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...