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Background and Aims: The earliest-diverging orchid lineage, Apostasioideae, consists only of two genera: Apostasia and Neuwiedia. Previous reports of Apostasia nipponica indicated a symbiotic association with an ectomycorrhiza-forming Ceratobasidiaceae clade and partial utilization of fungal carbon during the adult stage. However, the trophic strategy of Neuwiedia throughout its development remains unidentified. To further improve our understanding of mycoheterotrophy in the Apostasioideae, this study focused on Neuwiedia malipoensis examining both the mycorrhizal association and the physiological ecology of this orchid species across various development stages., Methods: We identified the major mycorrhizal fungi of N. malipoensis protocorm, leafy seedling and adult stages using molecular barcoding. To reveal nutritional resources utilized by N. malipoensis, we compared stable isotope natural abundances (δ13C, δ15N, δ2H, δ18O) of different developmental stages with those of autotrophic reference plants., Key Results: Protocorms exhibited an association with saprotrophic Ceratobasidiaceae rather than ectomycorrhiza-forming Ceratobasidiaceae and the 13C signature was characteristic of their fully mycoheterotrophic nutrition. Seedlings and adults were predominantly associated with saprotrophic fungi belonging to the Tulasnellaceae. While 13C and 2H stable isotope data revealed partial mycoheterotrophy of seedlings, it is unclear to what extent the fungal carbon supply is reduced in adult N. malipoensis. However, the 15N enrichment of mature N. malipoensis suggests partially mycoheterotrophic nutrition. Our data indicated a transition in mycorrhizal partners during ontogenetic development with decreasing dependency of N. malipoensis on fungal nitrogen and carbon., Conclusions: The divergence in mycorrhizal partners between N. malipoensis and A. nipponica indicates different resource acquisition strategies and allows various habitat options in the earliest-diverging orchid lineage, Apostasioideae. While A. nipponica relies on the heterotrophic carbon gain from its ectomycorrhizal fungal partner and thus on forest habitats, N. malipoensis rather relies on own photosynthetic carbon gain as an adult, allowing it to establish in habitats as widely distributed as those where Rhizoctonia fungi occur., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background and Aims: Many terrestrial orchids have an obligate dependence on their mycorrhizal associations for nutrient acquisition, particularly during germination and early seedling growth. Though important in plant growth and development, phosphorus (P) nutrition studies in mixotrophic orchids have been limited to only a few orchid species and their fungal symbionts. For the first time, we demonstrate the role of a range of fungi in the acquisition and transport of inorganic P to four phylogenetically distinct green-leaved terrestrial orchid species (Diuris magnifica, Disa bracteata, Pterostylis sanguinea and Microtis media subsp. media) that naturally grow in P-impoverished soils., Methods: Mycorrhizal P uptake and transfer to orchids was determined and visualized using agar microcosms with a diffusion barrier between P source (33P orthophosphate) and orchid seedlings, allowing extramatrical hyphae to reach the source., Key Results: Extramatrical hyphae of the studied orchid species were effective in capturing and transporting inorganic P into the plant. Following 7 d of exposure, between 0.5 % (D. bracteata) and 47 % (D. magnifica) of the P supplied was transported to the plants (at rates between 0.001 and 0.097 fmol h-1). This experimental approach was capable of distinguishing species based on their P-foraging efficiency, and highlighted the role that fungi play in P nutrition during early seedling development., Conclusions: Our study shows that orchids occurring naturally on P-impoverished soils can obtain significant amounts of inorganic P from their mycorrhizal partners, and significantly more uptake of P supplied than previously shown in other green-leaved orchids. These results provide support for differences in mycorrhiza-mediated P acquisition between orchid species and fungal symbionts in green-leaved orchids at the seedling stage. The plant-fungus combinations of this study also provide evidence for plant-mediated niche differentiation occurring, with ecological implications in P-limited systems., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: As in most land plants, the roots of orchids (Orchidaceae) associate with soil fungi. Recent studies have highlighted the diversity of the fungal partners involved, mostly within Basidiomycotas. The association with a polyphyletic group of fungi collectively called rhizoctonias (Ceratobasidiaceae, Tulasnellaceae and Serendipitaceae) is the most frequent. Yet, several orchid species target other fungal taxa that differ from rhizoctonias by their phylogenetic position and/or ecological traits related to their nutrition out of the orchid roots (e.g. soil saprobic or ectomycorrhizal fungi). We offer an evolutionary framework for these symbiotic associations., Scope: Our view is based on the 'Waiting Room Hypothesis', an evolutionary scenario stating that mycorrhizal fungi of land flora were recruited from ancestors that initially colonized roots as endophytes. Endophytes biotrophically colonize tissues in a diffuse way, contrasting with mycorrhizae by the absence of morphological differentiation and of contribution to the plant's nutrition. The association with rhizoctonias is probably the ancestral symbiosis that persists in most extant orchids, while during orchid evolution numerous secondary transitions occurred to other fungal taxa. We suggest that both the rhizoctonia partners and the secondarily acquired ones are from fungal taxa that have broad endophytic ability, as exemplified in non-orchid roots. We review evidence that endophytism in non-orchid plants is the current ecology of many rhizoctonias, which suggests that their ancestors may have been endophytic in orchid ancestors. This also applies to the non-rhizoctonia fungi that were secondarily recruited by several orchid lineages as mycorrhizal partners. Indeed, from our review of the published literature, they are often detected, probably as endophytes, in extant rhizoctonia-associated orchids., Conclusion: The orchid family offers one of the best documented examples of the 'Waiting Room Hypothesis': their mycorrhizal symbioses support the idea that extant mycorrhizal fungi have been recruited among endophytic fungi that colonized orchid ancestors., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Aims: The aim of this study was to assess the effects of beneficial micro-organisms on the growth, nutrient accumulation and root-associated fungal species composition of pot orchids grown in the greenhouse., Methods and Results: A greenhouse pot experiment was conducted to investigate the beneficial effects of a mycorrhizal fungus, Epulorhiza repens isolate ML01, an endophytic fungus, Umbelopsis nana isolate ZH3A-3 and a mixed commercial inoculum Rem, alone or in combination. Nested PCR assays showed that both isolates ML01 and ZH3A-3 can successfully establish in inoculated soil. All the inoculants significantly increased the plant total dry weight of Cymbidium hybridum 'Golden Boy', whereas only co-inoculation with the endophytic fungus ZH3A-3 and the Rem enhanced the fresh weight and height of host plants. The mycorrhizal fungus positively affected P, K, Ca, Mg content in shoots and Zn content in roots, while the endophytic fungus improved N, P, Ca accumulation in shoots and roots. Co-inoculation with the Rem and ML01 improved root to shoot translocation of Fe and Zn. In addition, inoculation with ZH3A-3, ML01+Rem and ZH3A-3+Rem decreased the relative frequency of Fusarium sp. on orchid roots. Trichoderma sp. were isolated from the roots treated with ML01, ML01+Rem and ZH3A-3+Rem., Conclusions: Both mycorrhizal and endophytic fungi had the potential to create favourable microflora in the orchid roots and stimulate the growth of transplanted plantlets under greenhouse condition., Significance and Impact of the Study: The newly isolated endophytic strain ZH3A-3 showed significant application value in orchid production., (© 2020 The Society for Applied Microbiology.)

