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.

Do fungal associates of co-occurring orchids promote seed germination of the widespread orchid species Gymnadenia conopsea?

Interactions with mycorrhizal fungi have been increasingly recognized as one of the most important ecological factors determining the distribution and local abundance of orchids. While some orchid species may interact with a variety of fungal associates, others are more specific in their choice of mycorrhizal partners. Moreover, orchids that co-occur at a given site, often associate with different partners, possibly to avoid competition and to allow stable coexistence. However, whether differences in mycorrhizal partners directly affect seed germination and subsequent protocorm formation remains largely unknown. In this research, we used in vitro germination experiments to investigate to what extent seed germination and protocorm formation of Gymnadenia conopsea was affected by the origin and identity of fungal associates. Fungi were isolated from G. conopsea and three other co-occurring orchid species (Dactylorhiza viridis (Coeloglossum viride), Herminium monorchis, and Platanthera chlorantha). In total, eight fungal associates, belonging to Tulasnellaceae, Ceratobasidiaceae, and Serendipitaceae, were successfully isolated and cultured. While all eight fungal strains were able to promote early germination of G. conopsea seeds, only fungal strain GS2, a member of the Ceratobasidiaceae isolated from G. conopsea itself, was able to promote protocorm formation and subsequent growth to a seedling. Two other fungal strains isolated from G. conopsea only supported seed germination until the protocorm formation stage. The other five fungal strains isolated from the co-occurring orchid species did not support seed germination beyond the protocorm stage. We conclude that, although G. conopsea is considered a mycorrhizal generalist that associates with a wide range of fungi during its adult life, it requires specific fungi to promote protocorm formation and growth to a seedling.

Are fungi from adult orchid roots the best symbionts at germination? A case study.

We studied mycobionts from advanced seedlings and adult mycorrhizal roots of the terrestrial orchid Arundina graminifolia. Fungi were isolated, identified by ITS sequencing, and tested for their impact on seed germination, protocorm formation, and development of advanced seedlings (emergence of first leaf) in vitro. Among the six fungal species isolated, four were not standard orchid mycorrhizal fungi (Fusarium solani, Cylindrocarpon sp., Acremonium sp., and Phlebiopsis flavidoalba) and did not support germination beyond imbibition and greening of the seeds during a span of 35 days. Over the same time, one Tulasnella species isolated from adult mycorrhiza allowed protocorm formation but not further development. However, another Tulasnella species isolated from advanced seedlings facilitated development to the advanced seedling stage. Our results support (i) the inability of occasional orchid root colonizers to support late seed germination, and (ii) the growing literature showing that fungal associates can change over orchid development. Functionally, we show that mycorrhizal taxa isolated from advanced seedlings can be more efficient than those from adults in supporting germination in some species, leading to recommendations for ex situ orchid conservation.

Pyrola japonica, a partially mycoheterotrophic Ericaceae, has mycorrhizal preference for russulacean fungi in central Japan.

Mycorrhizal symbiosis often displays low specificity, except for mycoheterotrophic plants that obtain carbon from their mycorrhizal fungi and often have higher specificity to certain fungal taxa. Partially mycoheterotrophic (or mixotrophic, MX) plant species tend to have a larger diversity of fungal partners, e.g., in the genus Pyrola (Monotropoideae, Ericaceae). Preliminary evidence however showed that the Japanese Pyrola japonica has preference for russulacean fungi based on direct sequencing of the fungal internal transcribed spacer (ITS) region from a single site. The present study challenges this conclusion using (1) sampling of P. japonica in different Japanese regions and forest types and (2) fungal identification by ITS cloning. Plants were sampled from eight sites in three regions, in one of which the fungal community on tree ectomycorrhizal (ECM) tips surrounding P. japonica was also analyzed. In all, 1512 clone sequences were obtained successfully from 35 P. japonica plants and 137 sequences from ECM communities. These sequences were collectively divided into 74 molecular operational taxonomic units (MOTUs) (51 and 33 MOTUs, respectively). MOTUs from P. japonica involved 36 ECM taxa (96 % of all clones), and 17 of these were Russula spp. (76.2 % of all clones), which colonized 33 of the 35 sampled plants. The MOTU composition significantly differed between P. japonica and ECM tips, although shared species represented 26.3 % of the ECM tips community in abundance. This suggests that P. japonica has a preference for russulacean fungi.

Progress and Prospects of Mycorrhizal Fungal Diversity in Orchids.

Orchids form mycorrhizal symbioses with fungi in natural habitats that affect their seed germination, protocorm growth, and adult nutrition. An increasing number of studies indicates how orchids gain mineral nutrients and sometime even organic compounds from interactions with orchid mycorrhizal fungi (OMF). Thus, OMF exhibit a high diversity and play a key role in the life cycle of orchids. In recent years, the high-throughput molecular identification of fungi has broadly extended our understanding of OMF diversity, revealing it to be a dynamic outcome co-regulated by environmental filtering, dispersal restrictions, spatiotemporal scales, biogeographic history, as well as the distribution, selection, and phylogenetic spectrum width of host orchids. Most of the results show congruent emerging patterns. Although it is still difficult to extend them to all orchid species or geographical areas, to a certain extent they follow the "everything is everywhere, but the environment selects" rule. This review provides an extensive understanding of the diversity and ecological dynamics of orchid-fungal association. Moreover, it promotes the conservation of resources and the regeneration of rare or endangered orchids. We provide a comprehensive overview, systematically describing six fields of research on orchid-fungal diversity: the research methods of orchid-fungal interactions, the primer selection in high-throughput sequencing, the fungal diversity and specificity in orchids, the difference and adaptability of OMF in different habitats, the comparison of OMF in orchid roots and soil, and the spatiotemporal variation patterns of OMF. Further, we highlight certain shortcomings of current research methodologies and propose perspectives for future studies. This review emphasizes the need for more information on the four main ecological processes: dispersal, selection, ecological drift, and diversification, as well as their interactions, in the study of orchid-fungal interactions and OMF community structure.