Variation in mycorrhizal diversity and specificity within the Orchidaceae is of special interest considering the intimate involvement of orchid mycorrhizal fungi (OMF) in germination and development of individuals. The nature of orchid mycorrhizal symbiosis also impacts conservation efforts including in vitro and ex situ culture of orchids, and habitat restoration although the diversity and specificity of OMF in these environments remains poorly understood. Further, the succession of OMF communities during the phenological development of plants is not known but can impact the interpretation and application of orchid-fungal symbiosis. We present the first comparison of OMF communities within the roots of asymbiotically cultured plants growing in ex situ conditions and those occurring naturally in situ across three developmental stages. The temperate terrestrial orchid taxon Platanthera chapmaniiwas used to test the hypothesis that plants generated via asymbiotic germination methods and subsequently cultured ex situ will have a different suite of OMF in comparison to plants occurring in situ and that OMF communities in plants emerging from dormancy, during vegetative phase, and during anthesis will be distinct. Nuclear ribosomal internal transcribed spacer (nrITS) region was used for mycorrhizal fungal identification from roots collected during seven sampling events between 2012 and 2015.
From seven sampling events across three years, 120 sequences were generated from 32 plants which clustered into 12 operational taxonomic units (OTUs). Eleven OTUs represented fungal family Tulasnellaceae while one belonged to Ceratobasidiaceae. Shannon-Wiener (H) and Simpson diversity (D) indices were similar (H, p=0.31; D, p=0.31) for both ex situ and in situ mycorrhizal communities, and across the three phenological stages (H, p=0.36; D, p=0.36). Permutational Multivariate ANOVA (PERMANOVA) based on Bray-Curtis index showed differences in OTU abundances between in situ and ex situ environments (p=0.02), whereas no differences were detected in OTU abundances either among the seven individual sampling events (p=1.00) or among the three phenological stages (p=0.91). Bayesian and Maximum Likelihood (ML) phylograms clustered ex situ and in situ environment derived fungal OTUs into the same clades. Our data suggest that: 1) P. chapmanii is specific toward fungi from the Tulasnellaceae in both ex situ and in situ environments, 2) its preferred OTUs exhibit narrow phylogenetic breadth and are restricted to a few narrow clades, and 3) phenological stage of the plants is unrelated to mycorrhizal specificity in either growing environment.