Untangling above‐ and belowground mycorrhizal fungal networks in tropical orchids

Orchids typically depend on fungi for establishment from seeds, forming mycorrhizal associations with basidiomycete fungal partners in the polyphyletic group rhizoctonia from early stages of germination, sometimes with very high specificity. This has raised important questions about the roles of plant and fungal phylogenetics, and their habitat preferences, in controlling which fungi associate with which plants. In this issue of Molecular Ecology, Martos et al. (2012) report the largest network analysis to date for orchids and their mycorrhizal fungi, sampling a total of over 450 plants from nearly half the 150 tropical orchid species on Reunion Island, encompassing its main terrestrial and epiphytic orchid genera. The authors found a total of 95 operational taxonomic units of mycorrhizal fungi and investigated the architecture and nestedness of their bipartite networks with 73 orchid species. The most striking finding was a major ecological barrier between above‐ and belowground mycorrhizal fungal networks, despite both epiphytic and terrestrial orchids often associating with closely related taxa across all three major lineages of rhizoctonia fungi. The fungal partnerships of the epiphytes and terrestrial species involved a diversity of fungal taxa in a modular network architecture, with only about one in ten mycorrhizal fungi partnering orchids in both groups. In contrast, plant and fungal phylogenetics had weak or no effects on the network. This highlights the power of recently developed ecological network analyses to give new insights into controls on plant–fungal symbioses and raises exciting new hypotheses about the differences in properties and functioning of mycorrhiza in epiphytic and terrestrial orchids.     

The impact of life form on the architecture of orchid mycorrhizal networks in tropical forest

Understanding the processes that determine the architecture of interaction networks represents a major challenge in ecology and evolutionary biology. One of the most important interactions involving plants is the interaction between plants and mycorrhizal fungi. While there is a mounting body of research that has studied the architecture of plant–fungus interaction networks, less is known about the potential factors that drive network architecture. In this study, we described the architecture of the network of interactions between mycorrhizal fungi and 44 orchid species that represented different life forms and co‐occurred in tropical forest and assessed the relative importance of ecological, evolutionary and co‐evolutionary mechanisms determining network architecture. We found 87 different fungal operational taxonomic units (OTUs), most of which were members of the Tulasnellaceae. Most orchid species associated with multiple fungi simultaneously, indicating that extreme host selectivity was rare. However, an increasing specificity towards Tulasnellaceae fungal associates from terrestrial to epiphytic and lithophytic orchids was observed. The network of interactions showed an association pattern that was significantly modular (M = 0.7389, M_random = 0.6998) and nested (NODF = 5.53, p < 0.05). Terrestrial orchids had almost no links to modules containing epiphytic or lithophytic orchids, while modules containing epiphytic orchids also contained lithophytic orchids. Within each life form several modules were observed, suggesting that the processes that organize orchid–fungus interactions are independent of life form. The overall phylogenetic signal for both partners in the interaction network was very weak. Overall, these results indicate that tropical orchids associate with a wide number of mycorrhizal fungi and that ecological rather than phylogenetic constraints determine network architecture.     

Mycorrhizal symbioses found in roots of fern and its relatives in Korea

Mycorrhizal symbioses were found in the roots of 45 out of 59 species of pteridophytes collected in Korea. The mycorrhizal fungi were colonized in the root cortical cells, primarily in terrestrial species, but rarely in epiphytic or aquatic pteridophytes. Mycorrhizae that are typically found in orchid colonized the roots of the epiphytic pteridophytes, but not in other species. These were the first observations of orchid mycorrhizae in pteridophytes. Arbuscular mycorrhizal fungi were examined after staining, then confirmed with PCR, using a specific primer. This is the first report of arbuscular mycorrhizal colonization in the roots of pteridophyte species in Asia.     

