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 single nucleotide polymorphsims (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 that 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.