A narrowly endemic photosynthetic orchid is non‐specific in its mycorrhizal associations

Mycorrhizal association is a common characteristic in a majority of land plants, and the survival and distribution of a species can depend on the distribution of suitable fungi in its habitat. Orchidaceae is one of the most species‐rich angiosperm families, and all orchids are fully dependent on fungi for their seed germination and some also for subsequent growth and survival. Given this obligate dependence, at least in the early growth stages, elucidating the patterns of orchid–mycorrhizal relationships is critical to orchid biology, ecology and conservation. To assess whether rarity of an orchid is determined by its specificity towards its fungal hosts, we studied the spatial and temporal variability in the host fungi associated with one of the rarest North American terrestrial orchids, Piperia yadonii. The fungal internal transcribed spacer region was amplified and sequenced by sampling roots from eight populations of P. yadonii distributed across two habitats, Pinus radiata forest and maritime chaparral, in California. Across populations and sampling years, 26 operational taxonomic units representing three fungal families, the Ceratobasidiaceae, Sebacinaceae and Tulasnellaceae, were identified. Fungi belonging to the Sebacinaceae were documented in orchid roots only at P. radiata forest sites, while those from the Ceratobasidiaceae and Tulasnellaceae occurred in both habitats. Our results indicate that orchid rarity can be unrelated to the breadth of mycorrhizal associations. Our data also show that the dominance of various fungal families in mycorrhizal plants can be influenced by habitat preferences of mycorrhizal partners.     

The evolutionary history of mycorrhizal specificity among lady's slipper orchids

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.     

High specificity generally characterizes mycorrhizal association in rare lady's slipper orchids, genus Cypripedium

Lady's slipper orchids (Cypripedium spp.) are rare terrestrial plants that grow throughout the temperate Northern Hemisphere. Like all orchids, they require mycorrhizal fungi for germination and seedling nutrition. The nutritional relationships of adult Cypripedium mycorrhizae are unclear; however, Cypripedium distribution may be limited by mycorrhizal specificity, whether this specificity occurs only during the seedling stage or carries on into adulthood. We attempted to identify the primary mycorrhizal symbionts for 100 Cypripedium plants, and successfully did so with two Cypripedium calceolus, 10 Cypripedium californicum, six Cypripedium candidum, 16 Cypripedium fasciculatum, two Cypripedium guttatum, 12 Cypripedium montanum, and 11 Cypripedium parviflorum plants from a total of 44 populations in Europe and North America, yielding fungal nuclear large subunit and mitochondrial large subunit sequence and RFLP (restriction fragment length polymorphism) data for 59 plants. Because orchid mycorrhizal fungi are typically observed without fruiting structures, we assessed fungal identity through direct PCR (polymerase chain reaction) amplification of fungal genes from mycorrhizally colonized root tissue. Phylogenetic analysis revealed that the great majority of Cypripedium mycorrhizal fungi are members of narrow clades within the fungal family Tulasnellaceae. Rarely occurring root endophytes include members of the Sebacinaceae, Ceratobasidiaceae, and the ascomycetous genus, Phialophora. C. californicum was the only orchid species with apparently low specificity, as it associated with tulasnelloid, ceratobasidioid, and sebacinoid fungi in roughly equal proportion. Our results add support to the growing literature showing that high specificity is not limited to nonphotosynthetic plants, but also occurs in photosynthetic ones.     

Three new species in the genus Tulasnella isolated from orchid mycorrhiza of Spiranthes sinensis var. amoena (Orchidaceae)

We isolated Tulasnella spp. from Spiranthes sinensis var. amoena, a Japanese native winter green terrestrial orchid collected in Tsukuba City, Ibaraki Pref., Japan. These isolates were classified into four morphotypes according to morphological characters, i.e., shape of monilioid cells and branch type of monilioid cell chains, while they were separated into five clades by molecular phylogenetic analysis based on the rDNA 5.8S and D1/D2 regions. The four morphotypes and five clades were correlated, and four morpho-phylogenetic groups were identified. Thus, Tulasnella deliquescens including two phylogenetic subgroups and three new species, Tulasnella dendritica sp. nov., Tulasnella ellipsoidea sp. nov., and Tulasnella cumulopuntioides sp. nov., were recognized in this study. In this study, the monilioid cell chain morphology is newly defined, and is suggested as a useful taxonomic characteristic in the asexual stage of Tulasnella spp.     

