Achlorophyllous Orchid Research Articles

Features of bacterial and fungal communities in the rhizosphere of Gastrodia elata cultivated in greenhouse for early harvest.

Symbiotic microbes are essential for developing and growing Gastrodia elata, an achlorophyllous orchid of high medicinal value. Recently, the cultivation of G. elata in greenhouses has been adopted in Korea to produce mature tubers in a short time. However, no studies have been conducted on the microbial community structure of G. elata cultivated in greenhouse environments. Therefore, we analyzed the temporal features of bacterial and fungal communities in the rhizosphere of G. elata at the juvenile [JT; 2 months after sowing (MAS)], young (YT; 6 MAS), and mature (MT; 11 MAS) tuber stages using culture-dependent and high-throughput sequencing technology. The richness and diversity of the bacterial and fungal communities decreased with tuber growth of G. elata. The symbiotic fungi Mycena sp. and Armillaria sp. as well as tuber extract inhibited the growth of various soil-inhabiting fungal and bacterial strains, indicating that G. elata and its symbiotic fungi play important roles in the selection of rhizosphere microbes. Mortierella rishikesha was the most abundant fungal species in the rhizosphere. We also identified the microorganisms potentially beneficial for G. elata development during greenhouse cultivation. Tubers and symbiotic fungi actively exert selective pressure on rhizosphere microbes, influencing the diversity and abundance of bacterial and fungal communities as G. elata grows. This study is a first report on the temporal microbial community structure of G. elata cultivated in greenhouse. The results on the associated microbiome of G. elata will help understand their beneficial interactions with G. elata and contribute to improvement in cultivation.

Transcriptomic heterochrony and completely cleistogamous flower development in the mycoheterotrophic orchid Gastrodia.

Cleistogamy, in which plants can reproduce via self-fertilization within permanently closed flowers, has evolved in > 30 angiosperm lineages; however, consistent with Darwin's doubts about its existence, complete cleistogamy - the production of only cleistogamous flowers - has rarely been recognized. Thus far, the achlorophyllous orchid genus, Gastrodia, is the only known genus with several plausible completely cleistogamous species. Here, we analyzed the floral developmental transcriptomes of two recently evolved, completely cleistogamous Gastrodia species and their chasmogamous sister species to elucidate the possible changes involved in producing common cleistogamous traits. The ABBA-BABA test did not support introgression and protein sequence convergence as evolutionary mechanisms leading to cleistogamy, leaving convergence in gene expression as a plausible mechanism. Regarding transcriptomic differentiation, the two cleistogamous species had common modifications in the expression of developmental regulators, exhibiting a gene family-wide signature of convergent expression changes in MADS-box genes. Our transcriptomic pseudotime analysis revealed a prolonged juvenile state and eventual maturation, a heterochronic pattern consistent with partial neoteny, in cleistogamous flower development. These findings indicate that transcriptomic partial neoteny, arising from changes in the expression of conserved developmental regulators, might have contributed to the rapid and repeated evolution of cleistogamous flowers in Gastrodia.

Spatial separation of mycobionts in the giant, differentiated root system of Cyrtosia septentrionalis, a fully myco-heterotrophic orchid

The rare achlorophyllous orchid Cyrtosia septentrionalis (Rchb.f.) Garay is thought to switch mycobiont from a Physisporinus species (‘TK-10’) to an Armillaria species as it develops. We conducted morphological, chemical, and molecular analyses of the root system of three adult plants growing sympatrically in Japan to assess reliance on different mycorrhizae. The root systems were found to comprise several long primary roots that radiate outwards from the emergent stem and many shorter lateral ones. Long roots branched most vigorously where they encountered Armillaria gallica in the soil, and the resulting lateral roots contained the highest density of intra-radical rhizomorphs. One plant was associated with ‘TK-10’ and the other two with A. gallica, but only the apical portion of long roots and a few lateral roots were colonized. Mycobionts isolated from the base of long roots proved incapable of germinating C. septentrionalis seeds. Whereas glucose was the dominant monosaccharide in non-mycorrhizal roots, galactose was in mycorrhizal ones. Lateral roots are specialized in mycorrhization and their velamen, and mucilage may play a key role in preventing desiccation. We hypothesize that C. septentrionalis can associate with either fungus from germination to adulthood, but that detection of A. gallica in the soil can prompt replacement of ‘TK-10’.

