Differences between males and females are usually more subtle in dioecious plants than animals, but strong sexual dimorphism has evolved convergently in the South African Cape plant genus Leucadendron. Such sexual dimorphism in leaf size is expected largely to be due to differential gene expression between the sexes. We compared patterns of gene expression in leaves among 10 Leucadendron species across the genus. Surprisingly, we found no positive association between sexual dimorphism in morphology and the number or the percentage of sex-biased genes (SBGs). Sex bias in most SBGs evolved recently and was species specific. We compared rates of evolutionary change in expression for genes that were sex biased in one species but unbiased in others and found that SBGs evolved faster in expression than unbiased genes. This greater rate of expression evolution of SBGs, also documented in animals, might suggest the possible role of sexual selection in the evolution of gene expression. However, our comparative analysis clearly indicates that the more rapid rate of expression evolution of SBGs predated the origin of bias, and shifts towards bias were depleted in signatures of adaptation. Our results are thus more consistent with the view that sex bias is simply freer to evolve in genes less subject to constraints in expression level.

Flowering plants utilize small RNA molecules to guide DNA methyltransferases to genomic sequences. This RNA-directed DNA methylation (RdDM) pathway preferentially targets euchromatic transposable elements. However, RdDM is thought to be recruited by methylation of histone H3 at lysine 9 (H3K9me), a hallmark of heterochromatin. How RdDM is targeted to euchromatin despite an affinity for H3K9me is unclear. Here we show that loss of histone H1 enhances heterochromatic RdDM, preferentially at nucleosome linker DNA. Surprisingly, this does not require SHH1, the RdDM component that binds H3K9me. Furthermore, H3K9me is dispensable for RdDM, as is CG DNA methylation. Instead, we find that non-CG methylation is specifically associated with small RNA biogenesis, and without H1 small RNA production quantitatively expands to non-CG methylated loci. Our results demonstrate that H1 enforces the separation of euchromatic and heterochromatic DNA methylation pathways by excluding the small RNA-generating branch of RdDM from non-CG methylated heterochromatin.