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 , involv- ing 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 ex- amined 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 , American Journal of Botany 102 ( 2 ): 312 – 324 , 2015 ; http://www.amjbot.org/ © 2015 Botanical Society of America 312 FRIEDMAN—FERTILIZATION OF WELWITSCHIA • VOL. 102 , NO. 2 FEBRUARY 2015 • 313 1998 ; Carmichael and Friedman, 1995 , 1996 ; Friedman and MATERIALS AND METHODS Carmichael, 1996 , 1998 ) deepened our understanding of the re- productive 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 pollina- tion. 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)