Ontogenetic and molecular analyses reveal the evolution of prolonged female gametophyte‐dominating reproductive cycles in Pinaceae

Land plants follow an evolutionary trajectory of 'gametophyte reduction' and 'sporophyte dominance'. As a major shift in gametophyte reduction, gymnosperms have evolved a unique female gametophyte (FG) development mode, associated with their prolonged reproductive cycles. However, the genetic programs underlying this process remain largely unknown. Here, we employed anatomical, transcriptomic, and genetic approaches to investigate the female gametogenesis, focusing on the divergent coenocytic free nuclear stage in three species (Cedrus deodara, Picea smithiana, and Pinus tabuliformis) from the largest gymnosperm family Pinaceae. We obtained a comprehensive anatomical profile of FG development, correlating variations in the timing of the free nuclear stage with the diverse reproductive cycles. We also revealed the transcriptional dynamics underlying each stereotypical stage of FG development, highlighting the involvement of cyclin-dependent kinase 2a, cyclin B genes, specific MADS-box genes, and other conserved homologous transcription factors. Moreover, a focused examination of the fascinating long reproductive cycle of Pinus, the largest genus of gymnosperms, further unveiled regulatory molecules for growth-defense trade-off and summer dormancy of FG. Our study highlights the molecular mechanisms underpinning heterochronic development of FG during the free nuclear stage in Pinaceae, offering crucial insights into the evolution of plant reproductive strategies.