Cell division and lineage dynamics during antheridium differentiation and male gametophyte development in Ceratopteris richardii

Sexual reproduction in land plants involves diverse strategies for gamete production and fertilization, significantly differing between seed plants, which deliver sperm via pollen, and seed-free plants such as ferns, which produce motile sperm within specialized multicellular structures, antheridia, on independently growing gametophytes. Despite their crucial roles in sexual reproduction, the cellular mechanisms governing antheridium differentiation and male gametophyte development in ferns remain largely unexplored. Here, using non-invasive, time-lapse confocal imaging combined with computational three-dimensional analysis, we reconstructed detailed lineage maps of antheridium initiation, proliferation, and differentiation in the fern Ceratopteris richardii. Our findings demonstrate that antheridium development begins with highly conserved asymmetric cell divisions, giving rise to distinct sterile and spermatogenous cell lineages. Spermatogenous cells undergo synchronized and continuous proliferation, followed by programmed differentiation, eventually forming motile sperm released from mature antheridia. In contrast, the sterile lineage undergoes limited cell divisions, forming structural support tissues surrounding the spermatogenous core. Furthermore, quantitative analyses reveal the cellular basis underlying the previously reported antagonistic effects of the pheromone antheridiogen and abscisic acid on antheridium initiation and spermatogenous cell proliferation. These findings elucidate both conserved and lineage-specific mechanisms regulating sexual differentiation, providing comparative insights into reproductive strategies and hormone-mediated developmental processes across land plants.