Comparative transcriptome analysis of tubers, stems, and flowers of Gastrodia elata Blume reveals potential genes involved in the biosynthesis of phenolics

Orchidaceae, well known for its fascinating flowers, is one of the largest and most diverse families of flowering plants. There are many kinds of plants in this family; these are distributed practically globally and have high ornamental and medicinal values. Gastrodia elata Blume, a traditional Chinese medicinal herb, is a rootless and leafless achlorophyllous orchid. Phenolic compounds are considered to be the major bioactive constituents in G. elata, with antioxidant, antiangiogenic, neuroprotective, antidepressant, anxiolytic, and sedative activities. In this study, we determined the contents of six main phenolic components in tubers, stems and flowers from G. elata. Meanwhile, the transcriptomes of the tuber, stem and flower tissues of G. elata were obtained using the BGISEQ-500 platform. A total of 58.29 Gb of data and 113,067 unigenes were obtained, of which 74,820 unigenes were functionally annotated against seven public databases. Differentially expressed genes between tuber, stem and flower tissues were identified. A total of 76 DEGs encoding eight key enzymes were identified as candidate genes involved in the biosynthesis of phenolics in G. elata. For further validation, the expression levels of unigenes were measured using quantitative real-time PCR. Our results greatly enrich the transcriptomic data of G. elata and provide valuable information for the identification of candidate genes involved in the biosynthesis of secondary metabolites.

Improved de novo Assembly of the Achlorophyllous Orchid Gastrodia elata.

Achlorophyllous plants are full mycoheterotrophic plants with no chlorophyll and they obtain their nutrients from soil fungi. Gastrodia elata is a perennial, achlorophyllous orchid that displays distinctive evolutionary strategy of adaptation to the non-photosynthetic lifestyle. Here in this study, the genome of G. elata was assembled to 1.12 Gb with a contig N50 size of 110 kb and a scaffold N50 size of 1.64 Mb so that it helped unveil the genetic basics of those adaptive changes. Based on the genomic data, key genes related to photosynthesis, leaf development, and plastid division pathways were found to be lost or under relaxed selection during the course of evolution. Thus, the genome sequence of G. elata provides a good resource for future investigations of the evolution of orchids and other achlorophyllous plants.

Molecular evidence supports simultaneous association of the achlorophyllous orchid Chamaegastrodia inverta with ectomycorrhizal Ceratobasidiaceae and Russulaceae

BackgroundAchlorophyllous orchids are mycoheterotrophic plants, which lack photosynthetic ability and associate with fungi to acquire carbon from different environmental sources. In tropical latitudes, achlorophyllous forest orchids show a preference to establish mycorrhizal relationships with saprotrophic fungi. However, a few of them have been recently found to associate with ectomycorrhizal fungi and there is still much to be learned about the identity of fungi associated with tropical orchids. The present study focused on mycorrhizal diversity in the achlorophyllous orchid C. inverta, an endangered species, which is endemic to southern China. The aim of this work was to identify the main mycorrhizal partners of C. inverta in different plant life stages, by means of morphological and molecular methods.ResultsMicroscopy showed that the roots of analysed C. inverta samples were extensively colonized by fungal hyphae forming pelotons in root cortical cells. Fungal ITS regions were amplified by polymerase chain reaction, from DNA extracted from fungal mycelia isolated from orchid root samples, as well as from total root DNA. Molecular sequencing and phylogenetic analyses showed that the investigated orchid primarily associated with ectomycorrhizal fungi belonging to a narrow clade within the family Ceratobasidiaceae, which was previously detected in a few fully mycoheterotrophic orchids and was also found to show ectomycorrhizal capability on trees and shrubs. Russulaceae fungal symbionts, showing high similarity with members of the ectomycorrhizal genus Russula, were also identified from the roots of C. inverta, at young seedling stage. Ascomycetous fungi including Chaetomium, Diaporthe, Leptodontidium, and Phomopsis genera, and zygomycetes in the genus Mortierella were obtained from orchid root isolated strains with unclear functional role.ConclusionsThis study represents the first assessment of root fungal diversity in the rare, cryptic and narrowly distributed Chinese orchid C. inverta. Our results provide new insights on the spectrum of orchid-fungus symbiosis suggesting an unprecedented mixed association between the studied achlorophyllous forest orchid and ectomycorrhizal fungi belonging to Ceratobasidiaceae and Russulaceae. Ceratobasidioid fungi as dominant associates in the roots of C. inverta represent a new record of the rare association between the identified fungal group and fully mycoheterotrophic orchids in nature.

The effects of illumination, temperature and 6-benzylaminoprine on asymbiotic seed germination and protocorm development in vitro in the achlorophyllous orchid Gastrodia pubilabiata Sawa

We examined the effects of culture conditions, namely, illumination, temperature and the addition of 6-benzylaminopurine (BAP) to the germination medium, on the asymbiotic germination of mature seeds of Gastrodia pubilabiata Sawa, an achlorophyllous orchid. Mature seeds were harvested from plants at two sites, one in Toyama Prefecture and one in Chiba Prefecture, Japan. We used gellan gum-solidified New Dogashima medium as the basal medium. The maximum frequency of germination of the seeds in darkness was approximately 65%. BAP had no stimulatory effect on seed germination, but accelerated protocorm development. Necessary conditions for successful germination of seeds and subsequent development of seedlings from mature seeds of Gastrodia pubilabiata clarified in this study are prerequisites not only for conservation of biodiversity but also for the elucidation of the life cycle of this achlorophyllous orchid.