Background and Aims: For symbiotic organisms, their colonization and spread across remote oceanic islands should favour generalists. Plants that form obligate symbiotic associations with microbes dominate island ecosystems, but the relationship between island inhabitance and symbiotic specificity is unclear, especially in the tropics. To fill this gap, we examined the mycorrhizal specificity of the Hawaiian endemic orchid Anoectochilus sandvicensis across multiple populations encompassing its entire geographic distribution., Methods: By molecular phylogenetic approaches we identified the mycorrhizal fungi associated with A. sandvicensis across its entire geographic distribution and determined the relationship of these fungi to others found elsewhere around the globe. With richness estimators, we assessed the mycorrhizal specificity of A. sandvicensis within and among islands. We then tested whether geographic proximity of orchid populations was a significant predictor for the presence of particular mycorrhizal fungi and their community composition., Key Results: We found that each population of A. sandvicensis forms specific associations with one of three fungi in the genus Ceratobasidium and that the closest relatives of these fungi are globally widespread. Based on diversity indices, A. sandvicensis populations were estimated to partner with one to four mycorrhizal taxa with an estimated total of four compatible mycorrhizal fungi across its entire distribution. However, the geographic proximity of orchid populations was not a significant predictor of mycorrhizal fungal community composition., Conclusions: Our findings indicate that the colonization and survival of plant species on even the most remote oceanic islands is not restricted to symbiotic generalists, and that partnering with few, but cosmopolitan microbial symbionts is an alternative means for successful island establishment. We suggest that the spatial distribution and abundance of symbionts in addition to island age, size and isolation should also be taken into consideration for predictions of island biodiversity., (© The Author(s) 2018. Published by Oxford University Press on behalf of the Annals of Botany Company.)

Background and Aims: An understanding of mycorrhizal variation, orchid seed germination temperature and the effect of co-occurring plant species could be critical for optimizing conservation translocations of endangered plants with specialized mycorrhizal associations., Methods: Focusing on the orchid Thelymitra epipactoides, we isolated mycorrhizal fungi from ten plants within each of three sites; Shallow Sands Woodland (SSW), Damp Heathland (DH) and Coastal Heathland Scrub (CHS). Twenty-seven fungal isolates were tested for symbiotic germination under three 24 h temperature cycles: 12 °C for 16 h-16 °C for 8 h, 16 °C for 16 h-24 °C for 8 h or 27 °C constant. Fungi were sequenced using the internal transcribed spacer (ITS), nuclear large subunit 1 (nLSU1), nLSU2 and mitochondrial large rRNA gene (mtLSU). Orchids were grown to maturity and co-planted with each of ten associated plant species in a glasshouse experiment with tuber width measured at 12 months after co-planting., Key Results: Two Tulasnella fungal lineages were isolated and identified by phylogenetic analyses, operational taxonomic unit 1 (OTU1) and 'T. asymmetrica'. Fungal lineages were specific to sites and did not co-occur. OTU1 (from the SSW site) germinated seed predominantly at 12-16 °C (typical of autumn-winter temperature) whereas 'T. asymmetrica' (from the DH and CHS sites) germinated seed across all three temperature ranges. There was no difference in the growth of adult orchids germinated with different OTUs. There was a significant reduction in tuber size of T. epipactoides when co-planted with six of the commonly co-occurring plant species., Conclusions: We found that orchid fungal lineages and their germination temperature can change with habitat, and established that translocation sites can be optimized with knowledge of co-occurring plant interactions. For conservation translocations, particularly under a changing climate, we recommend that plants should be grown with mycorrhizal fungi tailored to the recipient site.

