reviewers for mysteries of the reproductive biology of Welwitschia . H. H. W. their excellent suggestions for the improvement of the manuscript; Amy Pearson (1906 , 1909 , 1910 , 1929 ) and Pierre Martens (1959a , McPherson, Managing Editor for the American Journal of Botany, for all 1959b , 1961 , 1963 , 1971 , 1973 ; also Martens and Waterkeyn, manner of assistance with this submission; John Trager, Sean Lahmeyer, 1974 , 1975 ) produced a series of publications documenting and Karen Zimmerman (Huntington Botanical Gardens) and Dennis the reproductive morphology and embryological features of Walker and Mike Messler (California State University, Humboldt) for Welwitschia . Female and male gametophyte development were hand-pollinating and collecting Welwitschia cones; Dan Dvorkin for closely examined by Strasburger (1872), Pearson (1906, 1909 ), histological preparation of the plant materials; and Kate Morozova for digital microscopy imaging. This research was supported by a grant from and Martens and Waterkeyn (1974) . Nevertheless, throughout the National Science Foundation (IBN-9696013) to W.E.F. the last century and a half, the formation of female gametes and 2 E-mail: [email protected] the process of fertilization remained unseen. In the 1990s, a series of studies of Ephedra and Gnetum doi:10.3732/ajb.1400472 (Friedman, 1990a, 1990b , 1991 , 1992a , 1992b , 1994 , 1995 ,

1998 ; Carmichael and Friedman, 1995 , 1996 ; Friedman and MATERIALS AND METHODS Carmichael, 1996 , 1998 ) deepened our understanding of the reproductive biology of extant Gnetales. In two species of Ephe- Welwitschia plants grown in cultivation at the Huntington Botanical Garden dra ( E. nevadensis and E. trifurca ) and one species of Gnetum and California State University at Humboldt yielded the materials used as the ( G. gnemon ), these investigations documented the expression basis for this study of fertilization. Welwitschia is functionally dioecious, and of a distinctive and regular pattern of gnetalean double fertiliza- female and male strobili are formed once a year. Ovules in female cones form pollination droplets at the tips of the micropylar tubes and become receptive to tion events. Although female gametophyte structure and devel- pollination over a period of several weeks, beginning at the base of the cone and opment in Ephedra and Gnetum differ in many substantial ways proceeding acropetally. When pollination droplets were present on individual ( Friedman and Carmichael, 1998 ), double fertilization events in ovules, they were hand pollinated. Whole cones were collected in two succes- these two gymnospermous taxa are essentially similar (Friedman, sive years. Depending on the time of cone collection, and as a consequence of 1998 ). the acropetal pattern of pollination receptivity and ovule maturation, ovules In E. nevadensis ( Friedman, 1990a , b ), E. trifurca ( Friedman could be found at various developmental stages before and after hand pollination. Whole cones were overnight expressed to the laboratory for immediate 1991 , 1992b ) and G. gnemon (Carmichael and Friedman, 1995, dissection and chemical fi xation. 1996 ), two sperm nuclei from a binucleate sperm cell of a single pollen tube participate in double fertilization events with female Light microscopy — Ovules were trimmed before chemical fi xation. Integu- nuclei from a single female gametophyte. In Ephedra and Gne- ments as well as extraneous nucellar tissue surrounding the female gameto- tum, double fertilization events yield two diploid zygotes that phyte were carefully removed from each ovule. Dissected ovules were fi xed in each initiate embryo developmental programs (Friedman, 1994; 4% w/v acrolein dissolved in 100 mM PIPES buffer, pH 6.8, for 24 h at room Carmichael and Friedman, 1996 ) and are almost certainly evolu- temperature. The ovules were then rinsed three times in PIPES buffer, dehy- tionarily homologous, having been inherited from a common drated through an ethanol series (10%, 20%, 30%, 50%, 75%, 95%, 100%, 2 h per step), and infi ltrated with glycol methacrylate (JB-4 Embedding Kit, Poly- ancestor of extant Gnetales (Friedman and Carmichael, 1996; sciences, Warrington, Pennsylvania, USA). The samples were infi ltrated for Friedman, 1998 ). Although multiple double fertilization events 3 wk to ensure the complete displacement of ethanol with glycol methacrylate. may occur in each female gametophyte/ovule, only one embryo Ovules were then embedded, and the embedding medium was polymerized will ultimately fi ll the mature seed and the remainder will abort in an oxygen-free environment by fl ushing nitrogen gas through a closed (as is typically the case with all seed plants). chamber. In light of the compelling evidence of the monophyly of the Embedded ovules were serially sectioned on a Leica 2155 microtome at thick- nesses of 5 µm with a glass knife made from a microscope slide. Sections were Gnetales (fi rst elucidated by Hooker, 1863 ; confi rmed in early mounted on microscope slides, stained with toluidine blue ( O’Brien and McCulley, cladistic analyses by Crane, 1985a, b and Doyle and Donoghue, 1981 ), and preserved with mounting medium. Bright fi eld and differential inter- 1986a , b ; and fi rst confi rmed in molecular phylogenetic analyses ference contrast images were recorded with a Zeiss Axio Imager Z2 microscope by Hasebe et al., 1992), the obvious question remains: do gneta- equipped with a Zeiss HR Axiocam digital camera (Zeiss, Oberkochen, Ger- lean double fertilization events similar to those in Ephedra and many). Image manipulations were restricted to operations that were applied to the Gnetum also occur in Welwitschia ? Neither Pearson (1909) nor entire image, except where specifi cally noted in fi gure captions. Martens and Waterkeyn (1974), in the only two studies that examined developmental events surrounding the fertilization process, produced evidence of two fertilization products (zygotes) RESULTS involving the sperm from a single pollen tube in Welwitschia . However, Pearson (1909) suggested that double fertilization Prefertilization female gametophyte development— As pre- events involving a single pollen tube and pairs of female gametic viously reported (Martens, 1963), the female gametophyte of nuclei were at least theoretically possible. Welwitschia is tetrasporic in origin (Fig. 1). Initial development Clearly, a focused study of sexual reproduction in Welwitschia of the female gametophyte is free nuclear in Welwitschia , but was warranted to examine the structure of the gametes, document unlike all other seed plants, no central vacuole forms within the the fertilization process, and determine whether gnetalean double coenocyte, and the nuclei undergo successive mitotic divi- fertilization events do in fact occur in this unusual plant species. sions and maintain an evenly spaced distribution (Fig. 1). At Given that double fertilization is plesiomorphic within the Gne- the end of the free nuclear phase, more than a thousand nuclei tales ( Friedman, 1998 ), such events might be found upon closer ( Pearson, 1909 ; Martens and Waterkeyn, 1974 ) are spaced examination of the reproductive process in Welwitschia mirabi- within a relatively homogeneous and moderately dense cyto- lis . With this in mind, the developmental biology of the male and plasm ( Fig. 2A ). At this stage, the female gametophyte is female gametophytes of Welwitschia , as well as the events asso- roughly 600 µm long. ciated with the fertilization process, were closely examined. For Cellularization of the female gametophyte involves the ap- the fi rst time, formation of the (most unusual) egg cell and syn- parently simultaneous formation of cell walls throughout the gamy in Welwitschia , are described. single-celled coenocyte to create hundreds of compartments Finally, while basic aspects of female gametophyte develop- (cells), each of which encloses as many as a dozen free nuclei ment are known in Ephedra , Gnetum, and Welwitschia , there ( Fig. 2B ). At this point, the female gametophyte begins to dif- has never been a thorough analysis of the evolutionary history ferentiate into two distinct regions: a large chalazal domain that of diversifi cation of the female gametophytes of these three dis- is vegetative in nature and a smaller micropylar domain that parate taxa. How do the highly derived patterns of female ga- will ultimately supply female gametes to the fertilization pro- metophyte development in Gnetum and Welwitschia relate to cess. The cells of the micropylar domain typically contain each other, and to Ephedra and other nonfl owering seed plants? fewer nuclei (approximately three to six) than the cells of the As will be seen, Welwitschia continues to demonstrate its clear chalazal domain. tendency toward apomorphic biology within the Gnetales and Nuclei within each coenocytic cell of the chalazal domain seed plants as a whole—and fully justifi es Sir Joseph Hooker’s next become arrayed in rings and fuse to produce a single highly assigned specifi c epithet, mirabilis, meaning amazing, won- polyploid nucleus (Fig. 3), thus completing a transition from a drous, remarkable! portion of a single-celled coenocyte through a multicellular 314 • VOL. 102 , NO. 2 FEBRUARY 2015 • AMERICAN JOURNAL OF BOTANY

Fig. 1. Early female gametophyte development in Welwitschia . Each lower panel is a higher magnifi cation of the panel above it. (A, E) Ovule with megaspore mother cell. (B, F) A four-nucleate coenocytic megaspore. Black box indicates digital inset from adjacent serial section in position where nucleus would lie within the coenomegaspore. The fourth nucleus, inset bottom left in red box, is from an adjacent section, its relative position indicated by red arrow. (C, G) Female gametophyte after several rounds of mitosis. The nuclei are evenly spaced and no central vacuole forms. (D, H) Free nuclear female gametophyte with dense cytoplasm and hundreds of nuclei. cms = coenomegaspore, fng = free nuclear gametophyte (single cell), mmc = megaspore mother cell, n = nucellus. Scale bar = 50 µm. coenocytic stage to a uninucleate multicellular tissue ( Figs. 2, pollination, the male gametophyte consists of three cells: an 3). This polyploid uninucleate cellular portion of the female ga- ephemeral prothallial cell ( Waterkeyn, 1959 ; Martens and metophyte will continue to grow and develop into a large em- Waterkeyn, 1974 ), a spermatogenous cell, and a tube cell. Thus, bryo-nourishing tissue (mitosis is coupled with cytokinesis) the male gametophyte contains two viable cells after being that will fi ll much of the seed at maturity. withdrawn into the micropylar canal (Fig. 5A). During the time The micropylar-most portion of the female gametophyte fol- of retraction of the pollination droplet into the micropylar lows a signifi cantly different developmental path from the cha- canal, pollen typically germinates. Initial growth of the post- lazal domain. Within the micropylar domain, the nuclei within pollination male gametophyte does not appear to be associated each coenocytic cell remain separate and unfused (Fig. 3A–F). with tip growth. Instead, the male gametophyte swells and a Roughly a dozen of the micropylar-most cells initiate tubular spherical protrusion forms (also noted by Rydin and Friis, projections that penetrate into the overlying nucellus (Fig. 3A– 2005 ). Formation of a tubular axis of growth from the initially F). These tubes, termed “prothallial tubes” (see the Commen- spherical male gametophyte occurs quickly and eventually the tary in this issue for a historical analysis of the terminology pollen tube penetrates the apex of the nucellus (Fig. 5B–D). associated with these tubular structures [ Friedman, 2015]), were Within each pollen tube, mitosis without cytokinesis of the fi rst described by Hooker (1863). Each tube continues to grow spermatogenous cell gives rise to a binucleate sperm cell ( Fig. apically within the nucellus toward the micropyle. The several 5E–G ; fi rst observed by Pearson, 1909 ). A binucleate sperm nuclei originally isolated within each of the multinucleate cells cell is likely to be characteristic of all members of the Gnetales in the micropylar domain migrate upward (distally) within the studied to date (see Carmichael and Friedman, 1996 for discussion prothallial tubes ( Fig. 3A–F ). Initially, these female (presum- of fi ndings from earlier studies). Pollen tube growth extends ably haploid) nuclei migrate in clusters of two, three, or four approximately a third of the length to two thirds of the length of ( Fig. 4 ), but eventually appear to spread out ( Fig. 4F ). The pro- the nucellar tissue that lies micropylar to the vegetative portion thallial tubes ultimately progress from a quarter of the distance to of the female gametophyte ( Fig. 5D ). almost all of the way upward into the nucellus. The micropylar domain of the female gametophyte and some of the contents of its Single fertilization events in Welwitschia — Several pairs prothallial tubes will participate in fertilization events. of prothallial tubes and pollen tubes meet in the nucellus, and a fusion of the apices creates a continuous passage in each mating Male gametophyte development — Pollen capture is accom- set of gametophytic structures. After contact and continuity are plished by means of a pollination droplet secreted by the ovule. established between an individual pollen tube and prothallial The pollen exine is polyplicate and monosulcate. At the time of tube, entry of a binucleate sperm cell into the largely vacuolate FRIEDMAN—FERTILIZATION OF WELWITSCHIA • VOL. 102 , NO. 2 FEBRUARY 2015 • 315

perhaps the only group of extant seed plants that Gnetales was not closely related to was angiosperms ( Hansen et al., 1999 ; Qiu et al., 1999 ; Samigullin et al., 1999 ; Bowe et al., 2000 ; Chaw et al., 2000 ; Sanderson et al., 2000 ; for a review, see Mathews, 2009 ). Today, the consensus is that the interrelation- ships of extant seed plants remain as opaque as they have ever been ( Stevens, 2001 onwards ; M. Donoghue, personal commu- nication). Gnetales have been posited to be sister to all other extant seed plants, sister to other extant gymnosperms, sister to Pinaceae, sister to conifers, and sister to cupressophytes (for a thorough review of this literature, see Stevens, 2001 onwards ; Wang and Ran, 2014 ). Occasional analyses, typically using morphological data, continue to recover a close relationship of Gnetales with angiosperms ( Stevens, 2001 onwards ), but without strong support. Throughout all of these analyses, the one con- stant has been the recovery of extant Gnetales, Ephedra, Gnetum , and Welwitschia , as monophyletic—a result ﬁ rst illuminated by Joseph Hooker in 1863—with Ephedra sister to a Gnetum plus Welwitschia clade ( Price, 1996 ; Rydin and Korall, 2009 ).

