Weak population spatial genetic structure and low infraspecific specificity for fungal partners in the rare mycoheterotrophic orchid Epipogium aphyllum.

Some plants abandoned photosynthesis and developed full dependency on fungi for nutrition. Most of the so-called mycoheterotrophic plants exhibit high specificity towards their fungal partners. We tested whether natural rarity of mycoheterotrophic plants and usual small and fluctuating population size make their populations more prone to genetic differentiation caused by restricted gene flow and/or genetic drift. We also tested whether these genetic characteristics might in turn shape divergent fungal preferences. We studied the mycoheterotrophic orchid Epipogium aphyllum, addressing the joint issues of genetic structure of its populations over Europe and possible consequences for mycorrhizal specificity within the associated fungal taxa. Out of 27 sampled E. aphyllum populations, nine were included for genetic diversity assessment using nine nuclear microsatellites and plastid DNA. Population genetic structure was inferred based on the total number of populations. Individuals from 17 locations were included into analysis of genetic identity of mycorrhizal fungi of E. aphyllum based on barcoding by nuclear ribosomal DNA. Epipogium aphyllum populations revealed high genetic diversity (uHe = 0.562) and low genetic differentiation over vast distances (FST = 0.106 for nuclear microsatellites and FST = 0.156 for plastid DNA). Bayesian clustering analyses identified only two genetic clusters, with a high degree of admixture. Epipogium aphyllum genets arise from panmixia and display locally variable, but relatively high production of ramets, as shown by a low value of rarefied genotypic richness (Rr = 0.265). Epipogium aphyllum genotype control over partner selection was negligible as (1) we found ramets from a single genetic individual associated with up to 68% of the known Inocybe spp. associating with the plant species, (2) and partner identity did not show any geographic structure. The absence of mosaicism in the mycorrhizal specificity over Europe may be linked to preferential allogamous habit of E. aphyllum and significant gene flow, which tend to promote host generalism.

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.