Background and Aims: Wildfires are common in seasonally dry parts of the world with a Mediterranean climate. Prescribed burning is used to reduce fuel load and fire risk, but often without reliable information on its effects. This study investigated the effects of prescribed burns in different seasons on Pterostylis revoluta, an autumn-flowering Australian terrestrial orchid, and its orchid mycorrhizal fungi (OMFs) to find the least damaging season for a prescribed burn., Methods: Burns were conducted mid-season in spring and summer 2011 and autumn and winter 2012. Orchids were enumerated and measured during their flowering season in autumn 2011-2014 and mycorrhizal fungi were isolated before and after the burns in autumn 2011, 2012 and 2014. Micro-organisms isolated were characterized. DNA was extracted from the OMFs, and the internal transcribed spacer region was amplified by PCR. Amplicons were clustered by restriction fragment length polymorphism (RFLP), and representative amplicons were sequenced. OMF were tested for sensitivity to smoke water., Key Results: The number of plants increased up to 4-fold and 90 % of plants became vegetative during this study. Isolation of mycorrhizal fungi increased and isolation of bacteria decreased. Before the burns, the main OMF isolated was unexpectedly Tulasnella calospora (Boud.) Juel. By 2014, after the burns, the expected Ceratobasidium sp. D.P. Rogers was the only OMF isolated in most burnt quadrats, whereas T. calospora was confined to a minority of unburnt 'control' and the 'spring burn' quadrats, which were also the only ones with flowering plants., Conclusions: The decline in rainfall during 2010-2012 probably caused the switch from mainly flowering to mainly vegetative plants and the change in OMFs. Burning in spring to summer was less damaging to this orchid than burning in autumn to winter, which should be noted by authorities in fire management plans for fire-prone areas in which this orchid occurs., (© The Author(s) 2018. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background and Aims: Partially mycoheterotrophic plants are enriched in 13 C and 15 N compared to autotrophic plants. Here, it is hypothesized that the type of mycorrhizal fungi found in orchid roots is responsible for variation in 15 N enrichment of leaf tissue in partially mycoheterotrophic orchids., Methods: The genus Epipactis was used as a case study and carbon and nitrogen isotope abundances of eight Epipactis species, fungal sporocarps of four Tuber species and autotrophic references were measured. Mycorrhizal fungi were identified using molecular methods. Stable isotope data of six additional Epipactis taxa and ectomycorrhizal and saprotrophic basidiomycetes were compiled from the literature., Key Results: The 15 N enrichment of Epipactis species varied between 3·2 ± 0·8 ‰ ( E. gigantea ; rhizoctonia-associated) and 24·6 ± 1·6 ‰ ( E. neglecta ; associated with ectomycorrhizal ascomycetes). Sporocarps of ectomycorrhizal ascomycetes (10·7 ± 2·2 ‰) were significantly more enriched in 15 N than ectomycorrhizal (5·2 ± 4·0 ‰) and saprotrophic basidiomycetes (3·3 ± 2·1 ‰)., Conclusions: As hypothesized, it is suggested that the observed gradient in 15 N enrichment of Epipactis species is strongly driven by 15 N abundance of their mycorrhizal fungi; i.e. ɛ 15 N in Epipactis spp. associated with rhizoctonias < ɛ 15 N in Epipactis spp. with ectomycorrhizal basidiomycetes < ɛ 15 N in Epipactis spp. with ectomycorrhizal ascomycetes and basidiomycetes < ɛ 15 N in Epipactis spp. with ectomycorrhizal ascomycetes., (© The Author 2017. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com)

Background and Aims: The endangered leafless ghost orchid, Dendrophylax lindenii, one of the most renowned orchids in the world, is difficult to grow under artificial conditions. Published information on asymbiotic and symbiotic (co-culture with a mycobiont) seed germination, seedling anatomy and developmental morphology of this leafless orchid is completely lacking. This information is critical for the development of efficient procedures for ghost orchid production for successful reintroduction., Methods: Ghost orchid seedling early development stages were morphologically and anatomically defined to compare germination, embryo and protocorm maturation and seedling development during asymbiotic and symbiotic culture with one of two mycorrhizal strains (Dlin-379 and Dlin-394) isolated from ghost orchid roots in situ KEY RESULTS: Seeds symbiotically germinated at higher rates when cultured with fungal strain Dlin-394 than with strain Dlin-379 or asymbiotically on P723 medium during a 10-week culture period. Fungal pelotons were observed in protocorm cells co-cultured with strain Dlin-394 but not Dlin-379. Some 2-year-old seedlings produced multinode inflorescences in vitro Production of keikis from inflorescence nodes indicated the capacity for clonal production in the ghost orchid., Conclusions: Ghost orchid embryo and seedling development were characterized into seven stages. Fungal strain Dlin-394 was confirmed as a possible ghost orchid germination mycobiont, which significantly promoted seed germination and seedling development. Internal transcribed spacer sequencing data confirmed that Dlin-394 belongs within the genus Ceratobasidium These results offer the opportunity to examine the benefits of using a mycobiont to enhance in vitro germination and possibly ex vitro acclimatization and sustainability following outplanting., (© The Author 2016. Published by Oxford University Press on behalf of the Annals of Botany Company.)