Mycorrhizal Fungi Promote Growth and Nitrogen Utilization by Dendrobium nobile (Orchidaceae)

Mycorrhizal associations play a key role in the life cycle and evolutionary history of orchids. Although most orchid species are tropical and epiphytic, their mycorrhizae are poorly understood compared with those of temperate, terrestrial orchids. To investigate the influences of such fungi on photosynthetic, epiphytic orchids, we inoculated seedlings of Dendrobium nobile with Epulorhiza sp. (S1) or Tulasnella sp. (S3). These fungi had been identified based on their morphological and molecular characters. Both S1 and S3 formed symbiotic associations with our seedlings, promoting their growth and development to various degrees. Results from signature experiments with the 15N stable isotope suggested that the utilization of organic nitrogen by orchid seedlings was significantly improved by S1, but not by S3. Dendrobine contents were significantly higher in all inoculated seedlings. Our findings demonstrate that these mycorrhizal fungi enhance plant growth, their utilization of organic nitrogen, and the accumulation of secondary metabolites in this epiphytic orchid species.     

Influence of mycorrhizal fungi on seed germination and growth in terrestrial and epiphytic orchids

Epiphytes constitute over 70% of orchid diversity, but little is known about the functioning of their mycorrhizal associations. Terrestrial orchid seeds germinate symbiotically in soil and leaf litter, whereas epiphytic orchids may be exposed to relatively high light levels from an early stage of development and often produce green seeds. This suggests that seedlings of the two groups of orchids may differ in their responses to light and requirements for mycorrhiza-supplied carbon. The interactive effects of light, exogenous carbon and mycorrhizal status on germination and growth were investigated in vitro using axenic agar microcosms for one tropical epiphyte and three geophytic orchid species. The geophytic species strongly depended on their mycorrhiza for growth and this could not be substituted by exogenous sucrose, whereas the epiphytic species achieved 95% of the mycorrhizal seedling volume when supplied with exogenous sucrose in the dark. Mycorrhiza status strongly interacted with light exposure, enabling germination. Light inhibited or severely reduced growth, especially for the terrestrial orchids in the absence of mycorrhiza. For the first time, this study showed the parallel ecological importance of mycorrhizal fungi in overcoming light inhibition of seed germination and growth in both terrestrial and epiphytic orchids.     

菌根真菌促进金钗石斛的生长及氮利用

菌根在兰科的生命周期和进化史上起着关键作用。兰科中大多数是附生兰,但它们的菌根研究相对缺乏。为了探讨菌根对附生兰的影响,本研究用金钗石斛（Dendrobium nobile）与通过形态学特征和分子生物学鉴定的分属于瘤菌根菌属（Epulorhiza）的s1和胶膜菌属（Tulasnella）的S3真菌共培养。共培养结果表明,S1和S3与金钗石斛形成了共生关系,且不同程度地促进了其生长。15N稳定同位素标记实验证实,S1菌株显著促进了金钗石斛对有机氮的利用,而S3菌株没有显著的促进作用。同时．S1和S3真菌均能提高金钗石斛中石斛碱的含量。研究结果表明,菌根真菌能促进附生兰幼苗的生长、有机氮的利用和次生代射产物的积累。     

Atractiellomycetes belonging to the ‘rust’ lineage (Pucciniomycotina) form mycorrhizae with terrestrial and epiphytic neotropical orchids

Distinctive groups of fungi are involved in the diverse mycorrhizal associations of land plants. All previously known mycorrhiza-forming Basidiomycota associated with trees, ericads, liverworts or orchids are hosted in Agaricomycetes, Agaricomycotina. Here we demonstrate for the first time that Atractiellomycetes, members of the ‘rust’ lineage (Pucciniomycotina), are mycobionts of orchids. The mycobionts of 103 terrestrial and epiphytic orchid individuals, sampled in the tropical mountain rainforest of Southern Ecuador, were identified by sequencing the whole ITS1-5.8S-ITS2 region and part of 28S rDNA. Mycorrhizae of 13 orchid individuals were investigated by transmission electron microscopy. Simple septal pores and symplechosomes in the hyphal coils of mycorrhizae from four orchid individuals indicated members of Atractiellomycetes. Molecular phylogeny of sequences from mycobionts of 32 orchid individuals out of 103 samples confirmed Atractiellomycetes and the placement in Pucciniomycotina, previously known to comprise only parasitic and saprophytic fungi. Thus, our finding reveals these fungi, frequently associated to neotropical orchids, as the most basal living basidiomycetes involved in mycorrhizal associations of land plants.     

