Intraspecific chemical variation within the crustose lichen genus Haematomma: anthraquinone production in selected cultured mycobionts as a response to stress and nutrient supply

The mycobionts isolated from selected species of Haematomma (Haematomma africanum, Haematomma fenzlianum, Haematomma flourescens, Haematomma persoonii, Haematomma stevensiae) have been successfully cultured. The chemical profile of the mycobionts could be effectively influenced and modulated by varying the composition of the nutrient medium using alternative carbohydrates (glucose, sucrose, and polyols). Under artifical laboratory conditions and simulated environmental stress (exposure to UV light, desiccation, and lower temperatures) the mycobionts began producing typical secondary lichen metabolites after an incubation time of 5–6 months. Modified Lilly and Barnett medium (LBM) and Murashige Skoog Medium favoured the production of depsides such as sphaerophorin and isosphaeric acid. Surprisingly, the mycobiont from H. stevensiae in modified MS medium produced two anthraquinones in the mycelia, haematommone at the base and russulone in the upper parts of the mycelium. By contrast, the natural lichen only produced these anthraquinones in the reddish orange apothecia. The mycobiont from H. flourescens only produced the expected lichexanthone in LBM, enriched with the polyols, sorbitol and mannitol. Once the media requirements and environmental stress factors that trigger polyketide production in lichen mycobionts have been determined, it is possible to obtain a particular lichen product by a completely defined procedure. Using such knowledge, we should be able to study polyketide expression in mycobionts under optimized culture conditions for various genetic applications.     

Developmental stages and secondary compound biosynthesis of mycobiont and whole thallus cultures of <Emphasis Type="Italic">Buellia subsororioides</Emphasis>

Lichen species have unique culture media preferences, and established cultures are known for the synthesis of secondary metabolites. This paper reports observations on the developmental stages and secondary compound biosynthesis by the mycobiont and whole thallus cultures of Buellia subsororioides. It also investigates the suitable media compositions for the culture growth, the role (nutrient or stressor) of sucrose concentrations on the growth stages, biomass, secondary compound profiles, and the quantity of biosynthesized known compounds/g of culture biomass for each treatment using mycobiont cultures. The ascospore-derived mycobiont cultures and thallus macerate-derived whole thallus cultures of B. subsororioides were established and grown using malt yeast extract (MY) medium. Mycobiont cultures were subcultured in MY medium supplemented with sucrose and its concentrations ranging from 0 to 30 % (with 2 % increment between treatments) for 120 days. The molecular identity of cultures was confirmed using nuclear ribosomal Internal transcribed spacer (ITS) DNA sequences obtained from the cultured mycobiont and from the natural thallus. The ITS DNA sequences of the mycobiont showed 99 % similarity with the sequences of the natural thallus. The mycobiont cultures under varying sucrose concentrations initiated as white cottony stages and transformed to brown compact mycelia, with optimum biomass and biosynthesis of nine secondary compounds in MY 10 %. The number of compounds (1–9) varies according to treatments. The whole thallus cultures (MY 0 %) showed a profile of secondary compounds similar to that of the natural thalli along with a trace of one unknown compound. The obtained results are encouraging for the synthesis of the desired quantities of lichen secondary compounds through cultures for relevant applications.     

Secondary chemistry of cultured mycobionts: formation of a complete chemosyndrome by the lichen fungus of Lobaria spathulata

Abstract:The study aimed to optimize culture conditions and nutrient requirements for the production of secondary metabolites by the cultured mycobiont Lobaria spathulata. This species proved to be an excellent model system for such studies, as the complete chemosyndrome found in the natural lichen thallus was repeatedly formed in the cultured mycobiont with differentiated, aerial mycelia. Nutrient media containing the disaccharide, sucrose, were found to favour both rapid growth and the production of typical lichen substances. Higher proportions of the secondary compounds were detected in the developing mycobiont than in mature lichen thalli.     

Does aposymbiotically cultivated fungus Ramalina produce isolichenan?