Species of the Fusarium fujikuroi species complex (FFC) cause a wide spectrum of often devastating diseases on diverse agricultural crops, including coffee, fig, mango, maize, rice, and sugarcane. Although species within the FFC are difficult to distinguish by morphology, and their genes often share 90% sequence similarity, they can differ in host plant specificity and life style. FFC species can also produce structurally diverse secondary metabolites (SMs), including the mycotoxins fumonisins, fusarins, fusaric acid, and beauvericin, and the phytohormones gibberellins, auxins, and cytokinins. The spectrum of SMs produced can differ among closely related species, suggesting that SMs might be determinants of host specificity. To date, genomes of only a limited number of FFC species have been sequenced. Here, we provide draft genome sequences of three more members of the FFC: a single isolate of F. mangiferae, the cause of mango malformation, and two isolates of F. proliferatum, one a pathogen of maize and the other an orchid endophyte. We compared these genomes to publicly available genome sequences of three other FFC species. The comparisons revealed species-specific and isolate-specific differences in the composition and expression (in vitro and in planta) of genes involved in SM production including those for phytohormome biosynthesis. Such differences have the potential to impact host specificity and, as in the case of F. proliferatum, the pathogenic versus endophytic life style., (© The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Background and Aims: Mycoheterotrophy entails plants meeting all or a portion of their carbon (C) demands via symbiotic interactions with root-inhabiting mycorrhizal fungi. Ecophysiological traits of mycoheterotrophs, such as their C stable isotope abundances, strongly correlate with the degree of species' dependency on fungal C gains relative to C gains via photosynthesis. Less explored is the relationship between plant evolutionary history and mycoheterotrophic plant ecophysiology. We hypothesized that the C and nitrogen (N) stable isotope compositions, and N concentrations of fully and partially mycoheterotrophic species differentiate them from autotrophs, and that plant family identity would be an additional and significant explanatory factor for differences in these traits among species. We focused on mycoheterotrophic species that associate with ectomycorrhizal fungi from plant families Ericaceae and Orchidaceae., Methods: Published and unpublished data were compiled on the N concentrations, C and N stable isotope abundances (δ(13)C and δ(15)N) of fully (n = 18) and partially (n = 22) mycoheterotrophic species from each plant family as well as corresponding autotrophic reference species (n = 156). These data were used to calculate site-independent C and N stable isotope enrichment factors (ε). Then we tested for differences in N concentration, (13)C and (15)N enrichment among plant families and trophic strategies., Key Results: We found that in addition to differentiating partially and fully mycoheterotrophic species from each other and from autotrophs, C and N stable isotope enrichment also differentiates plant species based on familial identity. Differences in N concentrations clustered at the plant family level rather than the degree of dependency on mycoheterotrophy., Conclusions: We posit that differences in stable isotope composition and N concentrations are related to plant family-specific physiological interactions with fungi and their environments., (© The Author 2016. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background and Aims: Mycorrhizal associations are influenced by abiotic and biotic factors, including climate, soil conditions and the identity of host plants. However, the effect of environmental conditions on orchid mycorrhizal associations remains poorly understood. The present study examined how differences in soil nutrient availability are related to the diversity and composition of mycorrhizal fungi associated with two terrestrial orchid species from central Chile., Methods: For 12 populations of Bipinnula fimbriata and B. plumosa, OTU (operational taxonomic unit) richness, phylogenetic diversity and community composition of mycorrhizal fungi in root samples were estimated using internal transcribed spacer (ITS) sequences. Then, these mycorrhizal diversity variables were related to soil nutrients and host species using generalized linear models and non-metric multidimensional scaling., Key Results: Variation in OTU composition of mycorrhizal fungi among sites was explained mainly by orchid host species. Fungi in Tulasnellaceae and Ceratobasidiaceae were isolated from both orchid species, but the former were more frequent in B. fimbriata and the latter in B. plumosa. Soil nutrients and orchid host species had significant effects on OTU richness and phylogenetic diversity. Mycorrhizal diversity decreased in habitats with higher N in both species and increased with P availability only in B. fimbriata, Conclusions: The results suggest that soil nutrient availability modulates orchid mycorrhizal associations and provide support for the hypothesis that specialization is favoured by higher soil nutrient availability. Inter-specific differences in mycorrhizal composition can arise due to a geographical pattern of distribution of orchid mycorrhizal fungi, host preferences for fungal partners or differential performance of mycorrhizal fungi under different nutrient availabilities. Further experiments are needed to evaluate these hypotheses., (© The Author 2016. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background and Aims: In orchid species that have populations occurring in strongly contrasting habitats, mycorrhizal divergence and other habitat-specific adaptations may lead to the formation of reproductively isolated taxa and ultimately to species formation. However, little is known about the mycorrhizal communities associated with recently diverged sister taxa that occupy different habitats., Methods: In this study, 454 amplicon pyrosequencing was used to investigate mycorrhizal communities associating with Epipactis helleborine in its typical forest habitat and with its presumed sister species E. neerlandica that almost exclusively occurs in coastal dune habitats. Samples of the phylogenetically more distant E. palustris, which co-occurred with E. neerlandica, were also included to investigate the role of habitat-specific conditions on mycorrhizal communities., Results: A total of 105 operational taxonomic units (OTUs) of putative orchid mycorrhizal fungi were observed in the three studied species. The majority of these fungi were endophytic fungi of Helotiales and ectomycorrhizal fungi belonging to Thelephoraceae, Sebacinaceae and Inocybaceae. In addition, a large number of other ectomycorrhizal taxa were detected, including Cortinarius, Cenococcum, Tuber, Geopora, Wilcoxina, Meliniomyces, Hebeloma, Tricholoma, Russula and Peziza Mycorrhizal communities differed significantly between the three species, but differences were most pronounced between the forest species (E. helleborine) and the two dune slack species (E. neerlandica and E. palustris)., Conclusion: The results clearly showed that recently diverged orchid species that occupy different habitats were characterized by significantly different mycorrhizal communities and call for more detailed experiments that aim at elucidating the contribution of habitat-specific adaptations in general and mycorrhizal divergence in particular to the process of speciation in orchids., (© The Author 2016. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: Seedling recruitment is essential to the sustainability of any plant population. Due to the minute nature of seeds and early-stage seedlings, orchid germination in situ was for a long time practically impossible to observe, creating an obstacle towards understanding seedling site requirements and fluctuations in orchid populations. The introduction of seed packet techniques for sowing and retrieval in natural sites has brought with it important insights, but many aspects of orchid seed and germination biology remain largely unexplored., Key Considerations: The germination niche for orchids is extremely complex, because it is defined by requirements not only for seed lodging and germination, but also for presence of a fungal host and its substrate. A mycobiont that the seedling can parasitize is considered an essential element, and a great diversity of Basidiomycota and Ascomycota have now been identified for their role in orchid seed germination, with fungi identifiable as imperfect Rhizoctonia species predominating. Specificity patterns vary from orchid species employing a single fungal lineage to species associating individually with a limited selection of distantly related fungi. A suitable organic carbon source for the mycobiont constitutes another key requirement. Orchid germination also relies on factors that generally influence the success of plant seeds, both abiotic, such as light/shade, moisture, substrate chemistry and texture, and biotic, such as competitors and antagonists. Complexity is furthermore increased when these factors influence seeds/seedling, fungi and fungal substrate differentially., Conclusions: A better understanding of germination and seedling establishment is needed for conservation of orchid populations. Due to the obligate association with a mycobiont, the germination niches in orchid species are extremely complex and varied. Microsites suitable for germination can be small and transient, and direct observation is difficult. An experimental approach using several levels of environmental manipulation/control is recommended., (© The Author 2015. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background and Aims: Although mycorrhizal associations are predominantly generalist, specialized mycorrhizal interactions have repeatedly evolved in Orchidaceae, suggesting a potential role in limiting the geographical range of orchid species. In particular, the Australian orchid flora is characterized by high mycorrhizal specialization and short-range endemism. This study investigates the mycorrhizae used by Pheladenia deformis, one of the few orchid species to occur across the Australian continent. Specifically, it examines whether P. deformis is widely distributed through using multiple fungi or a single widespread fungus, and if the fungi used by Australian orchids are widespread at the continental scale., Methods: Mycorrhizal fungi were isolated from P. deformis populations in eastern and western Australia. Germination trials using seed from western Australian populations were conducted to test if these fungi supported germination, regardless of the region in which they occurred. A phylogenetic analysis was undertaken using isolates from P. deformis and other Australian orchids that use the genus Sebacina to test for the occurrence of operational taxonomic units (OTUs) in eastern and western Australia., Key Results: With the exception of one isolate, all fungi used by P. deformis belonged to a single fungal OTU of Sebacina. Fungal isolates from eastern and western Australia supported germination of P. deformis. A phylogenetic analysis of Australian Sebacina revealed that all of the OTUs that had been well sampled occurred on both sides of the continent., Conclusions: The use of a widespread fungal OTU in P. deformis enables a broad distribution despite high mycorrhizal specificity. The Sebacina OTUs that are used by a range of Australian orchids occur on both sides of the continent, demonstrating that the short-range endemism prevalent in the orchids is not driven by fungal species with narrow distributions. Alternatively, a combination of specific edaphic requirements and a high incidence of pollination by sexual deception may explain biogeographic patterns in southern Australian orchids., (© The Author 2015. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background and Aims: Many terrestrial orchids have an obligate requirement for mycorrhizal associations to provide nutritional support from germination to establishment. This study will investigate the ability of orchid mycorrhizal fungi (OMF) to utilize a variety of nutrient sources in the nutrient-impoverished (low organic) soils of the Southwest Australian Floristic Region (SWAFR) in order to effectively compete, survive and sustain the orchid host., Methods: Mycorrhizal fungi representing key OMF genera were isolated from three common and widespread species: Pterostylis recurva, Caladenia flava and Diuris corymbosa, and one rare and restricted species: Drakaea elastica. The accessibility of specific nutrients was assessed by comparing growth including dry biomass of OMF in vitro on basal CN MMN liquid media., Key Results: Each of the OMF accessed and effectively utilized a wide variety of nutrient compounds, including carbon (C) sources, inorganic and organic nitrogen (N) and inorganic and organic phosphorus (P). The nutrient compounds utilized varied between the genera of OMF, most notably sources of N., Conclusions: These results suggest that OMF can differentiate between niches (micro-niche specialization) in a constrained, highly resource-limited environment such as the SWAFR. Phosphorus is the most limited macronutrient in SWAFR soils and the ability to access phytate by OMF indicates a characterizing functional capacity of OMF from the SWAFR. Furthermore, compared with OMF isolated from the rare D. elastica, OMF associating with the common P. recurva produced far greater biomass over a wider variety of nutritional sources. This suggests a broader tolerance for habitat variation providing more opportunities for the common orchid for recruitment and establishment at a site.

