Extensive N4 cytosine methylation is essential for Marchantia sperm function.

N4-methylcytosine (4mC) is an important DNA modification in prokaryotes, but its relevance and even its presence in eukaryotes have been mysterious. Here we show that spermatogenesis in the liverwort Marchantia polymorpha involves two waves of extensive DNA methylation reprogramming. First, 5-methylcytosine (5mC) expands from transposons to the entire genome. Notably, the second wave installs 4mC throughout genic regions, covering over 50% of CG sites in sperm. 4mC requires a methyltransferase (MpDN4MT1a) that is specifically expressed during late spermiogenesis. Deletion of MpDN4MT1a alters the sperm transcriptome, causes sperm swimming and fertility defects, and impairs post-fertilization development. Our results reveal extensive 4mC in a eukaryote, identify a family of eukaryotic methyltransferases, and elucidate the biological functions of 4mC in reproductive development, thereby expanding the repertoire of functional eukaryotic DNA modifications.