Early female gametophyte development — While Hooker (1863) was the fi rst to describe the extraordinary micropylar tubular extensions of the female gametophyte in Welwitschia , a Fig. 2. Cellularization of female gametophyte of Welwitschia . (A) Single- half century would pass before Pearson (1906, 1909 ) published celled free nuclear gametophyte just before fi rst cellularization event. (B) Cel- detailed analyses of female gametophyte development in Wel- lularized female gametophyte with sets of nuclei compartmentalized into witschia , including free nuclear development, and cellular- multinucleate cells. In both ovules, the wavy purple layer just outside the fe- ization. While largely accurate, Pearson (1906 , 1909 , 1929 ) male gametophyte is likely to be the megaspore wall (with some distortion). This stage of female gametophyte development is extremely delicate, and the concluded that the female gametophyte was monosporic in ori- shrinkage of the gametophyte from contact with the nucellus is almost certainly gin, an error that would be set right by Martens (1963) . Martens a fi xation artifact. fng = free nuclear gametophyte, cg = cellularized gameto- and Waterkeyn (1974) provided additional details about early phyte, n = nucellus. Scale bar = 200 µm. female gametophyte development, most of it confi rmatory of the fi ndings of Pearson (1906 , 1909 ). In Welwitschia , the female gametophyte is tetrasporic in prothallial tube stimulates the rapid coagulation of vesiculate origin; Gnetum is also tetrasporic, while Ephedra and all other cytoplasm around a single female nucleus (Fig. 6). This nucleus extant gymnosperms are monosporic (Singh, 1978; Carmichael and its associated cytoplasm then act as a single female gametic and Friedman, 1996 ). As is true of all seed plants, initial de- cell (egg cell). The two sperm nuclei are released from the bi- velopment of the female gametophyte is free nuclear ( Singh, nucleate sperm cell (Figs. 5G, 6A), and one sperm nucleus en- 1978; Gifford and Foster, 1989; Friedman and Carmichael, ters the egg cytoplasm ( Fig. 6B ), while the second sperm 1998), but in Welwitschia, the coenocyte is densely cytoplas- nucleus remains outside, as does the tube nucleus. The result is mic, and the free nuclei are evenly distributed throughout the an initially binucleate zygote (Fig. 6B). Nuclear fusion within single cell ( Pearson, 1909 ; Martens and Waterkeyn, 1974 ; re- the zygote ensues (evidenced by a single nucleus with two sults presented herein). Thus, the cytoplasmic organization prominent nucleoli) and embryogenesis is initiated ( Fig. 6C ). of the single-celled coenocytic stage of female gametophyte Although many pairs of prothallial tubes and pollen tubes development in Welwitschia differs markedly from the pattern may fuse and undergo fertilization in a single ovule (in essence, found in cycads, Ginkgo , conifers, Ephedra, and a majority of the standard gymnosperm process of simple polyembryony, al- the female gametophyte of Gnetum (see below), where a large though without discrete archegonia), only one fertilization event central vacuole forms and the nuclei are confi ned to a thin occurs per pollen tube/prothallial tube pair. In one instance in this parietal band of cytoplasm ( Pearson, 1929; Gifford and Foster, study, a pair of zygotes was found in direct proximity to each 1989 ). other within what appeared to be a single prothallial tube fertil- Strikingly, formation of a densely cytoplasmic coenocyte in ization chamber (Fig. 7), suggesting that double fertilization Welwitschia is also found in a portion of the female gameto- events in Welwitschia might occur on rare occasions. phyte of Gnetum . Although the majority of the volume of the coenocytic female gametophyte in Gnetum resembles that of most other extant gymnosperms (large central vacuole with a DISCUSSION parietal band of cytoplasm housing the nuclei), the chalazal- most end of the free nuclear female gametophyte (prior to A quarter century ago, Gnetales was widely (although not fertilization) lacks a central vacuole and contains cytoplasm universally; see for example, Hasebe et al., 1992) believed to be with evenly distributed free nuclei ( Lotsy, 1899 ; Pearson, the most closely related extant seed plant clade to angiosperms 1915 , 1929 ; Waterkeyn, 1954 ; Maheshwari and Vasil, 1961 ; (Crane, 1985a, b ; Doyle and Donoghue, 1986a, b ; Rothwell Friedman and Carmichael, 1998 ), similar to the organization of and Serbet, 1994 ; Doyle, 1996 ). This view changed in 1999 the entirety of the single-celled coenocytic gametophyte of ( Donoghue and Doyle, 2000 ), when a series of molecular phylo- Welwitschia. Thus, Gnetum expresses early developmental genetic analyses of extant seed plants strongly suggested that characteristics found in Ephedra and other extant gymnosperms, 316 • VOL. 102 , NO. 2 FEBRUARY 2015 • AMERICAN JOURNAL OF BOTANY FRIEDMAN—FERTILIZATION OF WELWITSCHIA • VOL. 102 , NO. 2 FEBRUARY 2015 • 317

fuse to create uninucleate highly polyploid cells ( Pearson, 1909 ; Martens and Waterkeyn, 1974 ; results presented herein). This unusual process also occurs in the multinucleate cells of the chalazal-most region of the female gametophyte of Gnetum ( Lotsy, 1899 ; Pearson, 1915 ; Maheshwari and Vasil, 1961 ; Friedman and Carmichael, 1998 ), a similarity fi rst noted by Pearson (1909) . In both Gnetum (Friedman and Carmichael, 1998) and Welwitschia , proliferation of the resulting polyploid uninucleate cellular tissue will generate the overwhelming bulk of the vegetative embryo- nourishing tissue of the female gametophyte. In Welwitschia , roughly a dozen or more of the micropylar- most multinucleate cells of the female gametophyte do not undergo nuclear fusion. Rather, these cells remain coenocytic and initiate prothallial tubes. These tubular extensions of the female gametophyte have been variously referred to as “secondary embryo-sacs” and “secondary sacs” (Hooker, 1863), “Corpusculumschläuche” (Strasburger, 1872), “archegonial tubes” (Coulter and Chamberlain, 1901), “prothallial tubes” (Pearson, 1906), “embryo-sac tubes” (Pearson, 1909, Fig. 4. Female gametic nuclei in prothallial tubes. (A–E) Female nuclei in 1929 ), “Prothallienschläuche” ( Goebel, 1932 ), “Embryosack- younger prothallial tubes often travel in sets of two, three, or four. (F) Older schläuche” (Schnarf, 1933), “egg tubes” (Battaglia, 1951), prothallial tube. Nuclei in older prothallial tubes are often found singly. Fn = female nuclei, n = nucellus, ptt = prothallial tube. Scale bar = 50 µm. “tubes endospermiques” ( Martens and Waterkeyn, 1974 ), and “female gametophytic tubes” ( Gifford and Foster, 1989 ). These tubular extensions of the female gametophyte in Welwitschia grow up through the nucellus and carry the haploid nuclei that as well as Welwitschia : the female gametophyte produces a mi- will serve as female gametic nuclei. Eventually, these tubular cropylar coenocytic zone with a large central vacuole and a cha- extensions of the female gametophyte meet and fuse with lazal coenocytic zone that is densely cytoplasmic with evenly the tips of downward-growing pollen tubes ( Pearson, 1909 ; distributed nuclei. Notably in Gnetum (or at least G. gnemon , Martens, 1971 ; Martens and Waterkeyn, 1974; results presented Friedman and Carmichael, 1998 ), the plesiomorphic micropy- herein ). For the present study, the term “prothallial tube” has been lar zone of the coenocytic female gametophyte does not un- selected for these tubular