Background and Aims: Orchid mycorrhizas exhibit a unique type of mycorrhizal symbiosis that occurs between fungi and plants of the family Orchidaceae. In general, the roots of orchids are typically coarse compared with those of other plant species, leading to a considerably low surface area to volume ratio. As a result, orchids are often ill-adapted for direct nutrient acquisition from the soil and so mycorrhizal associations are important. However, the role of the fungal partners in the acquisition of inorganic and organic N by terrestrial orchids has yet to be clarified., Methods: Inorganic and amino acid N uptake by non-mycorrhizal and mycorrhizal Cymbidium goeringii seedlings, which were grown in pots in a greenhouse, was investigated using a (15)N-labelling technique in which the tracer was injected at two different soil depths, 2·5 cm or 7·5 cm. Mycorrhizal C. goeringii seedlings were obtained by inoculation with three different mycorrhizal strains isolated from the roots of wild terrestrial orchids (two C. goeringii and one C. sinense)., Key Results: Non-mycorrhizal C. goeringii primarily took up NO3(-) from tracers injected at 2·5-cm soil depth, whereas C. goeringii inoculated with all three mycorrhiza primarily took up NH4(+) injected at the same depth. Inoculation of the mycorrhizal strain MLX102 (isolated from adult C. sinense) on C. goeringii roots only significantly increased the below-ground biomass of the C. goeringii; however, it enhanced (15)NH4(+) uptake by C. goeringii at 2·5-cm soil depth. Compared to the uptake of tracers injected at 2·5-cm soil depth, the MLX102 fungal strain strongly enhanced glycine-N uptake by C. goeringii from tracers injected at 7·5-cm soil depth. Cymbidium goeringii inoculated with CLB113 and MLX102 fungal strains demonstrated a similar N uptake pattern to tracers injected at 2·5-cm soil depth., Conclusions: These findings demonstrate that mycorrhizal fungi are able to switch the primary N source uptake of a terrestrial orchid, in this case C. goeringii, from NO3(-) to NH4(+). The reasons for variation in N uptake in the different soil layers may be due to possible differentiation in the mycorrhizal hyphae of the C. goeringii fungal partner.