Highly diverse and spatially heterogeneous mycorrhizal symbiosis in a rare epiphyte is unrelated to broad biogeographic or environmental features

Symbiotic interactions are common in nature. In dynamic or degraded environments, the ability to associate with multiple partners (i.e. broad specificity) may enable species to persist through fluctuations in the availability of any particular partner. Understanding how species interactions vary across landscapes is necessary to anticipate direct and indirect consequences of environmental degradation on species conservation. We asked whether mycorrhizal symbiosis by populations of a rare epiphytic orchid (Epidendrum firmum) is related to geographic or environmental heterogeneity. The latter would suggest that interactions are governed by environmental conditions rather than historic isolation of populations and/or mycorrhizal fungi. We used DNA-based methods to identify mycorrhizal fungi from eleven E. firmum populations in Costa Rica. We used molecular and phylogenetic analyses to compare associations. Epidendrum firmum exhibited broad specificity, associating with diverse mycorrhizal fungi, including six Tulasnellaceae molecular operational taxonomic units (MOTUs), five Sebacinales MOTUs and others. Notably, diverse mycorrhizal symbioses formed in disturbed pasture and roadside habitats. Mycorrhizal fungi exhibited significant similarity within populations (spatial and phylogenetic autocorrelation) and significant differences among populations (phylogenetic community dissimilarity). However, mycorrhizal symbioses were not significantly associated with biogeographic or environmental features. Such unexpected heterogeneity among populations may result from complex combinations of fine-scale environmental factors and macro-evolutionary patterns of change in mycorrhizal specificity. Thus, E. firmum exhibits broad specificity and the potential for opportunistic associations with diverse fungi. We suggest that these characteristics could confer symbiotic assurance when mycorrhizal fungi are stochastically available, which may be crucial in dynamic or disturbed habitats such as tropical forest canopies.     

Evolution of host breadth in broad interactions: mycorrhizal specificity in East Asian and North American rattlesnake plantains (Goodyera spp.) and their fungal hosts

Host breadth is often assumed to have no evolutionary significance in broad interactions because of the lack of cophylogenetic patterns between interacting species. Nonetheless, the breadth and suite of hosts utilized by one species may have adaptive value, particularly if it underlies a common ecological niche among hosts. Here, we present a preliminary assessment of the evolution of mycorrhizal specificity in 12 closely related orchid species (genera Goodyera and Hetaeria) using DNA‐based methods. We mapped specificity onto a plant phylogeny that we estimated to infer the evolutionary history of the mycorrhiza from the plant perspective, and hypothesized that phylogeny would explain a significant portion of the variance in specificity of plants on their host fungi. Sampled plants overwhelmingly associated with genus Ceratobasidium, but also occasionally with some ascomycetes. Ancestral mycorrhizal specificity was narrow in the orchids, and broadened rarely as Goodyera speciated. Statistical tests of phylogenetic inertia suggested some support for specificity varying with increasing phylogenetic distance, though only when the phylogenetic distance between suites of fungi interacting with each plant taxon were taken into account. These patterns suggest a role for phylogenetic conservatism in maintaining suits of fungal hosts among plants. We stress the evolutionary importance of host breadth in these organisms, and suggest that even generalists are likely to be constrained evolutionarily to maintaining associations with their symbionts.     

Diversity and host specificity of endophytic Rhizoctonia-like fungi from tropical orchids

All orchids have an obligate relationship with mycorrhizal symbionts. Most orchid mycorrhizal fungi are classified in the form-genus Rhizoctonia. This group includes anamorphs of Tulasnella, Ceratobasidium, and Thanatephorus. Rhizoctonia can be classified according to the number of nuclei in young cells (multi-, bi-, and uninucleate). From nine Puerto Rican orchids we isolated 108 Rhizoctonia-like fungi. Our isolates were either bi- or uninucleate, the first report of uninucleate Rhizoctonia-like fungi as orchid endophytes. We sequenced the internal transcribed spacer (ITS) region of nuclear ribosomal DNA from 26 isolates and identified four fungal lineages, all related to Ceratobasidium spp. from temperate regions. Most orchid species hosted more than one lineage, demonstrating considerable variation in mycorrhizal associations even among related orchid species. The uninucleate condition was not a good phylogenetic character in mycorrhizal fungi from Puerto Rico. All four lineages were represented by fungi from Tolumnia variegata, but only one lineage included fungi from Ionopsis utricularioides. Tropical epiphytic orchids appear to vary in degree of specificity in their mycorrhizal interactions more than previously thought.     

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.     