extensions of the female gametophyte dergo alveolation (centripetal formation of anticlinal walls that (see the companion article [ Friedman, 2015 ] in this issue for are open at the inner periclinal face and house a single nu- a more thorough discussion of this nomenclatural debate con- cleus), and most reports indicate that development of this re- cerning homology assessment). gion of the female gametophyte is truncated at the free nuclear stage (Maheshwari and Vasil, 1961; Friedman and Carmichael, 1998 ). Prothallial tubes and possible female gamete competition Cellularization of the single-celled coenocytic female game- in Welwitschia — Prothallial tubes appear to have no counter- tophyte in Welwitschia results in the formation of many multi- part among the female gametophytes of Gnetum , Ephedra , or nucleate chambers (coenocytic cells), each delimited by a cell any other extant nonfl owering seed plants and are likely to be wall. The process is not centripetal and thus differs from the an apomorphy of Welwitschia . Hooker (1863) thought he saw pattern of alveolation expressed in the free nuclear to cellular similar structures in the female gametophyte of Gnetum scan- transition in female gametophytes of cycads, Ginkgo , conifers, dens, but these were probably fi lamentous embryos. Among and Ephedra ( Singh, 1978 ; Gifford and Foster, 1989 ). Cellular- angiosperms, several reports have documented tube-like exten- ization of the female gametophyte of Welwitschia is essentially sions of the female gametophyte (see Maheshwari, 1950 for a identical to the process in the chalazal densely cytoplasmic free review of earlier literature; also Haig, 1990), most recently in nuclear portion of the female gametophyte of Gnetum . There Trimenia, a member of the ancient fl owering plant clade Aus- too, cellularization results in the compartmentalization of sev- trobaileyales (Bachelier and Friedman, 2011). The analogous eral nuclei within individual cells (Pearson, 1915; Maheshwari occurrence of tubular extensions of female gametophytes in and Vasil, 1961 ; Friedman and Carmichael, 1998 ). Welwitschia and a few fl owering plants (e.g., Loranthus ) was Shortly after cellularization to create multinucleate cells fi rst pointed out by Hooker (1863) . throughout the female gametophyte of Welwitschia , in all but In angiosperms with tube-like growth of the female game- the most micropylar cells, the multiple nuclei within these cells tophyte, there is often more than one female gametophyte

← Fig. 3. Female gametophyte development after prothallial tube initiation. Upper panel of three images (A–C) are higher magniﬁ cations of middle three panels (D–F) of whole female gametophytes. Red arrows identify free female nuclei seen in upper three panels. Each lower panel (G–I) is a higher magniﬁ cation of the vegetative portion of the female gametophyte in the middle panel above it (D–G). (A–C) Young prothallial tubes with migrating sets of female nuclei. (D–F) Cellular gametophytes at three different stages. (D, G) Cellularized female gametophyte with sets of nuclei compartmentalized into multinucleate cells. Prothallial tubes have begun to grow from the micropylar apex of the female gametophyte. (E, H) Transitional cellularized female gametophyte with sets of nuclei in vegetative domain arrayed in rings that will fuse into individual polyploid nuclei. In several of these cells, the nuclei have fused and a single highly polyploid nucleus is evident. Prothal- lial tubes are evident at the micropylar apex of the female gametophyte. Nuclei in prothallial tubes do not fuse. (F, I) Cellularized female gametophyte containing mostly uninucleate polyploid cells and showing extended prothallial tubes. fn = female nuclei in prothallial tubes, ptt = prothallial tube, cg = cellularized gametophyte. Scale bars = 50 µm, A–C, G–I; 200 µm, D–F. 318 • VOL. 102 , NO. 2 FEBRUARY 2015 • AMERICAN JOURNAL OF BOTANY FRIEDMAN—FERTILIZATION OF WELWITSCHIA • VOL. 102 , NO. 2 FEBRUARY 2015 • 319

initiated per ovule, suggesting that individual meiotically re- monosporic initiation of the female gametophyte was lost in the lated (hence, not genetically identical) gametophytes may be common ancestor of Gnetum plus Welwitschia . The coeoncytic competing within an ovule to mate with the one pollen tube that pattern of early development with nuclei located in a parietal typically enters an angiosperm ovule, as in Paeonia californica band of cytoplasm surrounding a large central vacuole was (Walters, 1962), Cassytha fi liformis (Sastri, 1956), and Trimenia maintained in Gnetales through the common ancestor of Gne- moorei (Bachelier and Friedman, 2011). In members of the tum plus Welwitschia, but subsequently lost in Welwitschia Loranthaceae ( Davis, 1966 ; Bhatnagar and Johri, 1983 ), multi- (Fig. 8). The condition of densely cytoplasmic organization ple (meiotically related) female gametophytes grow up into the during free nuclear development, followed by compartmental- style, with an egg apparatus at each of their apices, and appear ization to create multiple multinucleate cells, and subsequent to compete for sires (Haig, 1990); only one embryo ultimately nuclear fusion events to yield polyploid uninucleate cells that will fi ll the seed within the fruit. develop into the embryo-nourishing tissue of the female game- In Welwitschia , all of the prothallial tubes are derived from tophyte, evolved in a common ancestor of Gnetum plus Wel- a single female gametophyte, but because the female gameto- witschia . In both taxa, this apomorphic pattern is expressed in phyte is tetrasporic, the nuclear contents of each prothallial the chalazal region of the female gametophyte. Both Gnetum tube may differ genetically. It is unclear whether each tube and Welwitschia lack a process of alveolation (centripetal com- contains a genetically homogeneous or heterogeneous set of partmentalization of single nuclei into individual cells). female nuclei or whether the genotypes of egg cells in different The lack of archegonia in Gnetum and Welwitschia strongly prothallial tubes are genetically identical or meiotically related. suggests that this ancient gametangial differentiation program Given that the female gametophyte is tetrasporic and that was lost in a shared common ancestor. In both Gnetum and roughly a thousand nuclei are formed before the initial pro- Welwitschia, female gametes appear to be created from free nu- cess of cellular compartmentalization, it seems unlikely that clei in a coenocytic cytoplasm. Although precise details of fe- each of the prothallial tubes and their egg cells would be male gamete formation are still lacking for most species of genetically identical. Thus, as is the case in angiosperms with Gnetum , in Gnetum gnemon ( Carmichael and Friedman, 1995 , more than one female gametophyte per ovule, the prothallial 1996 ), egg cells apparently are never formed. Rather, the two tubes of Welwitschia may be exhibiting a form of female sperm nuclei from a single pollen tube are released into a mul- gamete competition among meiotic relatives, a suggestion tinucleate cytoplasm that ensheaths the pollen tube, and two fi rst made for Welwitschia by Buchholz (1922) in an early zygotic nuclei are formed. Each zygotic nucleus, which initially paper on gametophytic and gametic competition (which he lacks a cellular boundary, is then walled off with proximate cy- termed “developmental selection”) in plants, and more recently toplasm to create a single-celled zygote. In Welwitschia ( Fig. by Haig (1987 , 1990 ). 