LEAFY demonstrates ancestral reproductive functions in the gametophyte and not the sporophyte of the fern Ceratopteris richardii

Abstract Flowers are a key reproductive innovation of the angiosperms. They evolved as a modification of the ancestral plant life cycle whereby the haploid gamete-producing generation (gametophyte) became enclosed within the diploid, spore-producing generation (sporophyte). The transcription factor LEAFY (LFY) initiates angiosperm floral development, yet its lineage predates flowers and is found across all land plants. LFY function outside angiosperms is known from the moss Physcomitrium patens, where they control the first division of the sporophyte, and from the model fern Ceratopteris richardii, a vascular plant without seeds or flowers, where CrLFY1 and CrLFY2 maintain vegetative meristem activity. However, how LFY’s reproductive role evolved remains unclear. Using over-expression, we uncover new roles for CrLFY1/2 in fern gametophyte reproduction, particularly in sperm cells and in the gametophyte’s multicellular notch meristem. No sporophytic reproductive function was detected, but over-expression supports a role in fern frond compounding and a conserved role in the zygote’s first division. Our findings highlight an ancestral LFY function in fern haploid-stage reproduction, which may have been co-opted into the sporophyte during the origin of the flower. Summary Statement The origin of LEAFY’s floral function is unknown, with only vegetative roles known from seedless plants. We identify ancestral reproductive roles for the first time, unexpectedly in the fern gametophyte. Competing Interest Statement The authors have declared no competing interest.