The main α-glucan synthesized by lichens of the genera Ramalina in the symbiotic state is isolichenan. This polysaccharide was not found in the aposymbiotically cultivated symbionts. It is still unknown if this glucan is produced by the mycobiont only in the presence of a photobiont, in a lichen thallus, or if the isolichenan suppression is influenced by the composition of culture medium used in its aposymbiotic cultive. Consequently, the latter hypothesis is tested in this study. Cultures of the mycobiont Ramalina complanata were obtained from germinated ascospores and cultivated on 4% glucose Lilly and Barnett medium. Freeze-dried colonies were defatted and their carbohydrates extracted successively with hot water and aqueous 10% KOH, each at 100 °C. The polysaccharides nigeran, laminaran and galactomannan were liberated, along with a lentinan-type β-glucan and a heteropolysaccharide (Man : Gal : Glc, 21 : 28 : 51). Nevertheless, the α-glucan isolichenan was not found in the extracts. It follows that it was probably a symbiotic product, synthesized by the mycobiont only in this particular microenvironment, in the presence of the photobiont in the lichen thallus. A discussion about polysaccharides found in the symbiotic thallus as well as in other aposymbiotic cultivated Ramalina mycobionts is also included.     

Symbiotic seed germination and evidence for <Emphasis Type="Italic">in vitro</Emphasis> mycobiont specificity in <Emphasis Type="Italic">Spiranthes brevilabris</Emphasis> (Orchidaceae) and its implications for species-level conservation

Orchid–mycobiont specificity in the Orchidaceae was considered controversial and not well understood for many years. Differences in mycobiont specificity during germination in vitro vs in situ have lead some to consider orchid–mycobiont specificity as being generally low; however, others have suggested that specificity, especially in vitro, is surprisingly high. Mycobiont specificity may be genus or species specific. An in vitro symbiotic seed germination experiment was designed to examine mycobiont specificity of the endangered Florida terrestrial orchid Spiranthes brevilabris using mycobionts isolated from both the study species and the endemic congener Spiranthes floridana. In a screen of mycobionts, isolates Sflo-305 (99.5%), Sflo-306 (99.5%), and Sflo-308 (89.9%) (originating from S. floridana) supported higher initial (stage 1) seed germination than isolate Sbrev-266 (32.4%) (originating from S. brevilabris) after 3 wk culture. However, only isolate Sbrev-266 supported advanced germination and protocorm development to stage 5 (53.1%) after 12 wk culture. These findings suggest that S. brevilabris maintains a high degree of mycobiont specificity under in vitro symbiotic seed germination conditions. High orchid–mycobiont specificity in S. brevilabris may be indicative of the rare status of this orchid in Florida.     

Two new guaiane-sesquiterpenes from the cultured lichen mycobiont of Diorygma pruinosum

Lichens and the mycobionts derived from lichen-source are believed to be a valuable source of bioactive compounds. Diorygma pruinosum is a native lichen in Vietnam. A mycobiont of Diorygma pruinosum was separated, then cultivated. Phytochemical data on this lichen and its cultured mycobionts are scarce. The present study described the isolation and structural elucidation of two new guaiane-type sesquiterpenes, namely pruinosone (1) and hydroxypruinosone (2). Their absolute chemical structures were elucidated by extensive 1D and 2D NMR analysis, high-resolution mass spectroscopy, electronic circular dichroism (ECD), and comparisons in the literatures. Compounds 1 and 2 were evaluated for alpha-glucosidase inhibition and antimicrobial activity.     

Callus induction and high-efficiency plant regeneration via somatic embryogenesis in <Emphasis Type="Italic">Papaver nudicaule</Emphasis> L., an ornamental medicinal plant

We describe culture conditions for a high-efficiency in vitro regeneration system of Papaver nudicaule through somatic embryogenesis and secondary somatic embryogenesis. The embryogenic callus induction rate was highest when petiole explants were cultured on Murashige and Skoog (MS) medium containing 1.0 mg l⁻¹ α-naphthaleneacetic acid (NAA) and 0.1 mg l⁻¹ 6-benzyladenine (BA) (36.7%). When transferred to plant growth regulator (PGR)-free medium, 430 somatic embryos formed asynchronously from 90 mg of embryogenic callus in each 100-ml flask. Early-stage somatic embryos were transferred to MS medium containing 1.0 mg l⁻¹ BA and 1.0 mg l⁻¹ NAA to germinate at high frequency (97.6%). One-third-strength MS medium with 1.0% sucrose and 1.0 mg l⁻¹ GA₃ had the highest frequency of plantlet conversion from somatic embryos (91.2%). Over 90% of regenerated plantlets were successfully acclimated in the greenhouse. Secondary somatic embryos were frequently induced directly when the excised hypocotyls of the primary somatic embryos were cultured on MS medium without PGRs. Sucrose concentration significantly affected the induction of secondary embryos. The highest induction rate (89.5) and number of secondary somatic embryos per explant (9.3) were obtained by 1% sucrose. Most secondary embryos (87.2–94.3%) developed into the cotyledonary stage on induction medium. All cotyledonary secondary embryos were converted into plantlets both in liquid and on semisolid 1/3-strength MS medium with 1.0% sucrose.     

