PKN is a sex- and species-specific fertilization factor in brown algae

Summary Fertilization, the fusion of male and female gametes, is fundamental to sexual reproduction, yet the molecular mechanisms that mediate gamete recognition and enforce species specificity remain poorly understood, and only a handful of proteins are known to act as core fertilization factors across eukaryotes. Here, we identify PICKINESS-ASSOCIATED PROTEIN (PKN), a female gamete-specific transmembrane protein, as an essential determinant of fertilization in brown algae. CRISPR-Cas-mediated knockout of PKN abolishes successful male-female gamete interactions and prevents fertilization without affecting earlier mating behaviors, such as gamete attraction. Remarkably, PKN also enforces reproductive isolation by preventing interspecific fertilization, establishing it as a molecular gatekeeper of species specificity. Structural analyses reveal extracellular β-propeller and mucin-like domains enriched in predicted glycosylation sites and displaying rapid sequence evolution. Functional and comparative analyses suggest that PKN-dependent recognition mechanisms are conserved across diverse brown algal lineages. Because PKN originated within brown algae, its dual role in mediating both male-female gamete recognition and species-specific fertilization reveals a striking conceptual parallel with fertilization factors described in animals, suggesting that evolution repeatedly converges on lineage-specific gamete-expressed membrane proteins as key arbiters of reproductive recognition.