Sex is determined by XY chromosomes across the radiation of dioecious Nepenthes pitcher plants

Species with separate sexes (dioecy) are a minority among flowering plants, but dioecy has evolved multiple times independently in their history. The sex determination system and sex-linked genomic regions are currently identified in a limited number of dioecious plants only. Here, we study the sex-determination system in a genus of dioecious plants that lack heteromorphic sex chromosomes and are not amenable to controlled breeding: Nepenthes pitcher plants. We genotyped wild populations of flowering males and females of three Nepenthes taxa using ddRAD-seq, and sequenced a male inflorescence transcriptome. We developed a statistical tool (privacy rarefaction) to distinguish true sex-specificity from stochastic noise in read coverage of sequencing data from wild populations and identified male-specific loci and XY-patterned SNPs in all three Nepenthes taxa, suggesting the presence of homomorphic XY sex chromosomes. The male-specific region of the Y chromosome showed little conservation among the three taxa, except for the essential pollen development gene DYT1 which was confirmed as male-specific by PCR in additional Nepenthes taxa. Hence, dioecy and part of the male-specific region of the Nepenthes Y-chromosomes likely have a single evolutionary origin.