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Flowers develop unisexually from inception (termed type II unisexual flowers).\n                        <\/jats:p>\n                      <\/jats:list-item>\n                      <jats:list-item>\n                        <jats:p>\n                          We used PacBio HiFi and Hi\u2010C sequencing to assemble a high\u2010quality haplotype\u2010resolved male genome of\n                          <jats:italic>E. ulmoides<\/jats:italic>\n                          , and identified this species' sex\u2010linked region (SLR). We aimed to find sex\u2010determining candidate genes, and test the suggestion that sex in dioecious plants with type II unisexual flowers can be determined by MADS\u2010box genes.\n                        <\/jats:p>\n                      <\/jats:list-item>\n                      <jats:list-item>\n                        <jats:p>\n                          Comparative genomic and population genetic analyses revealed a male heterogametic system (XY), with a roughly 32\u2009Mb Y\u2010linked region. An older evolutionary stratum (S1) includes a Y\u2010specific A\u2010class MADS\u2010box gene,\n                          <jats:italic>EuMADS65<\/jats:italic>\n                          , a duplicate of\n                          <jats:italic>EuMADS38<\/jats:italic>\n                          , which is in the pseudoautosomal recombining region. Expression and functional experiments suggest neofunctionalization of\n                          <jats:italic>EuMADS65<\/jats:italic>\n                          after the duplication, to act as the male\u2010determining factor by regulating the expression of another sex\u2010linked gene,\n                          <jats:italic>EuAP3<\/jats:italic>\n                          , a stamen organ identity B\u2010class gene.\n                        <\/jats:p>\n                      <\/jats:list-item>\n                      <jats:list-item>\n                        <jats:p>\n                          As dioecy is prevalent among other species of Garryaceae,\n                          <jats:italic>E. ulmoides<\/jats:italic>\n                          probably did not evolve dioecy from a cosexual ancestor, and we evaluate the possibility that this duplication was involved in a sex chromosome turnover.\n                        <\/jats:p>\n                      <\/jats:list-item>\n               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