6 ), formation of an egg cell precedes the fertilization event, as Certainly, competition between meiotically related female vesicles are progressively localized to a common region around gametes in Welwitschia could have driven the evolution of pro- the future egg nucleus. Finally, the creation of multinucleate thallial tubes that “race” to meet pollen tubes in the nucellus. prothallial tubes bearing potential female gametic nuclei in Like pollen tube competition (sensu Mulcahy, 1979 ) between Welwitschia appears to be an autapomorphy restricted to this partially related or unrelated male gametophytes, the vigor of single species. each prothallial tube might serve as a potential screen of the “quality” of the female gamete enclosed within each tube or provide an advantage to the earliest-formed zygote/embryo to Fertilization in Welwitschia, Ephedra, and Gnetum — As “win” the race to fi ll the seed (Buchholz, 1922; Haig, 1987, discussed below, fertilization in Welwitschia is truly unique 1990 ). Alternatively, given that each embryo must grow within among land plants. But, it is also worth noting that from the the passageway of a prothallial tube to reenter the vegetative perspective of the male gametophyte, the progamic phase be- embryo-nourishing portion of the female gametophyte of Wel- tween pollination and fertilization within the Gnetales is strik- witschia ( Pearson, 1909 ; Martens and Waterkeyn, 1974 ), the ingly diverse. In Ephedra , degeneration of the nucellar tissue overall vigor/genetic quality of each sperm donor or mating directly above (micropylar to) the archegonia creates a pollen pair (embryo) might also be put to the test (kin confl ict) and chamber where pollen is deposited and germinates. Pollen screened by the maternal sporophyte. As with most seed plants, tubes in Ephedra then grow through an archegonial neck (the only one embryo will ultimately fi ll the mature seed. archegonial necks in Ephedra are the longest of any extant gymnosperm) and thus never encounter sporophytic tissue as a growth medium ( Friedman, 1990b ). In Gnetum , the nucellus Female gametophyte character evolution within Gnetales— above the female gametophyte is well developed and pollen From an evolutionary perspective, Welwitschia has entirely tubes grow through the entirety of the nucellus that overlies the lost the plesiomorphic pattern of female gametophyte devel- micropylar end of the female gametophyte. Male gametophyte opment for Gnetales. The ancestral seed plant program for development in Gnetum is completed with the entry and growth

← Fig. 5. Male gametophyte development and spermatogenesis. (A) Ungerminated pollen grain in micropylar tube, with generative (spermatogenous) cell and tube nucleus. The exine is plicate. (B) Germinated pollen grain, with pollen tube growing into the nucellus. The generative (spermatogenous) cell is still inside the pollen grain, while the tube nucleus is close to the growing tip of the tube. (C) Pollen tube growing in the nucellus. The generative (spermatogenous) cell has migrated into the tube. (D) Mature pollen tube after fertilization reconstructed from several adjacent sections (black boxes). The zygote is apparent in bulbous tip of a prothallial tube that fused with the pollen tube. The pollen tube is more than a millimeter in length. (E–G) Differentiation of the spermatogenous cell into a binucleate sperm cell. (E) Generative (spermatogenous) cell with prominent single nucleus and nucleolus. (F) Binucleate sperm cell after mitosis of the generative (spermatogenous) cell nucleus. (G) Binucleate sperm cell in process of releasing the ﬁ rst sperm nucleus into a prothallial tube. Arrows indicate individual sperm nuclei (each with a prominent nucleolus). Black boxes indicate digital insets from adjacent serial sections in proper position. gc = generative cell, n = nucellus, pt = pollen tube, sc = sperm cell (binucleate), tn = tube nucleus, zyg = zygote. Scale bars = 50 µm, A, E–G; 100 µm, B, C; 500 µm, D. 320 • VOL. 102 , NO. 2 FEBRUARY 2015 • AMERICAN JOURNAL OF BOTANY FRIEDMAN—FERTILIZATION OF WELWITSCHIA • VOL. 102 , NO. 2 FEBRUARY 2015 • 321

confi rmed and amplifi ed in the current study. In Welwitschia , zygotes are always formed within the confi nes of the cytoplasm of prothallial tubes, and as seen in the present work, involve the formation of a discrete egg cell that is penetrated by a sperm nucleus. Hence fertilization occurs within the body of the female gametophyte. Like their predecessors Strasburger and Pearson, Martens and Waterkeyn (1974) did not catch a glimpse of the formation and structure of the female gamete nor the fertilization process itself: “Nous n’avons pas eu la chance de sur- prendre la pénétration des gamètes mâles et la caryogamie elle-même…” We have not had the chance to catch the penetra- tion of the male gametes [into prothallial tubes and female gametes] and karyogamy itself ( Martens and Waterkeyn, 1974 ). Although there were indications that double fertilization-like events might occur rarely or occasionally in Ephedra ( Land, 1907 ; Herzfeld, 1922 ; Maheshwari, 1935 ; Khan, 1940 , 1943 ; Mulay, 1941 ; Narang, 1955 ; Moussel, 1978 ) and Gnetum ( Lotsy, 1899 ; Waterkeyn, 1954 ; Vasil, 1959 ; Sanwal, 1962 ), it was not until the 1990s that a clear view of the fertilization process in Ephedra and Gnetum emerged. In two species of Ephedra ( E. nevadensis and E. trifurca), and in Gnetum gnemon, the gametic nuclei of a binucleate sperm cell (from a single pollen tube) regularly engage in separate fertilization events with female gametic nuclei ( Friedman, 1990a , 1990b , 1991 , 1992a , 1992b , 1994 , 1995 , 1998 ; Carmichael and Friedman, 1995 , 1996 ; Friedman and Carmichael, 1998 ). The production of binucleate egg cells in Ephedra (containing an egg nucleus and a ventral canal nucleus, each of which is fertilized by a sperm nucleus from a binucleate sperm cell) and the highly unusual coeno- Fig. 7. Possible gnetalean double fertilization event in Welwitschia . Two zygotes sit side by side within what appears to be the space of a single prothal- cytic female gametophyte cytoplasm with free nuclei that enve- lial tube suggesting that a rare double fertilization event may have occurred in lope the pollen tube in Gnetum ( Carmichael and Friedman, Welwitschia. It is also possible that a second pollen tube could have fused with 1995 , 1996 ) and behave as gametes, clearly are permissive of a second prothallial tube and separately fertilized a female gamete from the regular double fertilization events in these two gnetalean gen- second prothallial tube, but the walls between the two prothallial tubes either era. In each of these taxa, the product of the second (extra) fer- could not be detected or have broken down. z1 = zygote 1, z2 = zygote 2, ptt = tilization event is a diploid supernumerary zygote (Friedman, prothallial tube. Scale bar = 50 µm. 1992b; Carmichael and Friedman, 1996). Importantly, in Ephe- dra and Gnetum , double fertilization events always involve of the pollen tube into the coenocytic micropylar portion of the sperm from a single pollen tube and are distinct from the wide- female gametophyte ( Carmichael and Friedman, 1996 ). In Wel- spread phenomenon among gymnosperms of multiple single witschia , pollen is deposited on the nucellar apex (as in Gne- fertilization events involving individual sperm from different tum), but the pollen tube only grows about halfway through the pollen