Seed plant female gametophytes are focal points for the evolutionary modification of development. From a structural perspective, the most divergent female gametophytes among all seed plants are found in Gnetum, a clade within Gnetales. Coenocytic organization at sexual maturity, absence of defined egg cells (free nuclei are fertilized), lack of centripetal cellularization, and postfertilization development of embryo-nourishing tissues are features of the female gametophytes of Gnetum unparalleled among seed plants. Although the female gametophyte of Gnetum retains the three basic phases of somatic development common to female gametophytes of plesiomorphic seed plants (free nuclear development, cellularization, cellular growth), the timing of fertilization has been accelerated relative to the rate of somatic development. As a consequence, the female gametophyte of Gnetum matures sexually (is fertilized) at a juvenile (compared with the ancestral somatic ontogeny) and free nuclear stage of somatic development, thereby precluding differentiation of egg cells. Unlike progenetic animals, where truncation of somatic ontogeny evolves in tandem with acceleration in the timing of sexual maturation, the female gametophyte of Gnetum completes the entire ancestral somatic ontogeny after precocious sexual maturation. This results in the evolution of postfertilization development of embryo-nourishing female gametophyte tissues, a phenomenon unique among seed plants. Nonheterochronic developmental innovations have also played important roles in the evolution of the female gametophyte ofGnetum. Centripetal cellularization, which is always associated with the phase change from coenocytic to cellular organization among plesiomorphic seed plant female gametophytes, is lacking in Gnetum. Instead, during early phases of development, apomorphic free nuclear organization is coupled with a highly anomalous pattern of cellularization. Stage-specific innovations during early development in the female gametophyte of Gnetum do not affect plesiomorphic aspects of later phases of development. Thus, a complex array of heterochronic and nonheterochronic developmental innovations have played critical roles in the ontogenetic evolution of the highly apomorphic female gametophyte of Gnetum.

Gnetum gnemon, a nonflowering seed plant and member of the Gnetales, expresses a rudimentary pattern of double fertilization that results in the formation of two zygotes per pollen tube. The process of double fertilization in G. gnemon was examined with light and fluorescence micmscopy, and the DNA content of various nuclei involved in sexual reproduction was quantified with 4',6-diamidino9-phenylindole microspectrofluommetry. Male and female gamete nuclei pass through the synthesis phase of the cell cycle and increase their DNA content from 1C to 2C before fertilization. Each of the two zygotes found in association with a pollen tube is diploid and contains the 4C quantity of DNA at inception. Based on these results as well as previous studies of nuclear DNA content in plant sperm, eggs, and zygotes, three fundamental and distinct patterns of gamete karyogamy among seed plants can be circumscribed: (1) G1 karyogamy, in which male and female gametes contain the 1C quantity of DNA throughout karyogamy and the zygote undergoes DNA replication; (2) S-phase karyogamy, in which gamete nuclei initiate fusion at 1C but pass through the S phase of the cell cycle before completely fusing; and (3) G2 karyogamy, in which male and female gamete nuclei pass through the S phase of the cell cycle before the onset of fertilization. Our results show definitively a pattern of G2 karyogamy in G. gnemon.

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The fertilization process in Gnetum is critical to our understanding of the evolution of sexual reproduction within the Gnetales, a monophyletic group of nonflowering seed plants that are the closest living relatives to flowering plants. Although much is known about the fertilization process in Ephedra, which is basal within the Gnetales, little is known about sexual reproduction in the derived sister groups Gnetum and Welwitschia. Ovules of Gnetum gnemon were col1ected at various stages after hand pol1ination and processed for light, fluorescence, and electron microscopy. Approximately 5 d after pollination, pol1en tubes reach sexual1y mature female gametophytes, which are coenocytic. At that time, a binucleate sperm cel1 is found within each pollen tube. Within 7 d of pollination, double fertilization events occur when each of two sperm nuclei released from a pol1en tube fuses with a separate, undifferentiated female nucleus within the free nuclear female gametophyte, which lacks differentiated egg cel1s. The products of double fertilization are two viable zygotes; endosperm is not formed. The lack of differentiated egg cel1s in Gnetum gnemon is unparal1eled among land plants and the documentation of a regularly occurring process of double fertilization is congruent with the hypothesis that a rudimentary process of double fertilization evolved in a common ancestor of angiosperms and Gnetales.