High diversity of root-associated fungi isolated from three epiphytic orchids in southern Ecuador

Knowledge of fungal root-associates is essential for effective conservation of tropical epiphytic orchids. We investigated the diversity of root-associated fungi of Cyrtochilum myanthum, Scaphyglottis punctulata and Stelis superbiens from a tropical mountain rainforest in southern Ecuador, using a culture dependent approach. We identified 115 fungal isolates, corresponding to 49 fungal OTUs, based on sequences of the nrDNA ITS and partial 28S region. Members of Ascomycota were unambiguously dominant (37 OTUs), including Trichoderma sp. as the most frequent taxon. Members of Basidiomycota (Agaricales and Polyporales) and Mucoromycota (Umbelopsidales and Mortierellales) were also identified. Four potential mycorrhizal OTUs of Tulasnellaceae and Ceratobasidiaceae were isolated from C. myanthum and S. superbiens. Fungal community composition was examined using Sørensen and Jaccard indices of similarity. Alfa diversity was significantly different between C. myanthum and S. superbiens. No difference in beta diversity of the fungal communities between the 3 orchid species and the collecting sites was detected. The study revealed a high diversity of fungi associated with orchid roots. Our results contribute to a better understanding of specific relationships between epiphytic orchids and their root-associated fungi.     

Molecular identifications uncover diverse fungal symbionts of Pleione (Orchidaceae)

Fungal mutualisms are essential for the evolution and diversification of Orchidaceae, yet the fungal symbionts of Pleione orchids are poorly understood because molecular data are unavailable for this genus. Based on ITS-rDNA sequencing for mycobionts of 15 Pleione species (both wild and cultivated plants were included), we conducted phylogenetic analyses for the most dominant mycobionts, and compared the operational taxonomic units (OTUs) of mycorrhizal fungi among species within Pleione. Tulasnellaceae, Ceratobasidiaceae, Serendipitaceae (Sebacinales), Atractiellales, and Auriculariales were reported as putative mycobionts of Pleione. In particular, the mycorrhizal associations between subtropical orchids and Atractiellales have not been observed before. For the dominant mycobionts in the roots of Pleione and its related genera, Bletilla and Coelogyne, we detected no fungal OTU that was shared. Within Pleione, species with a sympatric distribution showed preferences for different fungi. Epiphytic and lithophytic individuals of Pleione albiflora shared OTUs of Tulasnellaceae but harbored different OTUs of Sebacinales, indicating some degree of fungal specificity toward certain habitats. These findings provide new insights into the ecological adaptation and evolution of orchids, and will contribute to the conservation and utilization of species resources.     

Can orchid mycorrhizal fungi be persistently harbored by the plant host?

The environmental distribution of non-obligate orchid mycorrhizal (OM) symbionts belonging to the ‘rhizoctonia’ complex remains elusive. Some of these fungi, indeed, are undetectable in soil outside the host rhizosphere. A manipulation experiment was performed to assess the importance of neighbouring non-orchid plants and soil as possible reservoirs of OM fungi for Spiranthes spiralis, a widespread photosynthetic European terrestrial orchid species. Fungi of S. spiralis roots were identified by DNA metabarcoding before and 4 months after the removal of the surrounding vegetation and soil. Although such a treatment significantly affected fungal colonization of newly-formed orchid roots, most OM fungi were consistently associated with the host roots. Frequency patterns in differently aged roots suggest that these fungi colonize new orchid roots from either older roots or other parts of the same plant, which may thus represent an environmental source for the subsequent establishment of the OM symbiosis.