Background and Aims: Mycorrhizal specialization has been shown to limit recruitment capacity in orchids, but an increasing number of orchids are being documented as invasive or weed-like. The reasons for this proliferation were examined by investigating mycorrhizal fungi and edaphic correlates of Microtis media, an Australian terrestrial orchid that is an aggressive ecosystem and horticultural weed., Methods: Molecular identification of fungi cultivated from M. media pelotons, symbiotic in vitro M. media seed germination assays, ex situ fungal baiting of M. media and co-occurring orchid taxa (Caladenia arenicola, Pterostylis sanguinea and Diuris magnifica) and soil physical and chemical analyses were undertaken., Key Results: It was found that: (1) M. media associates with a broad taxonomic spectrum of mycobionts including Piriformospora indica, Sebacina vermifera, Tulasnella calospora and Ceratobasidium sp.; (2) germination efficacy of mycorrhizal isolates was greater for fungi isolated from plants in disturbed than in natural habitats; (3) a higher percentage of M. media seeds germinate than D. magnifica, P. sanguinea or C. arenicola seeds when incubated with soil from M. media roots; and (4) M. media-mycorrhizal fungal associations show an unusual breadth of habitat tolerance, especially for soil phosphorus (P) fertility., Conclusions: The findings in M. media support the idea that invasive terrestrial orchids may associate with a diversity of fungi that are widespread and common, enhance seed germination in the host plant but not co-occurring orchid species and tolerate a range of habitats. These traits may provide the weedy orchid with a competitive advantage over co-occurring orchid species. If so, invasive orchids are likely to become more broadly distributed and increasingly colonize novel habitats.

Background and Aims: The maintenance of species boundaries in sympatric populations of closely related species requires some kind of reproductive isolation that limits gene flow among species and/or prevents the production of viable progeny. Because in orchids mycorrhizal fungi are needed for seed germination and subsequent seedling establishment, orchid-mycorrhizal associations may be involved in acting as a post-mating barrier., Methods: We investigated the strength of post-mating barriers up to the seed germination stage acting between three closely related Orchis species (Orchis anthropophora, O. militaris and O. purpurea) and studied the role of mycorrhizal fungi in hybridization by burying seed packets of pure and hybrid seeds. After retrieval and assessment of seed germination, the fungi associating with protocorms originating from hybrid and pure seeds were determined and compared with those associating with adult individuals using DNA array technology., Results: Whereas pre-zygotic post-mating barriers were rather weak in most crosses, post-zygotic post-mating barriers were stronger, particularly when O. purpurea was crossed with O. anthropophora. Germination trials in the field showed that seed germination percentages of hybrid seeds were in most cases lower than those originating from pure crosses. In all species pair combinations, total post-mating reproductive isolation was asymmetric. Protocorms associated with a smaller range of fungal symbionts than adult plants, but there was considerable overlap in mycorrhizal associations between protocorms and their respective parents., Conclusions: Our results suggest that mycorrhizal associations contribute little to reproductive isolation. Pre-mating barriers are probably the main factors determining hybridization rates between the investigated species.