Recognition mechanisms during the pre-contact state of lichens: I. Mycobiont-photobiont interactions of the mycobiont of Fulgensia bracteata

Lichens are an association of a photoautotrophic alga/cyanobacteria (photobiont) and a heterotrophic fungus (mycobiont) constituting the lichen thallus as a complex phenotype. Many mycobionts reproduce sexually and the ascospores are dispersed without the photobiont. For successful re-lichenization the specific photobiont must be recognized, contacted, and incorporated by the mycobiont. A so-called pre-contact stage has been postulated as the initial step of a gradual recognition process. In the present study, the effect of the specific Trebouxia photobiont, an unspecific Asterochloris photobiont and the non-lichenizing green alga Myrmecia bisecta on the development of the mycobiont Fulgensia bracteata was assessed by pre-contact assays. Three hypotheses were confirmed: (i) the pre-contact stage exists, (ii) it is characterized by morphological reactions in the development of the mycobiont, and (iii) the reactions depend on the interacting alga. Control conditions revealed a mycelial growth arrest but this effect was not observed in the presence of any of the three algae. Different algae induce distinct growth patterns with respect to hyphal length, morphological characteristics, and formation of mucilage. The specific Trebouxia photobiont had a positive impact on hyphal growth, branching frequency, and mucilage formation. These effects were less explicit with the non-specific Asterochloris photobiont. Myrmecia bisecta induced uncharacteristic growth patterns with pronounced hyphal growth and high numbers of aerial hyphae but less formation of mucilage. These results indicate that symbiont recognition mechanisms are established before physical contact. Pre-contact reactions may be an evolutionary advantage that supports the persistence of the mycobiont on newly colonized sites and improves the probability of re-lichenization.     

Growth and triterpenic acid accumulation of Cyclocarya paliurus cell suspension cultures

Cyclocarya paliurus is a unique plant growing in central China with hypoglycaemic and hypolipaemia effects. To make better use of this functional food resource, cell suspension cultures and triterpenic acid accumulation were studied. Stable and uniform cell suspension cultures were established in liquid basal Murashige and Skoog medium supplemented with 2,4-dichlorophenoxy acetic acid (0.5 mg/L), naphthalene acetic acid (0.3 mg/L) and cytokinin (1.0 mg/L). According to the growth curve and triterpenic acid accumulation curve, the 8 ～ 10^th day postinoculation was the optimum time for subculture, and the 14^th day was the optimum time for harvest. Murashige and Skoog medium and woody plant medium were suitable for both cell growth and triterpenic acid accumulation. 3% sucrose (w/v), 60 mM total nitrogen (NO₃ ^?/NH₄ ⁺ = 2/1), 1.25 mM KH₂PO₄, 2 mM CaCl₂, and 2 mM MgSO₄ were all found to be fit for cell growth and triterpenic acid accumulation in a cell suspension culture of Cyclocarya paliurus. Total triterpenic acid, ursolic acid and oleanolic acid content in suspended cultured cells were all significantly higher than that of leaves and calluses (P ? 0.01), with levels up to 6.24, 2.28, and 0.94% (of dry weight), respectively. The betulinic acid content of suspended cultured cells also reached 0.82%, which was significantly higher than that of calluses. These results suggest that suspended cultured cells of Cyclocarya paliurus were rich in triterpenic acids and could be used for the production of total triterpenic acid, ursolic acid, oleanolic acid and betulinic acid.     

GROWTH STUDIES OF THREE CHLOROPHYLLOUS CALLUS PHENOTYPES OF GLYCINE MAX

The establishment of three chlorophyllous callus phenotypes, Glycine max (L.) Merrill, cultured on a modified Miller's medium is described. Experiments were designed to determine the hormonal requirement necessary to maintain an adequate callus growth rate that would allow for the phenotypical accumulation of chlorophyll in all phenotypes. Addition of α-naphthaleneacetic acid and kinetin, both at 1 mg/liter, to the basal medium fulfilled this requirement. However, callus growth for these phenotypes required only an exogenous supply of cytokinin. All callus phenotypes, when maintained on 3% sucrose, were shown to possess similar growth curves; however, optimal growth rates of these cultured phenotypes occurred on different levels of exogenous sucrose (NG, 2%; LG, 1%; Y, 2%). Sucrose (filtered and autoclaved) and, in most cases, fructose (filtered), when employed as a carbon source in the basal medium, maintained adequate growth rates. Glucose (filtered) supported only minimal callus growth. These callus phenotypes, after two years in culture, showed a certain degree of cell type differentiation as indicated by the formation of isolated tracheoids and in some cases organized tracheoid development. The chromosome complement (2n = 40) was observed to be polyploid.     