Dynamics of fungal communities during Gastrodia elata growth

BackgroundGastrodia elata is a widely distributed achlorophyllous orchid and is highly valued as both medicine and food. Gastrodia elata produces dust-like seeds and relies on mycorrhizal fungi for its germination and growth. In its life cycle, G. elata is considered to switch from a specific single-fungus relationship (Mycena) to another single-fungus relationship (Armillaria). However, no studies have investigated the changes in the plant-fungus relationship during the growth of G. elata in the wild. In this study, high-throughput sequencing was used to characterize the fungal community of tubers in different growth phases as well as the soils surrounding G. elata.ResultsThe predominant fungi were Basidiomycota (60.44%) and Ascomycota (26.40%), which exhibited changes in abundance and diversity with the growth phases of G. elata. Diverse basidiomycetes in protocorms (phase P) were Hyphodontia, Sistotrema, Tricholoma, Mingxiaea, Russula, and Mycena, but the community changed from a large proportion of Resinicium bicolor (40%) in rice-like tubers (phase M) to an unidentified Agaricales operational taxonomic unit 1(OTU1,98.45%) in propagation vegetation tubers (phase B). The soil fungi primarily included Simocybe, Psathyrella, Conocybe, and Subulicystidium. Three Mycena OTUs obtained in this study were differentially distributed among the growth phases of G. elata, accounting for less than 1.0% of the total reads, and were phylogenetically close to Mycena epipterygia and M. alexandri.ConclusionsOur data indicated that G. elata interacts with a broad range of fungi beyond the Mycena genus. These fungi changed with the growth phases of G. elata. In addition, these data suggested that the development of the fungal community during the growth of G. elata was more complex than previously assumed and that at least two different fungi could be involved in development before the arrival of Armillaria.

Achlorophyllous orchid can utilize fungi not only for nutritional demands but also pollinator attraction.

Achlorophyllous orchid can utilize fungi not only for nutritional demands but also pollinator attraction.

Divergence Times and Phylogenetic Patterns of Sebacinales, a Highly Diverse and Widespread Fungal Lineage.

Patterns of geographic distribution and composition of fungal communities are still poorly understood. Widespread occurrence in terrestrial ecosystems and the unique richness of interactions of Sebacinales with plants make them a target group to study evolutionary events in the light of nutritional lifestyle. We inferred diversity patterns, phylogenetic structures and divergence times of Sebacinales with respect to their nutritional lifestyles by integrating data from fossil-calibrated phylogenetic analyses. Relaxed molecular clock analyses indicated that Sebacinales originated late Permian within Basidiomycota, and their split into Sebacinaceae and Serendipitaceae nom. prov. likely occurred during the late Jurassic and the early Cretaceous, coinciding with major diversifications of land plants. In Sebacinaceae, diversification of species with ectomycorrhizal lifestyle presumably started during the Paleocene. Lineage radiations of the core group of ericoid and cavendishioid mycorrhizal Sebacinales started probably in the Eocene, coinciding with diversification events of their hosts. The diversification of Sebacinales with jungermannioid interactions started during the Oligocene, and occurred much later than the diversification of their hosts. Sebacinales communities associated either with ectomycorrhizal plants, achlorophyllous orchids, ericoid and cavendishioid Ericaceae or liverworts were phylogenetically clustered and globally distributed. Major Sebacinales lineage diversifications started after the continents had drifted apart. We also briefly discuss dispersal patterns of extant Sebacinales.

Gastrodia madagascariensis (Gastrodieae, Orchidaceae): from an historical designation to a description of a new species from Madagascar

Gastrodia madagascariensis, a leafless achlorophyllous orchid, is described and illustrated here. The epithet was originally coined by Perrier de la Bâthie in 1939 for fruiting material found on the eastern coastal plain of Madagascar more than a century ago, but the name was never validly published. This new species is closely related to G. similis from Reunion Island, from which it can be distinguished by the perianth tube spreading towards the apex, the shape of the column and stigma, and the flower colour. The achlorophyllous genus Gastrodia currently comprises five species in the tropical parts of the Afro-Madagascan region, one of which, G. africana, is possibly extinct. We provide an artificial key to distinguish them. In addition, there is also an extratropical species in continental Africa, the introduced G. sesamoides (very local near Cape Town, South Africa).

Highly diversified fungi are associated with the achlorophyllous orchid Gastrodia flavilabella.