Studies of Mycorrhizal Fungi of Chinese Orchids and Their Role in Orchid Conservation in China—A Review

China has over 1,200 species of native orchids in nearly 173 genera. About one fourth of native species are of horticultural merit. Some species are of Chinese medicinal value. In fact, the demand on orchid species with high Chinese medicinal values such as Gastrodia elata, Dendrobium offcinale, along with demands on species of cultural importance, such as those in the genus of Cymbidium, is a major factor causing wild populations to diminish and in some cases, drive wild populations to the brink of extinction. These market demands have also driven studies on the role of mycorrhizal fungi in orchid seed germination, seedling and adult growth, and reproduction. Most of these mycorrhizal studies of Chinese orchids, however, are published in Chinese, some in medical journals, and thus overlooked by the mainstream orchid mycorrhizal publications. Yet some of these studies contained interesting discoveries on the nature of the mycorrhizal relationships between orchids and fungi. We present a review of some of these neglected publications. The most important discovery comes from the mycorrhizal studies on G. elata, in which the researchers concluded that those fungi species required to stimulate seed germination are different from those that facilitate the growth of G. elata beyond seedling stages. In addition, presence of the mycorrhizal fungi associated with vegetative growth of post-seedling G. elata hindered the germination of seeds. These phenomena were unreported prior to these studies. Furthermore, orchid mycorrhizal studies in China differ from the mainstream orchid studies in that many epiphytic species (in the genus of Dendrobium, as medicinal herbs) were investigated as well as terrestrial orchids (mostly in the genus Cymbidium, as traditional horticultural species). The different responses between epiphytic and terrestrial orchid seeds to fungi derived from roots suggest that epiphytic orchids may have a more general mycorrhizal relationship with fungi than do terrestrial orchid species during the seed germination stage. To date, orchid mycorrhizal research in China has had a strongly commercial purpose. We suggest that this continuing research on orchid mycorrhizal relationships are a solid foundation for further research that includes more rare and endangered taxa, and more in-situ studies to assist conservation and restoration of the endangered orchids. Knowledge on the identities and roles of mycorrhizal fungi of orchids holds one of the keys to successful restoration and sustainable use of Chinese orchids.     

Fungal symbioses in hornworts: a chequered history

Hornworts are considered the sister group to vascular plants, but their fungal associations remain largely unexplored. The ancestral symbiotic condition for all plants is, nonetheless, widely assumed to be arbuscular mycorrhizal with Glomeromycota fungi. Owing to a recent report of other fungi in some non-vascular plants, here we investigate the fungi associated with diverse hornworts worldwide, using electron microscopy and molecular phylogenetics. We found that both Glomeromycota and Mucoromycotina fungi can form symbioses with most hornworts, often simultaneously. This discovery indicates that ancient terrestrial plants relied on a wider and more versatile symbiotic repertoire than previously thought, and it highlights the so far unappreciated ecological and evolutionary role of Mucoromycotina fungi.     

Analysis of network architecture reveals phylogenetic constraints on mycorrhizal specificity in the genus Orchis (Orchidaceae)

The specificity of orchids for their fungi can vary substantially, from highly specialist interactions to more generalist interactions, but little is known about the evolutionary history of the mycorrhizal specificity of orchids. Here, we used a network analysis approach to investigate orchid mycorrhizal associations in 16 species of the genus Orchis sampled across 11 different regions in Europe. We first examined in detail the structure of the network of associations and then tested for a phylogenetic signal in mycorrhizal specificity and identified the fungi with which the orchids associated. We found 20 different fungal lineages that associated with species of the genus Orchis, most of them being related to members of the Tulasnellaceae (84.33% of all identified associations) and a smaller proportion being related to members of the Ceratobasidiaceae (9.97%). Species associations formed a nested network that is built on asymmetric links among species. Evolution of mycorrhizal specificity in Orchis closely resembles a Brownian motion process, and the interaction between Orchis and Tulasnellaceae fungi is significantly influenced by the phylogenetic relationships between the Orchis species. Our results provide evidence of the presence of phylogenetic conservatism in mycorrhizal specificity in orchids and demonstrate that evolutionary processes may be an important factor in generating patterns of mycorrhizal associations.     