Establishment of Gymnema sylvestre hairy root cultures for the production of gymnemic acid

Gymnema sylvestre is an important medicinal plant that bears bioactive compound namely gymnemic acid. In the present study, G. sylvestre was transformed by Agrobacterium rhizogenes. Seedling explants namely roots, stems, hypocotyls, cotyledonary nodal segments, cotyledons and young leaves were inoculated with A. rhizogenes strain KCTC 2703. Transformed (hairy) roots were induced from cotyledons and leaf explants. Six transgenic clones of hairy roots were established and confirmed by polymerase chain reaction (PCR) and RT-PCR using rolC specific primers. Hairy roots cultured using MS liquid medium supplemented with 3 % sucrose showed highest accumulation of biomass (97.63 g l^?1 FM and 10.92 g l^?1 DM) at 25 days, whereas highest accumulation of gymnemic acid content (11.30 mg g^?1 DM) was observed at 20 days. Nearly 9.4-fold increment of biomass was evident in suspension cultures at 25 days of culture and hairy root biomass produced in suspension cultures possessed 4.7-fold higher gymnemic acid content when compared with the untransformed control roots. MS-based liquid medium was superior for the growth of hairy roots and production of gymnemic acid compared with other culture media evaluated (B5, NN and N6), with MS-based liquid medium supplemented with 3 % sucrose was optimal for secondary metabolite production. The current results showed great potentiality of hairy root cultures for the production of gymnemic acid.     

High frequency of shoot multiplication and bulblet formation of garlic in liquid cultures

In vitro shoot proliferation and bulblet production of garlic (Allium sativum L.) was studied in liquid cultures. Shoots grown in vitro were used as explants and were cultured in MS medium supplemented with 2% (w/v) sucrose and 0.5 mg l^–1 2-iP. Three culture methods (semi-solid, liquid-immersion and raft) were compared for shoot proliferation. Explants in liquid (immersion) culture exhibited an increased multiplication rate and fresh weight of shoots after 3 weeks of culture as compared with the other treatments. Bulblet formation and growth were studied in liquid medium with different concentrations of sucrose (2–13%). MS medium containing 11% (w/v) sucrose was optimal for bulblet development and bulblets developed in this medium within 9 weeks in culture. The highest multiplication rate was (135 bulblets/explant) found when explants were cultured in bulbing medium (MS medium containing 0.1 mg l^–1 NAA+11% (w/v) sucrose) supplemented with 10 M JA. Growth retardants CCC, B-9, ABA also promoted induction and growth of bulblets. Darkness promoted the bulblet induction and growth compared to light conditions (16-h photoperiod of 50 mol m^–2 s^–1). The dormancy of bulblets was broken by cold treatment at 4 °C for 8 weeks.     

Protoplast isolation from cultured lichen <Emphasis Type="Italic">Usnea ghattensis</Emphasis>, their fusion with protoplasts of <Emphasis Type="Italic">Aspergillus nidulans</Emphasis>, fusant regeneration and production of usnic acid

Protoplasts isolated from the mycobiont of a cultured lichen Usnea ghattensis were fused with protoplasts of the fungus Aspergillus nidulans in order to increase the growth rate of the cultured lichen mycobiont in vitro. The maximum protoplast yield (102 × 10⁴/g fresh cell mass) was reached in citrate buffer with 50 mmol/L 2-sulfanylethanol (‘2-mercaptoethanol’) containing 0.1 % Novozym after 1.5 h at pH 5 and ≤25 °C. The increase in the concentration of the above effectors or the addition of others (e.g., MgSO₄) as well as increase in time, shaking frequency, etc. caused the lower yield of protoplasts. The fused protoplasts were regenerated after transfer to malt extract-yeast extract medium and produced, after a 45-d cultivation, a fresh cell mass of 0.232 g (from starting 0.3 g) along with the lichen substance usnic acid.     