Background and Aims: Nutritional changes associated with the evolution of achlorophyllous, mycoheterotrophic plants have not previously been inferred with robust phylogenetic hypotheses. Variations in heterotrophy in accordance with the evolution of leaflessness were examined using a chlorophyllous-achlorophyllous species pair in Cymbidium (Orchidaceae), within a well studied phylogenetic background., Methods: To estimate the level of mycoheterotrophy in chlorophyllous and achlorophyllous Cymbidium, natural (13)C and (15)N contents (a proxy for the level of heterotrophy) were measured in four Cymbidium species and co-existing autotrophic and mycoheterotrophic plants and ectomycorrhizal fungi from two Japanese sites., Key Results: δ(13)C and δ(15)N values of the achlorophyllous C. macrorhizon and C. aberrans indicated that they are full mycoheterotrophs. δ(13)C and δ(15)N values of the chlorophyllous C. lancifolium and C. goeringii were intermediate between those of reference autotrophic and mycoheterotrophic plants; thus, they probably gain 30-50 % of their carbon resources from fungi. These data suggest that some chlorophyllous Cymbidium exhibit partial mycoheterotrophy (= mixotrophy)., Conclusions: It is demonstrated for the first time that mycoheterotrophy evolved after the establishment of mixotrophy rather than through direct shifts from autotrophy to mycoheterotrophy. This may be one of the principal patterns in the evolution of mycoheterotrophy. The results also suggest that the establishment of symbiosis with ectomycorrhizal fungi in the lineage leading to mixotrophic Cymbidium served as pre-adaptation to the evolution of the mycoheterotrophic species. Similar processes of nutritional innovations probably occurred in several independent orchid groups, allowing niche expansion and radiation in Orchidaceae, probably the largest plant family.

Aims: To develop and evaluate a new and reliable real-time PCR detection protocol on chromosomal DNA of the contagious plant pathogenic bacterium Erwinia amylovora, the causal agent of fire blight., Methods and Results: A Taqman minor-groove-binder real-time PCR assay targeting a hypothetical protein coding gene of Erw. amylovora has been developed. Colony PCR of 113 bacterial strains from different taxa was performed to prove specificity. Serial decimal dilutions of Erw. amylovora showed a consistent detection sensitivity of 2 bacterial units per microl. All strains of Erw. amylovora could be identified, and there were no cross-reactions with matrices or other bacteria also testing naturally contaminated samples., Conclusions: Rapid, reliable and sensitive detection of Erw. amylovora is important to avoid the spread of the disease within orchards, and the distribution by contaminated plant material or vectors carrying the pathogen. The selected conserved target gene allows relative quantitative detection of Erw. amylovora from different sources and host taxa. The newly developed protocol also enables the detection of recently found natural strains that lack the species-specific plasmid pEA29, which was so far widely used as target for detection and identification of this plant pathogen by PCR., Significance and Impact of the Study: This study demonstrates that the newly developed and evaluated real-time assay can specifically be used for identifying all known strains of the EU quarantine plant pathogen Erw. amylovora. Low concentrations of the bacteria can be detected and relatively quantified using a different target area than other real-time PCRs designed so far.

Background and Aims: Mycorrhizal associations are essential to the plant kingdom. The largest flowering plant family, the Orchidaceae, relies on mycorrhizal fungi for germination, growth and survival. Evidence suggests varying degrees of fungal-host specificity based on a single fungal isolate from a single plant. This paper shows for the first time the diversity of endophytes colonizing in a single plant over consecutive years and the functional significance of this diversity., Methods: Stem-collars of Caladenia formosa were collected in different seasons and years. Mycorrhizal fungi isolated were tested for their efficacy to induce leafing and genetically determined using ITS-RFLP and sequencing., Results: Multiple mycorrhizal fungi were repeatedly isolated from a single collar that displayed varying effectiveness in germination percentages and adult leaf length. Additional factors contributed to the isolation of effective mycorrhizal fungi; fungal collection season, year of collection and individual isolates. Surface sterilization only improved the number of isolated mycorrhizal fungi. Dual inoculation did not increase germination. All 59 mycorrhizal fungi effective in germinating seed belonged to one clearly defined ITS (internal transcribed spacer) clade and clustered close to Sebacina vermifera (79-89 % homology). Isolates resulting in the greatest germination were not necessarily those resulting in the greatest survival and growth 1 year after germination., Conclusion: Single orchid plants contained multiple mycorrhizal fungal strains of one species that had diverse functional differences. These results suggest that our current knowledge of fungal-host specificity may be incomplete due to experimental and analytical limitations. It also suggests that the long-term effectiveness of a mycorrhizal fungus or fungi could only be found by germination and longer-term growth tests rather than genetically.

Background and Aims: Epipogium aphyllum is a Eurasian achlorophyllous, mycoheterotrophic forest orchid. Due to its rarity, it is often protected, and its biology is poorly known. The identity and pattern of colonization of fungal associates providing carbon to this orchid have not been studied previously., Methods: Using samples from 34 individuals from 18 populations in Japan, Russia and France, the following were investigated: (a) colonization patterns of fungal associates of E. aphyllum by microscopy; (b) their identity by PCR amplification of nuclear ribosomal ITS carried out on rhizome fragments and hyphal pelotons., Results and Conclusions: Microscopic investigations revealed that thick rhizomes were densely colonized by fungi bearing clamp-connections and dolipores, i.e. basidiomycetes. Molecular analysis identified Inocybe species as exclusive symbionts of 75 % of the plants investigated and, more rarely, other basidiomycetes (Hebeloma, Xerocomus, Lactarius, Thelephora species). Additionally, ascomycetes, probably endophytes or parasites, were sometimes present. Although E. aphyllum associates with diverse species from Inocybe subgenera Mallocybe and Inocybe sensu stricto, no evidence for cryptic speciation in E. aphyllum was found. Since basidiomycetes colonizing the orchid are ectomycorrhizal, surrounding trees are probably the ultimate carbon source. Accordingly, in one population, ectomycorrhizae sampled around an individual orchid revealed the same fungus on 11.2 % of tree roots investigated. Conversely, long, thin stolons bearing bulbils indicated active asexual multiplication, but these propagules were not colonized by fungi. These findings are discussed in the framework of ecology and evolution of mycoheterotrophy.