Widespread mycorrhizal specificity correlates to mycorrhizal function in the neotropical, epiphytic orchid Ionopsis utricularioides (Orchidaceae)

Tropical orchids constitute the greater part of orchid diversity, but little is known about their obligate mycorrhizal relationships. The specificity of these interactions and associated fungal distributions could influence orchid distributions and diversity. We investigated the mycorrhizal specificity of the tropical epiphytic orchid Ionopsis utricularioides across an extensive geographical range. DNA ITS sequence variation was surveyed in both plants and mycorrhizal fungi. Phylogeographic relationships were estimated for the mycorrhizal fungi. Orchid functional outcomes were determined through in vitro seed germination and seedling growth with a broad phylogenetic representation of fungi. Most fungal isolates derived from one clade of Ceratobasidium (anamorphs assignable to Ceratorhiza), with 78% within a narrower phylogenetic group, clade B. No correlation was found between the distributions of orchid and fungal genotypes. All fungal isolates significantly enhanced seed germination, while fungi in clade B significantly enhanced seedling growth. These results show that I. utricularioides associates with a phylogenetically narrow, effective fungal clade over a broad distribution. This preference for a widespread mycorrhizae may partly explain the ample distribution and abundance of I. utricularioides and contrasts with local mycorrhizal diversification seen in some nonphotosynthetic orchids. Enhanced orchid function with a particular fungal subclade suggests mycorrhizal specificity can increase orchid fitness.     

Strong phylogenetic congruence between Tulasnella fungi and their associated Drakaeinae orchids

The study of congruency between phylogenies of interacting species can provide a powerful approach for understanding the evolutionary history of symbiotic associations. Orchid mycorrhizal fungi can survive independently of orchids making cospeciation unlikely, leading us to predict that any congruence would arise from host-switches to closely related fungal species. The Australasian orchid subtribe Drakaeinae is an iconic group of sexually deceptive orchids that consists of approximately 66 species. In this study, we investigated the evolutionary relationships between representatives of all six Drakaeinae orchid genera (39 species) and their mycorrhizal fungi. We used an exome capture dataset to generate the first well-resolved phylogeny of the Drakaeinae genera. A total of 10 closely related Tulasnella Operational Taxonomic Units (OTUs) and previously described species were associated with the Drakaeinae orchids. Three of them were shared among orchid genera, with each genus associating with 1–6 Tulasnella lineages. Cophylogenetic analyses show Drakaeinae orchids and their Tulasnella associates exhibit significant congruence (p < 0.001) in the topology of their phylogenetic trees. An event-based method also revealed significant congruence in Drakaeinae–Tulasnella relationships, with duplications (35), losses (25), and failure to diverge (9) the most frequent events, with minimal evidence for cospeciation (1) and host-switches (2). The high number of duplications suggests that the orchids speciate independently from the fungi, and the fungal species association of the ancestral orchid species is typically maintained in the daughter species. For the Drakaeinae–Tulasnella interaction, a pattern of phylogenetic niche conservatism rather than coevolution likely explains the observed phylogenetic congruency in orchid and fungal phylogenies. Given that many orchid genera are characterized by sharing of fungal species between closely related orchid species, we predict that these findings may apply to a wide range of orchid lineages.     

Mycorrhizal fungi of Vanilla: diversity, specificity and effects on seed germination and plant growth

Mycorrhizal fungi are essential for the germination of orchid seeds. However, the specificity of orchids for their mycorrhizal fungi and the effects of the fungi on orchid growth are controversial. Mycorrhizal fungi have been studied in some temperate and tropical, epiphytic orchids, but the symbionts of tropical, terrestrial orchids are still unknown. Here we study diversity, specificity and function of mycorrhizal fungi in Vanilla, a pantropical genus that is both terrestrial and epiphytic. Mycorrhizal roots were collected from four Vanilla species in Puerto Rico, Costa Rica and Cuba. Cultured and uncultured mycorrhizal fungi were identified by sequencing the internal transcribed spacer region of nuclear rDNA (nrITS) and part of the mitochondrial ribosomal large subunit (mtLSU), and by counting number of nuclei in hyphae. Vanilla spp. were associated with a wide range of mycorrhizal fungi: Ceratobasidium, Thanatephorus and Tulasnella. Related fungi were found in different species of Vanilla, although at different relative frequencies. Ceratobasidium was more common in roots in soil and Tulasnella was more common in roots on tree bark, but several clades of fungi included strains from both substrates. Relative frequencies of genera of mycorrhizal fungi differed significantly between cultured fungi and those detected by direct amplification. Ceratobasidium and Tulasnella were tested for effects on seed germination of Vanilla and effects on growth of Vanilla and Dendrobium plants. We found significant differences among fungi in effects on seed germination and plant growth. Effects of mycorrhizal fungi on Vanilla and Dendrobium were similar: a clade of Ceratobasidium had a consistently positive effect on plant growth and seed germination. This clade has potential use in germination and propagation of orchids. Results confirmed that a single orchid species can be associated with several mycorrhizal fungi with different functional consequences for the plant.     