Embryogenesis and haploid induction using anther culture in lovage (Levisticum officinale W.D.J. Koch)

An efficient and robust protocol to induce embryogenesis in lovage (Levisticum officinale W.D.J. Koch) has been developed. Immature anthers, with most of the microspores at the late uninucleate stage, were used as explants, and embryogenesis was induced in medium with combinations of plant growth regulators including α-naphthalene acetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), and 6-benzylaminopurine (BAP). The frequencies of in vitro embryogenesis ranged from 0.42 to 18.25% depending on the combinations of plant growth regulators in the induction medium. Induced globular embryos successfully developed into heart and torpedo-staged embryos. Fresh anther explants produced the highest embryo formation rate (17.75%). Anthers treated at 4?ºC for 3, 5, or 8 d, significantly reduced the embryogenic response (to 3.52–7.85%). More embryos were induced when the sucrose content in the medium was increased from 3 to 6% (w/v), but significantly fewer embryos were produced when sucrose was 8% or more. Nearly 20% of fresh anthers were able to produce embryogenic structures when cultured on Murashige and Skoog medium supplemented with 10.74 μM NAA, 8.80 μM BAP, 9.05 μM 2,4-D, and 6% sucrose. Furthermore, when silver nitrate was added to the embryo induction medium at 90 μM, the frequency of anther browning decreased by 30% and the embryo formation rate increased to 24.75% of anthers cultured. In total, 418 plants were regenerated and cytological analysis confirmed 11 haploid lines from 187 samples randomly selected.     

Perfusion strategy for rosmarinic acid production by Anchusa officinalis

The production of an intracellular secondary metabolite rosmarinic acid (RA) by plant cell suspensions of Anchusa officinalis cultivated with intermittent medium exchange is investigated. Initially, a two-stage perfusion culture method was employed. After being cultured in the batch mode for ca. 6 days in B5 medium plus 3% sucrose, 1 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.1 mg/L kinetin (2,4-D B5 medium), Anchusa culture was cultivated to high cell density by perfusion during the growth stage using a hormone-free Gamborg B5 medium supplemented with 6% sucrose. This was followed by a production stage, in which a complete medium exchange into B5 medium plus 3% sucrose and 0.25 mg/L naphthleneacetic acid (NAA) was conducted. The two-stage perfusion culture had a higher maximum culture RA concentration but a lower RA content per cell than the batch stock culture maintained in the 2,4-D B5 medium. Higher culture RA concentration was due primarily to high cell density. The high packed cell volume, however, seemed to reduce the synergistic effect of NAA on RA synthesis. Subsequently, a single-stage perfusion culture method was investigated. The best result was obtained by growing the culture in the batch mode for ca. 10 days using B5 medium supplemented with 3% sucrose and 0.25 mg/L NAA, followed by perfusing the culture with B5 medium plus 6% sucrose and 0.25 mg/L NAA at a constant perfusion rate of 0.1/day. A maximum cell dry weight of 35 g/L and a RA concentration of almost 4 g/L were achieved. This is the highest RA concentration ever reported in the Anchusa culture. (c) 1993 John Wiley & Sons, Inc.     

Recognition mechanisms during the pre-contact state of lichens: II. Influence of algal exudates and ribitol on the response of the mycobiont of Fulgensia bracteata

Successful re-lichenization between the two bionts of the lichen symbiosis, the fungal mycobiont and its specific photobiont, is a process that is not well understood yet. To assess potential signalling between the two bionts during initial pre-contact, exudates of the Trebouxia photobionts of Fulgensia bracteata, Fulgensia fulgens, and Xanthoria elegans, of the Asterochloris photobiont of Lecidea lurida, and of the non-lichenizing green alga Myrmecia bisecta were investigated. The compounds identified in these exudates were tested with respect to their influence on germination and early development of the Fulgensia bracteata mycobiont. Additionally, carbohydrates (glucose, sucrose, ribitol) were tested to appraise their effect on the mycobiont growth patterns. Three hypotheses were confirmed: (i) photobionts exude various substances, (ii) the photobiont exudation pattern varies with the identity of the photobiont, and (iii) a pre-contact influence induces changes in the early development of the mycobiont of F. bracteata. This study gives comparative insight to exudates of lichen photobionts. In vitro photobionts differentially release compounds belonging to several substance classes which include indole-3-carbaldehyde, two cyclic dipeptides, and rhamnose. Two compounds had inhibitory effects on germination and germ-tube growth of the mycobiont and one other enhanced spore germination. Additionally, ribitol was found to elicit a strong effect on the mycobiont’s growth. In general, photobiont-exudation, its effect on the mycobiont, and the response to ribitol suggest that complex pre-contact signalling has a crucial role in lichen biont recognition.     

