Although coevolution is acknowledged to occur in nature, coevolutionary patterns in symbioses not involving species‐to‐species relationships are poorly understood. Mycorrhizal plants are thought to be too generalist to coevolve with their symbiotic fungi; yet some plants, including some orchids, exhibit strikingly narrow mycorrhizal specificity. Here, we assess the evolutionary history of mycorrhizal specificity in the lady's slipper orchid genus, Cypripedium. We sampled 90 populations of 15 taxa across three continents, using DNA methods to identify fungal symbionts and quantify mycorrhizal specificity. We assessed phylogenetic relationships among sampled Cypripedium taxa, onto which we mapped mycorrhizal specificity. Cypripedium taxa associated almost exclusively with fungi within family Tulasnellaceae. Ancestral specificity appears to have been narrow, followed by a broadening after the divergence of C. debile. Specificity then narrowed, resulting in strikingly narrow specificity in most of the taxa in this study, with no taxon rewidening to the same extant as basal members of the genus. Sympatric taxa generally associated with different sets of fungi, and most clades of Cypripedium‐mycorrhizal fungi were found throughout much of the northern hemisphere, suggesting that these evolutionary patterns in specificity are not the result of biogeographic lack of opportunity to associate with potential partners. Mycorrhizal specificity in genus Cypripedium appears to be an evolvable trait, and associations with particular fungi are phylogenetically conserved.

Background: Orchids maintain a symbiotic relationship with mycorrhizal fungi in the lifecycle. Previous reports indicated that diverse mycobionts may have different roles during orchid growth and development. Although various mycorrhizal fungi have been isolated fromDendrobiumroots and protocorms, little is known about their specific effects on seed germination and seedling growth. To understand the specific role of isolated fungal strains (i.e.,TulasnellaandSebacina), we used symbiotic culture to compare the effect of 6 fungal strains on seed germination and seedling growth ofDendrobium officinale, an important Chinese medicinal orchid.Results: In symbiotic germination tests, 6 fungal strains (4Tulasnellastrains and 2Sebacinastrains) promoted seed germination with different efficiencies. Seeds inoculated withTulasnellastrains S6 and S7 conferred higher germination percentage and faster protocorm development than other fungal strains. In symbiotic cultures, seedlings inoculated withSebacinastrain S3 had optimal fresh and dry matter yield. Also,Tulasnellastrains S6 and S7 promoted seedling growth with good fresh and dry matter yield.Sebacinastrain S2 inoculation greatly enhanced root and tiller production and the content of total crude polysaccharides, although seedlings were smaller with less fresh and dry matter yield than other seedlings.Conclusions: TulasnellaandSebacinastrains could promote seed germination and seedling growth ofD. officinalewith different efficiencies. Our results suggest a non-specific mycorrhizal association and development-dependent preference. Our data provide the basic knowledge for use of different fungal strains in conservation and/or production practices ofD. officinale.

Background and Aims Although abscisic acid (ABA) is commonly recognized as a primary cause of seed dormancy, there is a lack of information on the role of ABA during orchid seed development. In order to address this issue, the localization and quantification of ABA were determined in developing seeds of Cypripedium formosanum. Methods The endogenous ABA profile of seeds was measured by enzyme-linked immunosorbent assay (ELISA). Temporal and spatial distributions of ABA in developing seeds were visualized by immunohistochemical staining with monoclonal ABA antibodies. Fluoridone was applied to test the causal relationship between ABA content and seed germinability. Key Results ABA content was low at the proembryo stage, then increased rapidly from 120 to 150 days after pollination (DAP), accompanied by a progressive decrease in water content and seed germination. Immunofluorescence signals indicated an increase in fluorescence over time from the proembryo stage to seed maturation. From immunogold labelling, gold particles could be seen within the cytoplasm of embryo-proper cells during the early stages of seed development. As seeds approached maturity, increased localization of gold particles was observed in the periplasmic space, the plasmalemma between embryo-proper cells, the surface wall of the embryo proper, and the inner walls of inner seed-coat cells. At maturity, gold particles were found mainly in the apoplast, such as the surface wall of the embryo proper, and the shrivelled inner and outer seed coats. Injection of fluoridone into capsules resulted in enhanced germination of mature seeds. Conclusions The results indicate that ABA is the key inhibitor of germination in C. formosanum. The distinct accumulation pattern of ABA suggests that it is synthesized in the cytosol of embryo cells during the early stages of seed development, and then exported to the apoplastic region of the cells for subsequent regulatory processes as seeds approach maturity.

Background: Gastrodia elatais a fully mycoheterotrophic orchid and has long been used in traditional Chinese medicine. The life cycle ofG. elatarequires an association with two different fungi-Mycenafor seed germination andArmillariafor tuber growth. The association withArmillariais representative of the phytophagous type of orchid mycorrhiza: the intracellular hyphae are lysed without forming condensed pelotons. However, whether the association withMycenaduring seed germination belongs to the same type of orchid mycorrhiza is unknown.Results: Histological and ultrastructural studies revealed several notable features in different developmental stages. First, a thickened cell wall with papillae-like structures appeared during fungal penetration in the suspensor end cell, epidermal cells and cortical cells of germinating embryos. In addition, the formation of two distinctive cell types in the colonized region of a protocorm (i.e., the passage canal cell filled with actively growing fungal hyphae) can be observed in the epidermal cell, and the distinctive digestion cell with a dense cytoplasm appears in the cortex. Finally, within the digestion cell, numerous electron-dense tubules form a radial system and attach to degrading fungal hyphae. The fungal hyphae appear to be digested through endocytosis.Conclusions: The present study provides important structural evidence for the phytophagous type of orchid mycorrhiza in the symbiotic germination ofG. elatawithMycena. This case demonstrates a particular nutrient transfer network betweenG. elataand its litter-decaying fungal partner.