菌根真菌在改善附生兰干旱耐受性中的作用：以禾叶贝母兰为例

干旱胁迫是在多种生态系统中影响植物生存、发育及产量的最主要的非生物因素之一。菌根共生已被证明可以提高植物对干旱的耐受性。兰科植物对菌根真菌有非常高的依赖性,但是有关兰科菌根真菌是否可以提高宿主植物的耐旱性以及能提高到什么程度还少有报道。在本研究中,我们检测了一株分离自附生型兰科植物禾叶贝母兰Coelogyne viscosa的胶膜菌属真菌Tullasnella sp. hy-111对宿主植物幼苗生长及耐旱性的影响,并从转录组水平检测了该菌根真菌对禾叶贝母兰幼苗基因表达的影响。结果显示,接种hy-111不仅能显著提高幼苗的生物量、与耐旱相关的酶活性以及渗透调节物质的富集,而且还能显著诱导植物抗性途径相关基因的上调表达。本研究表明菌根真菌能改善生长于胁迫的附生生境中的兰科植物对于干旱的耐受性,并可能在兰科植物的生态适应中起到重要作用。     

Ecological,historical and evolutionary determinants of modularity in weighted seed‐dispersal networks

Modularity is a recurrent and important property of bipartite ecological networks. Although well‐resolved ecological networks describe interaction frequencies between species pairs, modularity of bipartite networks has been analysed only on the basis of binary presence–absence data. We employ a new algorithm to detect modularity in weighted bipartite networks in a global analysis of avian seed‐dispersal networks. We define roles of species, such as connector values, for weighted and binary networks and associate them with avian species traits and phylogeny. The weighted, but not binary, analysis identified a positive relationship between climatic seasonality and modularity, whereas past climate stability and phylogenetic signal were only weakly related to modularity. Connector values were associated with foraging behaviour and were phylogenetically conserved. The weighted modularity analysis demonstrates the dominating impact of ecological factors on the structure of seed‐dispersal networks, but also underscores the relevance of evolutionary history in shaping species roles in ecological communities.     

Rangewide analysis of fungal associations in the fully mycoheterotrophic Corallorhiza striata complex (Orchidaceae) reveals extreme specificity on ectomycorrhizal Tomentella (Thelephoraceae) across North America

Fully mycoheterotrophic plants offer a fascinating system for studying phylogenetic associations and dynamics of symbiotic specificity between hosts and parasites. These plants frequently parasitize mutualistic mycorrhizal symbioses between fungi and trees. Corallorhiza striata is a fully mycoheterotrophic, North American orchid distributed from Mexico to Canada, but the full extent of its fungal associations and specificity is unknown. Plastid DNA (orchids) and ITS (fungi) were sequenced for 107 individuals from 42 populations across North America to identify C. striata mycobionts and test hypotheses on fungal host specificity. Four largely allopatric orchid plastid clades were recovered, and all fungal sequences were most similar to ectomycorrhizal Tomentella (Thelephoraceae), nearly all to T. fuscocinerea. Orchid-fungal gene trees were incongruent but nonindependent; orchid clades associated with divergent sets of fungi, with a clade of Californian orchids subspecialized toward a narrow Tomentella fuscocinerea clade. Both geography and orchid clades were important determinants of fungal association, following a geographic mosaic model of specificity on Tomentella fungi. These findings corroborate patterns described in other fully mycoheterotrophic orchids and monotropes, represent one of the most extensive plant-fungal genetic investigations of fully mycoheterotrophic plants, and have conservation implications for the >400 plant species engaging in this trophic strategy worldwide.