The development of unisexual flowers depends on the intricate coordination of flower organ identity and sex determination mechanisms. Unisexual flowering has emerged several times throughout evolution, being observed in 10% of flowering plants, and is thought to increase adaptive fitness by promoting cross-breeding and gene flow. Oaks (genus Quercus) are generally considered as strictly monoecious, with each individual tree displaying fully segregated male and female flowers in separate inflorescences. However, previous reports of atypical flowering across the genus contrast with this classical view, suggesting that oak flowering may be more flexible than traditionally assumed. The molecular mechanisms that coordinate floral development have been extensively characterized in model species, with most flower-promoting cues being integrated in the expression of FLOWERING LOCUS T (FT) in the leaves. The FT protein migrates to the meristems, and orchestrates the conversion from a vegetative to a reproductive state. Following floral initiation, the identity of each floral organ is specified by different combinations of transcription factors, as outlined by the ABCDE-model. However, despite the great economic and ecological importance of the Quercus genus, studies on the processes that control flower development in oaks are scarce.

In this work, recurrent hermaphroditic flowering in Quercus orocantabrica and Q. ilex is reported, occurring in several individuals and over several years. Unseasonal flowering events throughout the growth season were also detected, generally associated with an increased frequency of hermaphroditic flowers. In addition, a differential expression of B- and C-class genes in male and female flowers was detected, with B-class genes being predominantly expressed in male flowers. The B-class gene PISTILLATA is exclusively expressed in male flowers of Q. suber and Q. ilex, while being expressed in both male and hermaphroditic flowers of Q. orocantabrica, tightly associating the expression of this gene to the initiation and development of stamens. The C-class gene SHATTERPROOF is expressed in both flowers of Q. suber and Q. ilex, but is not detected in Q. orocantabrica male flowers, despite the absence of significant morphological differences between the flowers of these species. Collectively, these results support a reinterpretation of oak reproductive biology, based on a versatile and responsive system reliant on distinctly complex regulatory processes.