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

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.