tubes (known as simple polyembryony). nucellus to a point where it meets a prothallial tube. Thus, pol- At the time of these discoveries in the 1990s, Gnetales was len tubes in Ephedra only encounter gametophytic tissue, pol- widely hypothesized to be the most closely related extant seed len tubes in Gnetum encounter sporophytic and gametophytic plant clade to angiosperms. As such, parsimonious interpreta- tissues, and pollen tubes in Welwitschia only grow within spo- tion of these data suggested that this pattern of gnetalean double rophytic tissue. fertilization might well be evolutionarily homologous with the Pearson (1909) concluded that following fusion of a pollen process of double fertilization in angiosperms (Friedman and tube and prothallial tube, fertilization occurred within the con- Floyd, 2001 ). However, a seismic shift in favored phylogenetic fi nes of the male gametophyte: a free female nucleus from a hypotheses for seed plants, associated with the transition from prothallial tube was posited to migrate into the pollen tube and morphological cladistic analyses to DNA sequence-based fuse with a sperm nucleus within the cytoplasm of a binucleate analyses, indicated (and continues to indicate—see above) that sperm cell. Martens and Waterkeyn (1974) showed that this Gnetales are not closely related to angiosperms, even if their rather astounding report of a fertilization event within the con- true phylogenetic affi nities remain opaque. In light of these fi nes of the male gamete/gametophyte was erroneous, a fi nding fi ndings, the homology of double fertilization events in Gnetales

← Fig. 6. Developmental aspects of egg formation and the fertilization process in Welwitschia . Each horizontal row is a set of three serial sections of an individual fertilization event. (A) Before fertilization, both sperm nuclei are visible outside the forming egg cell with dense cytoplasm coagulating around a single female nucleus. The tube (vegetative) nucleus of the pollen tube is visible in the ﬁ rst and second panels at upper left. (B) Shortly after fertilization, a sperm nucleus has entered the egg cell, but the two gametic nuclei have not yet fused into a zygote nucleus. The second sperm nucleus and tube nucleus from the pollen tube are also present, but excluded from the incipient zygote. (C) A zygote. The male and female gametic nuclei have fused and a single zygotic nucleus is present. The tube nucleus and second sperm nucleus from the pollen tube remain just outside of the zygote. en = egg nucleus, sp1 = sperm nucleus 1, sp2 = sperm nucleus 2, tn = tube nucleus, zyg = zygote. Scale bar = 20 µm. 322 • VOL. 102 , NO. 2 FEBRUARY 2015 • AMERICAN JOURNAL OF BOTANY

with fertilization in Welwitschia . Examination of the camera lu- cida and micrographic fi gures showing zygotes in Pearson (1909) and Martens and Waterkeyn (1974) does not reveal an instance where two zygotes were housed in direct proximity to each other, as might be expected if double fertilization events involving a single pollen tube and prothallial tube occurred regularly in Welwitschia. Pearson (1909) did suggest that double fertilization events involving a single pollen tube and pairs of female gametic nuclei were theoretically possible. Similarly, Martens (1971) believed that all of the female nuclei within an individual prothallial tube could potentially serve in a gametic role. Although the pollen tubes of Welwitschia produce binucleate sperm cells similar to Gnetum and Ephedra , only one of the two sperm nuclei regularly engages in a fertilization process with a female gamete. This is so, even though each prothallial tube that emanates from the micropylar end of the female gametophyte contains several nuclei that appear to be identical in form and might individually participate in separate fertilization events. As shown here (Figs. 5, 6), following fusion of a pollen tube and prothallial tube, coagulation of cytoplasm around a single female nucleus in a prothallial tube creates a single egg cell capable of being fertilized. Only a single zygote is formed per mating pair of male and female gametophytic tubes, with Fig. 8. Character evolution of female gametophytes in Gnetales. The com- the second sperm nucleus and tube nucleus orphaned outside of mon ancestor of extant Gnetales is hypothesized to be monosporic, with the newly formed zygote. Thus, unlike Ephedra and Gnetum , a typical and plesiomorphic gymnosperm pattern of female gametophyte where both nuclei of a binucleate sperm cell regularly come development. A free nuclear phase with parietal cytoplasm surrounding a large central vacuole is followed by alveolation (centripetal cellularization) and sub- into contact with two fecundible female nuclei and double fer- sequent cellular development of the embryo-nourishing tissue. Archegonia are tilization events ensue, the second sperm nucleus in Welwitschia present, and double fertilization events involving the two sperm nuclei from an typically lacks a fecundible partner. individual pollen tube occur regularly. This pattern of reproductive biology Interestingly, the potential for double fertilization events characterizes extant species of Ephedra . In the common ancestor of Gnetum appears to have been retained in Welwitschia (as suggested by plus Welwitschia , tetrasporic initiation of female gametophytes evolves from a Pearson and Martens) each time a pollen tube fuses with a pro- monosporic condition, archegonia are lost, and the female gametes are initially free nuclei that become cellularized just before (as in Welwitschia) or just after thallial tube, should a second egg cell be formed. In one in- (as in Gnetum ) fertilization events take place. A novel female gametophyte cy- stance (Fig. 7) of the fertilization process observed in this study, tological organization also evolved in this common ancestor of Gnetum plus two zygotes were found in direct contact with each other and Welwitschia, in which a densely cytoplasmic coenocytic tissue is formed that within what appeared to be the confi nes of a single prothallial then cellularizes into smaller multinucleate cells with subsequent fusion of the tube. Thus, although the more distant gnetalean ancestors of multiple nuclei resulting in formation of polyploid uninucleate cells. In both Welwitschia engaged in regular double fertilization events, this Gnetum and Welwitschia , the polyploid uninucleate cellular tissue will prolifer- ate into the embryo-nourishing portion of the female gametophyte. Centripetal (regular) process has been lost in Welwitschia , although perhaps alveolation is lost in the common ancestor of Gnetum and Welwitschia , al- not entirely. though a free nuclear organization with central vacuole is present in a portion of the female gametophyte of Gnetum , suggesting that this was present in the com- Concluding thoughts — As embryological features are common ancestor of the Gnetum plus Welwitschia clade. Welwitschia has lost free pared across the extant members of the Gnetales, it remains a nuclear organization with a central vacuole (entirely), gained prothallial tubes remarkable fact that Joseph Hooker appears to have immedi- and lost the regular gnetalean process of double fertilization. ately detected the alliance of Welwitschia with Gnetum and Ephedra . At that time, the embryology of these two other gen- and angiosperms, could no longer be supported in light of phy- era was poorly understood. Nevertheless, as Hooker wrote to T. logenetically based analyses