Evidence for mixed sexual and asexual reproduction in the rare European mycoheterotrophic orchid Epipogium aphyllum, Orchidaceae (ghost orchid)

Background and Aims Despite their significant capacity to propagate vegetatively, most orchids reproduce via seeds. Sexual reproduction via seed is commonly reported, in contrast to apomixis, whereby seeds are clones of the mother. Although insect pollination and autonomous self- pollination exist in mycoheterotrophic plants, the reproductive embryology of these plants remains under- studied. This paper provides evidence for the co- occurrence of both sexual and apomictic reproduction in a population of mycoheterotrophic plants - Epipogium aphyllum. We investigated seed formation via open pollination, induced autogamy, autogamy sensu stricto and autonomous apomixis. Methods The study was performed on a population of E. aphyllum located in northern Poland. The research included studies of the micromorphology, histochemistry and embryology of four types of reproductive systems. Scanning, fluorescence and light microscopy accompanied by graphical and statistical analyses were employed. Key Results We observed gametophyte development, from the one-nucleate stage to maturity, in unpollinated flower buds. The lack of zygotes in flower buds indicated that fertilization did not occur at this stage. Manual self-pollination led to a zygote, followed by embryo formation. Fertilization and embryo development derived from embryogenesis via open pollination is delayed compared with hand pollination. Isolation from external pollination resulted only in structures resembling zygotes that may originate either sexually or independent of fertilization. Parthenogenetic structures that resembled zygotes were observed in flowers that were emasculated and isolated from pollination. Zygotes formed at significantly higher frequencies via open pollination and induced autogamy in comparison to the parthenogenetic structures formed in other treatments. Conclusions We showed the absence of pre-zygotic barriers for autogamy in E. aphyllum. Self-pollination and self-fertilization are possible; however, natural self-pollination is unlikely or rare due to the position of the pollinia. Incidental parthenogenesis in E. aphyllum is very likely, given the biology of ovule development of this mycoheterotrophic orchid. This species therefore has the potential to produce seeds via both sexual and asexual means, although the contribution of apomixis to this process appears largely negligible.