Generalism in the interaction of Tulasnellaceae mycobionts with orchids characterizes a biodiversity hotspot in the tropical Andes of Southern Ecuador

Biotic interactions play an important role in the assembly and stability of communities. All orchids depend on mycobionts for early establishment, but whether individual orchid species depend on a specific or broad spectrum of mycobionts is still a matter of debate. Tulasnellaceae (Basidiomycota) is the richest and most widespread mycobiont worldwide. We assessed Tulasnellaceae richness in epiphytic and terrestrial orchids in different habitats, and evaluated the degree of generalism in orchid-Tulasnellaceae interactions and the robustness of this mutualistic system to the extinction of mycobiont partners. We sampled 114 orchid individuals including all common and rare species in 56 plots of 1 m² in 3 habitats: pristine forest, regenerating forest and a landslide site in a tropical montane rainforest in Southern Ecuador. We found 52 orchid and 29 Tulasnellaceae species. The composition of Tulasnellaceae OTUs was moderately to highly similar across habitats and between orchid growth forms. A significantly nested network architecture indicated the existence of a core of generalist Tulasnellaceae OTUs interacting with both rare and common orchids. Terrestrial and epiphytic orchids showed significant differences in robustness to the extinction of their Tulasnellaceae mycobionts. Thus, generalist mycobionts may be relevant for the preservation of hyperdiverse orchid communities in the tropics.     

Studies on the in vitro regenerative competence of aerial roots of two horticultural important Cymbidium species

In the present study aerial roots were successfully used as explants source for in vitro propagation of Cymbidium aloifolium and Cymbidium iridioides. Aerial roots of ~5?C6?week old from axenic cultures were cultured on MS medium adjuncts with different additives. In C. aloifolium within 20?days of culture ~60% of explants responded positively on MS medium containing sucrose (3%, w/v) and Kn (3???M) and formed PLBs. While in C. iridioides ~50% root explants responded positively on medium enriched with sucrose (3%), AC (0.1%) and IAA (3???M) after 40?days of culture. The shoot buds/PLBs converted into plantlets when maintained on regeneration medium. Of the three basal media tested, MS medium supported optimum regeneration and culture proliferation in both the species. In C. aloifolium ~12 shoot buds developed on medium nourished with sucrose (3%) and BA (3???M) but in C. iridioides optimum regeneration was achieved when medium supplemented with sucrose (3%), CW (15%), CH (100?mg?L^?1) and ~20 shoot buds formed per subculture. The well rooted plantlets were acclimatized for 3?C4?week in 1/10th MS salt solution containing sucrose (1%), charcoal pieces, brick pieces and chopped mosses as support under normal laboratory conditions. The hardened plants were transferred to potting mix where 80 and 75% of transplants survived in C. aloifolium and C. iridioides respectively after 2?months of transfer.     

Mycobiont overlap between two mycoheterotrophic genera of Monotropoideae (Pterospora andromedea and Sarcodes sanguinea) found in the Greater Yellowstone Ecosystem

Pterospora andromedea, a mycoheterotroph, has been shown to form obligate symbioses with only three species of Rhizopogon in section Amylopogon: R. salebrosus, R. arctostaphyli and an undescribed molecular taxon. Sarcodes sanguinea, another my coheterotroph in Ericaceae, and sister taxon to Pterospora andromedea, has been found to form symbioses with two species of Rhizopogon in section Amylopogon: R. ellenae and R. subpurpurascens. To date no overlap has been recorded between these two achlorophyllous plants and their associated mycobionts. Tissue from Pterospora andromedea rootballs and Rhizopogon spp. basidiocarps were collected from the Greater Yellowstone Ecosystem. The mycobionts were identified using sequence analysis of the ITS locus and compared with sequences of Rhizopogon spp. section Amylopogon from GenBank. Sequences of two additional loci, ATP6 and RPB2 were also generated and analyzed. In addition to Rhizopogon salebrosus, Pterospora andromedea was found for the first time in association with a fourth mycobiont, Rhizopogon ellenae, a known associate of Sarcodes sanguinea. The discovery of a new symbiont may provide evidence for an undiscovered lineage of Pterospora andromedea inhabiting the Greater Yellowstone Ecosystem. In addition, overlap in obligate mycobionts between closely related mycoheterotrophs provides interesting new information on the phylogenetic history and coevolution of the mycoheterotrophs in the Monotropoideae (Ericaceae).