of character evolution ( Friedman and H. Huxley (20 January 1862): “[F]ancy my joy… at my being Floyd, 2001). Nevertheless, the presence of a gnetalean pro- able to show that though neither Dicot, Monocot, nor Gymno- cess of double fertilization involving two male gametes from a sperm in fl ower or Exogen or Endogen in structure of axis, single pollen tube and two female gametes from a female game- wood or bark (its cambium ring is facetious in the extreme), it tophyte to yield two diploid zygotes in Ephedra and Gnetum is still undoubtedly a member of the family Gnetaceae among suggests that this trait evolved in a common ancestor of extant Gymnosperms, as the structure of the ovule and development of Gnetales. To date, gnetalean double fertilization events to pro- the seed and embryo clearly show. It is out of all question the duce supernumerary embryos remains an intriguing process most wonderful plant ever brought to this country and the very whose biological benefi ts, if any, to the host organisms and spe- ugliest” (Huxley, 1918, pp. 24–25). Or, as only Darwin could cies are entirely unknown. put the case, upon reading Hooker’s (1863) classic monographic Long before the documentation of regular double fertiliza- work, “I am reading Wellwitschia [sic]: what a wonderful plant tion events in Ephedra and Gnetum , there had been suggestions it is; but the case requires more knowledge than I have fully to that double fertilization events might occur in Welwitschia . Nei- appreciate: those devilish ovules, embryos, sacks & membranes ther Pearson (1909) , Martens and Waterkeyn (1974) , nor any of drive my weakened brain half mad” (Charles Darwin to Joseph their predecessors, witnessed the developmental events associated Hooker, 5 March 1863, Darwin Correspondence Project). FRIEDMAN—FERTILIZATION OF WELWITSCHIA • VOL. 102 , NO. 2 FEBRUARY 2015 • 323

LITERATURE CITED F RIEDMAN , W. E. 1998 . The evolution of double fertilization and endosperm: An “historical” perspective. Sexual Plant Reproduction 11 : 6 – 16 . B ACHELIER , J. B. , AND W. E. FRIEDMAN . 2011 . Female gamete competi- F RIEDMAN , W. E. 2015 . Evolving words and the egg-bearing tubes of tion in an ancient angiosperm lineage. Proceedings of the National Welwitschia (Welwitschiaceae) . American Journal of Botany 102 : Academy of Sciences, USA 108 : 12360 – 12365 . 178 – 181 . B ATTAGLIA, E. 1951 . The male and female gametophytes of angiosperms— F RIEDMAN , W . E . , AND J. S. CARMICHAEL . 1996 . Double fertilization in An interpretation. Phytomorphology 1 : 87 – 116 . Gnetales: Implications for understanding reproductive diversifi cation B HATNAGAR , S. P. , AND B. M. JOHRI . 1983 . Embryology of Loranthaceae . among seed plants. International Journal of Plant Sciences 157 : S77 – S94 . In M. Calder and P. Bernhardt [eds.], The biology of mistletoes, 47– F RIEDMAN , W . E . , AND J. S. CARMICHAEL . 1998 . Heterochrony and devel- 68. Academic Press, Sydney, Australia. opmental innovation: Evolution of female gametophyte ontogeny in B OWE , L. M. , G. COAT , AND C . W . DEPAMPHILIS. 2000 . Phylogeny of seed Gnetum , a highly apomorphic seed plant. Evolution 52 : 1016 – 1030 . plants based on all three genomic compartments: Extant gymno- F RIEDMAN , W. E. , AND S. K. FLOYD. 2001 . Perspective: The origin of fl ow- sperms are monophyletic and Gnetales’ closest relatives are conifers. ering plants and their reproductive biology—A tale of two phylog- Proceedings of the National Academy of Sciences, USA 97 : 4092 – 4097 . enies. Evolution 55 : 217 – 231 . B UCHHOLZ, J. T. 1922 . Developmental selection in vascular plants. G IFFORD , E. M. , AND A. S. FOSTER . 1989 . Morphology and evolution of Botanical Gazette 73 : 249 – 286 . vascular plants, 3rd ed. W. H. Freeman, New York, New York, USA. C ARMICHAEL , J. S. , AND W. E. FRIEDMAN . 1995 . Double fertilization in G OEBEL , K. 1932 . Organographie der Pfl anzen: Insbesondere der Arche- Gnetum gnemon : The relationship between the cell cycle and sexual goniaten und Samenpfl anzen . G. Fischer, Jena, Germany. reproduction. Plant Cell 7 : 1975 – 1988 . H AIG , D. 1987 . Kin confl ict in seed plants. Trends in Ecology & Evolution C ARMICHAEL , J. S. , AND W. E. FRIEDMAN . 1996 . Double fertilization in 2 : 337 – 340 . Gnetum gnemon (Gnetaceae): Its bearing on the evolution of sexual H AIG , D. 1990 . New perspectives on the angiosperm female gametophyte. reproduction within the Gnetales and the anthophyte clade. American Botanical Review 56 : 236 – 274 . Journal of Botany 83 : 767 – 780 . H ANSEN , A . , S . H ANSMANN , AND T . S AMIGULLIN. 1999 . Gnetum and the an- C HAW , S.-M. , C. L. PARKINSON , Y . C HENG , T. M. VINCENT , AND J. D. PALMER . giosperms: Molecular evidence that their shared morphological char- 2000 . Seed plant phylogeny inferred from all three plant genomes: acters are convergent, rather than homologous. Molecular Biology Monophyly of extant gymnosperms and origin of Gnetales from co- and Evolution 16 : 1006 – 1009 . nifers. Proceedings of the National Academy of Sciences, USA 97 : H ASEBE , M . , M . I TO , R . K OFUJI , K . I WATSUKI , AND K . U EDA. 1992 . 4086 – 4091 . Phylogenetic relationships in gnetophyta deduced from rbcL gene C OULTER , J. M. , AND C. J. CHAMBERLAIN . 1901 . Morphlogy of spermato- sequences. Journal of Plant Research 105 : 385 – 391 . phytes . Appleton and Co., New York, New York, USA. H ERZFELD , S. 1922 . Ephedra campylopoda Mey. Morphologie der weib- C RANE , P. R. 1985a . Phylogenetic analysis of seed plants and the origin of lichen Blüte und Befruchtungvorgang. Denkschriften der Akademie angiosperms. Annals of the Missouri Botanical Garden 72 : 716 – 793 . der Wissenschaften in Wien Mathematisch-Naturwissenschaftliche C RANE , P. R. 1985b . Phylogenetic relationships in seed plants. Cladistics Klasse 98 : 243 – 268 . 1 : 329 – 348 . H OOKER , J. D. 1863 . I. On Welwitschia, a new genus of Gnetaceæ. D AVIS , G. L. 1966 . Systematic embryology of the angiosperms . John Transactions of the Linnean Society of London 24 : 1 – 48 . Wiley, New York, New York, USA. H UXLEY, L. 1918 . Life and letters of Sir Joseph Dalton Hooker based on D ONOGHUE , M. J. , AND J. A. DOYLE . 2000 . Seed plant phylogeny: Demise materials collected and arranged by Lady Hooker, volume II. John of the anthophyte hypothesis? Current Biology 10 : 106 – 109 . Murray, London, UK. D OYLE , J. A. 1996 . Seed plant phylogeny and the relationships of Gnetales. K HAN , R. 1940 . A note on “double fertilization” in Ephedra foliata. International Journal of Plant Sciences 157 : S3 – S39 . Current Science 9 : 323 – 325 . D OYLE , J . , AND M. J. DONOGHUE. 1986a . Seed plant phylogeny and the ori- K HAN , R. 1943 . Contributions to the morphology of Ephedra foliata gin of angiosperms—An experimental cladistic approach. Botanical Boiss. II. Fertilization and embryogeny. Proceedings of the National Review 52 : 321 – 431 . Academy of Sciences India 13 : 357 – 375 . D OYLE , J. A. , AND M. J. DONOGHUE . 1986b . Relationships of angiosperms L AND, W. 1907 . Fertilization and embryogeny in Ephedra trifurca . and Gnetales: A numerical cladistic approach. In R. A. Spicer and Botanical Gazette 44 : 273 – 292 . B. A. Thomas [eds.], Systematic and taxonomic approaches in pa- L OTSY , J. 1899 . Contributions to the life-history of the genus Gnetum . I. laeobotany, Systematics Association Special Volume 31, 177–199. The grosser morphology of production of Gnetum gnemon L. Annales Clarenden Press, Oxford, UK. du Jardin Botanique de Buitenzorg 16 : 46 – 114 . F RIEDMAN , W. E. 1990a . Double fertilization in Ephedra, a nonfl ower- M AHESHWARI , P. 1935 . Contributions to the morphology of Ephedra foli- ing seed plant: Its bearing on the origin of angiosperms. Science 247 : ata Boiss. 