Orchidaceae are the largest family of flowering plants, with at least 24,000 species, and perhaps better than any other family of flowering plants, orchids represent the extreme specializations that are possible. As a result, they have long fascinated luminaries of the botanical world including Linnaeus and Darwin, but the size of the family has historically been an impediment to their study. Specifically, the lack of detailed information about relationships within the family made it difficult to formulate explicit evolutionary hypotheses for such a large group, but the advent of molecular systematics has revolutionized our understanding of the orchids. Their complex life histories make orchids particularly vulnerable to environmental change, and as result many are now threatened with extinction. In this Special Issue we present a series of 20 papers on orchid biology ranging from phylogenetics, floral evolutionary development, taxonomy, mycorrhizal associations, pollination biology, population genetics and conservation.

Background: Conservation through reserves alone is now considered unlikely to achieve protection of plant species necessary to mitigate direct losses of habitat and the pervasive impact of global climate change. Assisted translocation/migration represent new challenges in the face of climate change; species, particularly orchids, will need artificial assistance to migrate from hostile environments, across ecological barriers (alienated lands such as farmlands and built infrastructure) to new climatically buffered sites. The technology and science to underpin assisted migration concepts are in their infancy for plants in general, and orchids, with their high degree of rarity, represent a particularly challenging group for which these principles need to be developed. It is likely that orchids, more than any other plant family, will be in the front-line of species to suffer large-scale extinction events as a result of climate change., Scope: The South West Australian Floristic Region (SWAFR) is the only global biodiversity hotspot in Australia and represents an ideal test-bed for development of orchid conservation principles. Orchids comprise 6 % of all threatened vascular plants in the SWAFR, with 76 out of the 407 species known for the region having a high level of conservation risk. The situation in the SWAFR is a portent of the global crisis in terrestrial orchid conservation, and it is a region where innovative conservation solutions will be required if the impending wave of extinction is to be averted. Major threatening processes are varied, and include land clearance, salinity, burning, weed encroachment, disease and pests. This is compounded by highly specialized pollinators (locally endemic native invertebrates) and, in the most threatened groups such as hammer orchids (Drakaea) and spider orchids (Caladenia), high levels of mycorrhizal specialization. Management and development of effective conservation strategies for SWAFR orchids require a wide range of integrated scientific approaches to mitigate impacts that directly influence ecological traits critical for survival., Conclusions: In response to threats to orchid species, integrated conservation approaches have been adopted (including ex situ and translocation principles) in the SWAFR with the result that a significant, multidisciplinary approach is under development to facilitate conservation of some of the most threatened taxa and build expertise to carry out assisted migration to new sites. Here the past two decades of orchid conservation research in the SWAFR and the role of research-based approaches for managing effective orchid conservation in a global biodiversity hotspot are reviewed.

Several key characteristics of the species-rich orchid family are due to its symbiotic relationships with pollinators and mycorrhizal fungi. The majority of species are insect pollinated and show strong adaptations for outcrossing, such as pollination by food- and sexual-deception, and all orchids are reliant on mycorrhizal fungi for successful seedling establishment. Recent studies of orchid pollination biology have shed light on the barriers to reproductive isolation important to diversification in different groups of deceptive orchids. Molecular identification of orchid mycorrhizal fungi has revealed high fungal specificity in orchids that obtain organic nutrients from fungi as adults. Both pollinator and fungal specificity have been proposed as drivers of orchid diversification. Recent findings in orchid pollination and mycorrhizal biology are reviewed and it is shown that both associations are likely to affect orchid distribution and population structure. Integrating studies of these symbioses will shed light on the unparalleled diversification of the orchid family.

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 and Aims: Mycorrhizal fungi play a vital role in providing a carbon subsidy to support the germination and establishment of orchids from tiny seeds, but their roles in adult orchids have not been adequately characterized. Recent evidence that carbon is supplied by Goodyera repens to its fungal partner in return for nitrogen has established the mutualistic nature of the symbiosis in this orchid. In this paper the role of the fungus in the capture and transfer of inorganic phosphorus (P) to the orchid is unequivocally demonstrated for the first time., Methods: Mycorrhiza-mediated uptake of phosphorus in G. repens was investigated using spatially separated, two-dimensional agar-based microcosms., Results: External mycelium growing from this green orchid is shown to be effective in assimilating and transporting the radiotracer (33)P orthophosphate into the plant. After 7 d of exposure, over 10 % of the P supplied was transported over a diffusion barrier by the fungus and to the plants, more than half of this to the shoots., Conclusions: Goodyera repens can obtain significant amounts of P from its mycorrhizal partner. These results provide further support for the view that mycorrhizal associations in some adult green orchids are mutualistic.