ABSTRACT: The origin of seed plants transformed land plant reproduction by protecting the embryo and enabling dispersal across diverse ecosystems. Yet many reproductive mechanisms remain poorly resolved, as research has focused predominantly on angiosperm models. Because Ginkgoales and Cycadales form a monophyletic clade sister to other living gymnosperms, Ginkgo biloba provides an informative system for investigating reproductive traits in early-diverging spermatophytes. Here, we integrated phenology, histology, RNA-seq, arabinogalactan-protein candidate profiling, and JIM8 immunolocalization to investigate male cone and pollen development. Male ontogeny was divided into three main developmental macro-stages, each characterized by a distinct transcriptional program: primordial maintenance in M1; microsporogenesis, tapetum activity, callose dynamics, and pollen wall formation in M2; and pollen maturation, endothecium reinforcement, and dehiscence in M3. A targeted candidate-gene workflow further identified structural and regulatory modules linked to tapetum differentiation, meiosis, sporopollenin biosynthesis, pollen maturation, microsporangial opening, and sex-determining-region-associated expression. JIM8-reactive epitopes showed stage- and tissue-specific redistribution across microsporocytes, tetrads, the tapetum, developing pollen, and the endothecium. A targeted comparison with a newly generated Cycas circinalis transcriptomic dataset recovered comparable reproductive modules, although with differences in their temporal deployment. These findings establish a comprehensive comparative framework for investigating shared and lineage-specific features of male reproduction in early-diverging gymnosperms.