1: The development of the male and female gametophytes. 951 – 954 . Proceedings of the Indiana Academy of Sciences 1 : 586 – 601 . F RIEDMAN , W. E. 1990b . Sexual reproduction in Ephedra nevadensis M AHESHWARI , P. 1950 . An introduction to the embryology of angio- (Ephedraceae): Further evidence of double fertilization in a nonfl ow- sperms . McGraw-Hill, New York, New York, USA. ering seed plant. American Journal of Botany 77 : 1582 – 1598 . M AHESHWARI , P . , AND V . V ASIL . 1961 . Gnetum . Council of Scientifi c & F RIEDMAN , W. E. 1991 . Double fertilization in Ephedra trifurca , a non- Industrial Research India, New Delhi, India. fl owering seed plant: The relationship between fertilization events and M ARTENS , P. 1959a . Études sur les Gnétales—III. Structure et ontogènese the cell cycle. Protoplasma 165 : 106 – 120 . du cône et de la fl eur femelles de Welwitschia mirabilis. La Cellule F RIEDMAN , W. E. 1992a . Double fertilization in nonfl owering seed plants. 60 : 169 – 286 . International Review of Cytology 140 : 319 – 355 . M ARTENS , P. 1959b . Structure et ontogènese de la fl eur [femelle] et du F RIEDMAN , W. E. 1992b . Evidence of a pre-angiosperm origin of endo- cône femelle de Welwitschia mirabilis Hooker. Comptes Rendus sperm: Implications for the evolution of fl owering plants. Science Academie Sciences Paris 248 : 2265 – 2268 . 255 : 336 – 339 . M ARTENS , P. 1961 . Études sur les Gnétales. V. Structure et ontogènese F RIEDMAN , W. E. 1994 . The evolution of embryogeny in seed plants and du cône et de la fl eur mâles de Welwitschia mirabilis. La Cellule 6 2 : the developmental origin and early history of endosperm. American 5 – 91 . Journal of Botany 81 : 1468 – 1486 . M ARTENS, P. 1963 . Études sur les Gnétales. VI. Recherches sur Welwitschia F RIEDMAN , W. E. 1995 . Organismal duplication, inclusive fi tness theory, mirabilis . III. L’ovule et le sac embryonnaire. Les sacs embryonnaires and altruism: Understanding the evolution of endosperm and the extra-fl oraux. La Cellule 63 : 309 – 329 . angiosperm reproductive syndrome. Proceedings of the National M ARTENS , P. 1971 . Les Gnétophytes. Encyclopedia of plant anatomy, vol. Academy of Sciences, USA 92 : 3913 – 3917 . 12, part 2 . Gebrueder Borntraeger, Berlin, Germany. 324 • VOL. 102 , NO. 2 FEBRUARY 2015 • AMERICAN JOURNAL OF BOTANY

M ARTENS , P. 1973 . Évolution ovulaire et embryogenèse. La Cellule 70 : R OTHWELL , G. W. , AND R . S ERBET . 1994 . Lignophyte phylogeny and 165 – 258 . the evolution of spermatophytes: A numerical cladistic analysis. M ARTENS , P . , AND L . W ATERKEYN. 1974 . Études sur les Gnétales. XIII. Systematic Botany 19 : 443 – 482 . Recherches sur Welwitschia mirabilis . 5. Évolution ovulaire et em- R YDIN , C . , AND E . F RIIS . 2005 . Pollen germination in Welwitschia mira- bryogenèse. La Cellule 70 : 163 – 258 . bilis Hook. f.: Differences between the polyplicate pollen producing M ARTENS , P. , AND L . W ATERKEYN . 1975 . Some new facts in the embryogeny of genera of the Gnetales. Grana 44 : 137 – 141 . Welwitschia mirabilis Hooker . In H. Y. Mohan Ram, J. J. Shah, and C. K. R YDIN , C . , AND P . K ORALL . 2009 . Evolutionary relationships in Ephedra Shah [eds.], Form, structure and function in plants, 195–196. Johri, B. M. (Gnetales), with implications for seed plant phylogeny. International Commemoration Volume. Sarita Prakashan, Nauchandi, Meerut, India. Journal of Plant Sciences 170 : 1031 – 1043 . M ATHEWS , S. 2009 . Phylogenetic relationships among seed plants: Persis- S AMIGULLIN , T. K. , W. F. MARTIN , A. V. TROITSKY , AND A. S. ANTONOV . tent questions and the limits of molecular data. American Journal of 1999 . Molecular data from the chloroplast rpo C1 gene suggest a Botany 96 : 228 – 236 . deep and distinct dichotomy of contemporary spermatophytes into M OUSSEL , B. 1978 . Double fertilization in the genus Ephedra. Phytomor- two monophyla: Gymnosperms (including Gnetales) and angio- phology 28 : 336 – 345 . sperms. Journal of Molecular Evolution 49 : 310 – 315 . M ULAY , B. N. 1941 . A study of the female gametophyte and a tropho- S ANDERSON , M. J. , M. F. WOJCIECHOWSKI , J. M. HU , T. S. KHAN , AND S. G. phyte in Ephedra foliata. Journal of Bombay University 10 : 56 – 89 . B RADY. 2000 . Error, bias, and long-branch attraction in data for two M ULCAHY, D. L. 1979 . The rise of the angiosperms: A genecological fac- chloroplast photosystem genes in seed plants. Molecular Biology and tor. Science 206 : 20 – 23 . Evolution 17 : 782 – 797 . N ARANG , N. 1955 . Contributions to the life history of Ephedra campy- S ANWAL , M. 1962 . Morphology and embryology of Gnetum gnemon L. lopoda. II. Fertilization and embryogeny. Proceedings of the Indian Phytomorphology 12 : 243 – 264 . Science Congress 3: 224. S ASTRI , R. L. N. 1956 . Embryo sac haustoria in Cassytha ﬁ liformis Linn. O ’ B RIEN , T. P. , AND M. E. MCCULLY. 1981 . The study of plant structure. Current Science 25 : 401 – 402 . Termarcarphi, Wantirna, Australia. S CHNARF, K. 1933 . Embryologie der Gymnospermen. Gebrüder Borntraeger, P EARSON , H. H. W. 1906 . Some observations on Welwitschia mirabilis , Berlin, Germany. Hooker, f. Philosophical Transactions of the Royal Society of London, S INGH , H. 1978 . Embryology of Gymnosperms . Encyclopedia of plant B, Biological Sciences 198 : 265 – 304 . anatomy, vol. 12. Gebruder Borntraeger, Berlin, Germany. P EARSON , H. H. W. 1909 . Further observations on Welwitschia. S TEVENS , P. F. 2001 [onward] . Angiosperm phylogeny website, version Philosophical Transactions of the Royal Society of London, B, 12, July 2012 [and more or less continuously updated since]. Biological Sciences 200 : 331 – 402 . S TRASBURGER, E. 1872 . Die Coniferen und die Gnetaceen. Hermann P EARSON , H. H. W. 1910 . On the embryo of Welwitschia. Annals of Dabis, Jena, Germany. Botany 4 : 759 – 766 . V ASIL , V. 1959 . Morphology and embryology of Gnetum ula Brongn. P EARSON , H. H. W. 1915 . IX. Notes on the morphology of certain struc- Phytomorphology 9 : 167 – 215 . tures concerned in reproduction in the genus Gnetum. Transactions of W ALTERS , J. 1962 . Megasporogenesis and gametophyte selection in the Linnean Society of London, 2nd Series, Botany 8 : 311 – 332 . Paeonia californica. American Journal of Botany 49 : 787 – 794 . P EARSON , H. H. W. 1929 . Gnetales . Cambridge University Press, London, W ANG , X.-Q. , AND J.-H. RAN . 2014 . Evolution and biogeography of gym- UK. nosperms. Molecular Phylogenetics and Evolution 75 : 24 – 40 . P RICE , R. A. 1996 . Systematics of the Gnetales: A review of morphologi- W ATERKEYN, L. 1954 . Études sur les Gnétales I: Le strobile femelle, cal and molecular evidence. International Journal of Plant Sciences l’ovule et la graine de Gnetum africanum Welw. La Cellule 5 6 : 157 ( supplement ): S40 – S49 . 103 – 146 . Q IU , Y.-L. , J. LEE , F . B ERNASCONI-QUADRONI , D. E. SOLTIS , P. S . S OLTIS , M . W ATERKEYN , L. 1959 . Études sur les Gnétales. II. Le strobile mâle, la mi- Z ANIS , AND E . Z IMMER . 1999 . The earliest angiosperms: Evidence from crosporogenèse et le gametophyte mâle de Gnetum africanum Welw. mitochondrial, plastid and nuclear genomes. Nature 402 : 404 – 407 . La Cellule 60 : 1 – 78 .