WOX neofunctionalization following an ancient duplication in mosses

Gene duplication generates new genetic material and provides a basis for evolutionary innovation.¹ In land plants, an ancient divergence gave rise to bryophytes and vascular plants, with each group following independent evolutionary trajectories.²WOX (WUSCHEL-like homeobox) gene activity is required for sporophyte development in angiosperms,³ and a gene duplication predating the origin of land plants generated T1 (AtWOX13) and T2/T3 (AtWOX9/WUSCHEL) WOX clades.⁴ Bryophytes only have T1 WOXes,⁴ and in the moss Physcomitrium patens, PpWOX13LA and PpWOX13LB are expressed broadly and promote post-zygotic sporophyte development.⁵ A third gene, PpWOX13LC, was presumed to be a pseudogene due to structural truncation and undetectable vegetative expression.⁵ Here, we demonstrate that PpWOX13LC has a reproductive function in suppressing sporophyte development. Using phylogenetic and structural analyses, we identify a large moss-specific clade of WOX13LC genes originating from an ancient gene duplication. While homeodomain structures are conserved between WOX13L sequences, WOX13LCs have a 3' homeodomain extension. We report that PpWOX13LC is expressed during gametangium and egg development, and Ppwox13lc mutants develop supernumerary sporophytes on reproductive shoot apices. We speculate that PpWOX13LC suppresses the activation of post-zygotic development, thus opposing the function of PpWOX13LA and PpWOX13LB in sporophyte growth activation. As the suppression of multiple sporophytes per reproductive shoot apex is a key life-history trait affecting maternal resource allocation and fitness, the innovation of WOX13LC function was likely adaptive,^6,7 leading to WOX13LC retention over hundreds of millions of years. Thus, an ancient WOX13L gene duplication and divergence of function contributed to reproductive innovation during the radiation of mosses.