In contrast to most angiosperms, in which fertilization occurs 1 or 2 days after pollination, in some plant orders, including the Fagales, fertilization is delayed from 4 days to more than 1 year, raising questions regarding why fertilization is delayed and where and how pollen tubes remain in the pistil during the delay. To answer these questions, we investigated pollen-tube growth in pistils of Fagus japonica (Fagaceae), which are tricarpellate and have six ovules, using light, fluorescence, and scanning electron microscopy. The ovules were immature at the time of pollination and required 5 weeks to become fully developed. During this 5 weeks, pollen tubes grew from the stigma to the embryo sac in association with the development of ovules and intermittently in three steps with two growth-cessation sites, i.e., on the funicle and near the micropyle. The number of pollen tubes was gradually reduced from many to one at the two growth-cessation sites, and fertilization occurred in one ovule that apparently developed earlier than the others in the pistil. Thus, delayed fertilization plays an important role in gametophyte competition and selection leading to nonrandom fertilization. Intermittent pollen-tube growth is also likely widespread in angiosperms because it is known in other Fagales and an unrelated order Garryales.

In alders, where fertilization occurs Ϸ8 weeks after pollination, the pollen tube (male gametophyte) grows intermittently in four steps in close association with the development of the ovary and its ovules. Pollen tubes stop growing in the style, at the ovarian locule, and at the chalaza (ovule), before reaching an embryo sac for fertilization. At the stage when the ovary develops an ovule primordium in each of the two locules, many pollen tubes germinate on the stigma, and a few of them reach the style, where they remain for Ϸ7 weeks. Thereafter, a single tube resumes growing; with a short stop in the upper space of the ovarian locule, it reaches the older of the two ovules when it has developed a two-nucleate embryo sac. Except in the last step, where the tube grows from the chalaza to an embryo sac (female gametophyte), an eight-nucleate mature embryo sac is not necessary for pollen-tube guidance in the pistil. Although the intermittent pollen-tube growth appears to play an important role in the selection of a single pollen tube from many and one ovule from two, its detection provides insight into the study of the mechanism of pollen-tube guidance.Betulaceae ͉ chalazogamy ͉ Fagales ͉ fertilization ͉ ovule I n flowering plants, fertilization occurs when the pollen tube (male gametophyte) reaches the embryo sac (female gametophyte) through the tissue of the pistil (sporophyte), unifying a sperm nucleus with an egg nucleus. This process suggests interaction not only between the pollen tube and the embryo sac, but also between the pollen tube (and embryo sac) and an intervening tissue, i.e., the tissue of the pistil. Recent studies using mutants of Arabidopsis thaliana that lacked normally developed ovules or embryo sacs showed that the pollen tubes lost their way to the target within the pistil (1-3). However, these studies mainly paid attention to interaction between pollen tubes and the embryo sac, and analysis of the correlation between the pollen-tube growth and the developmental stage of the pistils is still scarce.In a majority of cases, the length of time between pollination and fertilization ranges from 24 to 48 h, and in some plants it is even shorter (4-8). In fagalean species, however, the pollen tube spends Ͼ1 month in the pistil before fertilization occurs, because the ovules are not yet mature when the pistil receives pollen grains on the stigma (9-11). We recently reported the pollentube growth pattern in Casuarinaceae (Fagales), where a unique mode of fertilization (i.e., chalazogamy) in place of the ordinary mode (i.e., porogamy) was reported (12, 13) (for details regarding chalazogamy and porogamy, see Fig. 5A, which is published as supporting information on the PNAS web site). In Casuarina equisetifolia (12), fertilization occurs in five discontinuous steps of pollen tube growth from the stigma to the embryo sac. We suggested that the pollen tube exhibited repeated steps of growth in accordance with the development of the ovary and ovule(s). However, C. equisetifolia develops four to eight emb...

For a better understanding of pollen-tube guidance in relation to pollen-pistil interaction, we investigated the mode of pollen-tube growth in pistils of Casuarina equisetifolia, a monoecious, wind-pollinated species that undergoes chalazogamous fertilization. The pistil is bicarpellate, but only one of the two carpels develops with two ovules. One of these ovules develops more than four embryo sacs. Pistils usually require more than 1 month to reach maturity after pollen grains have been deposited on the stigmas. During that period, pollen-tube growth proceeds discontinuously in five distinct steps that lead up to fertilization: (1) from the stigma to the upper region of the style, (2) from the upper region of the style to a septum in the ovary, (3) from the septum to the surface of the funiculus, (4) from the funiculus to chalaza in the ovule, and (5) from the chalaza to an egg apparatus. Probably because of competitive interaction between male and female gametophytes (or ovules), one pollen tube is selected from among many during the first step (just before the second step), one ovule from the two during the second and third steps, and one embryo sac from more than four during the fourth and fifth steps. On the basis of our results, erroneous drawings and explanations reported in earlier publications on chalazogamy in Casuarinaceae should be brought into question.

Myrica differs from other families in that the pollen tubes arrest their growth on the nucellar surface, probably digesting nutrient from nucellar cells. There is little information on five other families of Fagales. An extensive study is needed to better understand the diversity and function of the mode of pollen-tube growth within the order.

We present the first overall molecular phylogenetic study of Casuarinaceae on the basis of sequences of two chloroplast genes, rbcL (1310 bp) and matK (1014 bp), using 15 species representing the family. The study included analyses of Ticodendron (Ticodendraceae) and three species of Betulaceae as close relatives, and one species each of Juglandaceae and Myricaceae as outgroups. Analyses based on matK gene sequences, which provided a much better resolution than the analyses based on rbcL gene sequences alone, resulted in a single most parsimonious tree whose topology is almost identical with the strict consensus tree generated by the combined data set of rbcL and matK gene sequences. Results showed that Casuarinaceae are monophyletic, comprising four distinct genera, A//ocasuarina, Casuarina, Ceuthostoma and Gymnostoma, which were not recognized until recently. Within the family, Gymnostoma is positioned at the most basal position and sister to the remainder. Within the remainder Ceuthostoma is sister to the A//ocasuarina-Casuarina clade. Morphologically the basalmost position of Gymnostoma is supported by plesiomorphies such as exposed stomata in the shallow longitudinal furrows of the branchlets, a basic chromosome number x=8 and the gynoecium composed of two fertile, biovulate carpels. The three other genera, A//ocasuarina, Casuarina, and Ceuthastoma, have invisible stomata in the deep longitudinal furrows of the branchlets, a higher basic chromosome number x=9 or 10-14 (unknown in Ceuthostoma), the gynoecium composed of one fertile and one sterile carpel with a single ovule (unknown in Ceuthastoma). The diversity of infructescence morphology found in the latter three genera suggests that they may have evolved in close association with the elaboration of fruit dispersal mechanisms.