BackgroundMycoheterotrophic orchids are achlorophyllous plants that obtain carbon and nutrients from their mycorrhizal fungi. They often show strong preferential association with certain fungi and may obtain nutrients from surrounding photosynthetic plants through ectomycorrhizal fungi. Gastrodia is a large genus of mycoheterotrophic orchids in Asia, but Gastrodia species’ association with fungi has not been well studied. We asked two questions: (1) whether certain fungi were preferentially associated with G. flavilabella, which is an orchid in Taiwan and (2) whether fungal associations of G. flavilabella were affected by the composition of fungi in the environment.ResultsUsing next-generation sequencing, we studied the fungal communities in the tubers of Gastrodia flavilabella and the surrounding soil. We found (1) highly diversified fungi in the G. flavilabella tubers, (2) that Mycena species were the predominant fungi in the tubers but minor in the surrounding soil, and (3) the fungal communities in the G. flavilabella tubers were clearly distinct from those in the surrounding soil. We also found that the fungal composition in soil can change quickly with distance.ConclusionsG. flavilabella was associated with many more fungi than previously thought. Among the fungi in the tuber of G. flavilabella, Mycena species were predominant, different from the previous finding that adult G. elata depends on Armillaria species for nutritional supply. Moreover, the preferential fungus association of G. flavilabella was not significantly influenced by the composition of fungi in the environment.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1422-7) contains supplementary material, which is available to authorized users.

An efficient in vitro propagation, antioxidant and antimicrobial activities of Aphyllorchis montana Rchb.f.

An in vitro plant regeneration protocol was successfully established in Aphyllorchis montana, a saprophytic achlorophyllous orchid by culturing immature seeds. Among the six basal media evaluated for seed germination, BM-1-Terrestrial Orchid medium was found to be the best followed by Knudson C medium. Half-strength BM-1-Terrestrial Orchid medium was supplemented with different growth regulators either individually or in combinations for multiplication of shoots induction. Of the five cytokinins tested, thidiazuron at 6.8 μM was found to be most effective for multiple shoot induction yielding 17.24 ± 0.27 shoots after 10 weeks of culture. Addition of low concentration of α-Naphthaleneacetic acid (1.3 μM) in MS medium supplemented with the cytokinin thidiazuron (6.8 μM) favored shoot multiplication. The addition of organic additives to the medium containing thidiazuron enhanced the number of regenerated shoots. The plantlets were acclimatized, and the survival rate was 100%. Screening of the antioxidant and antimicrobial activities and estimation of total phenolics and flavonoid content of methanolic extracts of micropropagated plants were also carried out and compared with that of the wild-grown plants. In all the tests, methanolic extract from wild-grown plants showed higher antioxidant, antimicrobial activity, total phenolics and flavonoid content than in vitro propagated plants.

수확시기와 자마의 특성별 천마의 아미노산 함량 변화

Gastrodia elata Blume, an achlorophyllous orchid plant, has been used in traditional medicine and harvests in spring and fall. Here we investigated the quantitative changes of amino acids in G. elata by harvest times and seed tubers. In the results, we found that there was not big difference in contents of total amino acids but the distribution of amino acids differed depending on harvest times. 19% of total amino acids were asparagin and valine, an essential amino acid, occupied 10% within total amino acids in the tubers harvested in October. Among amino acids in the tubers harvested in April, serine occupied 23% and arginine, an essential amino acid, occupied 10% within total amino acids in tuber. Interestingly, the use of sexually propagated seed tubers produced high concentration of total amino acids compared to vegetatively propagated seed tubers. As for sexually propagated seed tubers, essential amino acids contents similar to the tubes harvested in October and nonessential amino acids contents similar to the tubers harvested in April. In this study, we found that amino acids contents in G. elata tubers altered depending on various cultivation practices. Therefore if these results can be applied to food industry, the value of G. elata as a natural food resource will be enhanced to a great extent.

An Efficient In Vitro Propagation, Antioxidant and Antimicrobial Activities of Aphyllorchis Montana (Reichenb.f.)

An in vitro plant regeneration protocol was successfully established in Aphyllorchis Montana , a saprophytic achlorophyllous orchid by culturing immature seeds. Among the six basal media evaluated for seed germination, BM-TM medium was found to be the best followed by KC medium. After 40 days, all the media turned brown and the growths of the protocorms were arrested. Activated charcoal, 1 g/l in half strength BM-TM was found to be suitable for further development of protocorms. Half strength BM-TM medium was supplemented with different growth regulators either individually or in combinations for multiplication of shoots. Of the five cytokinins tested, TDZ at 6.8 μM was found to be most effective for multiple shoot induction yielding 17.24 ± 0.27 shoots after 10 weeks of culture. Addition of low concentration of NAA (1.3 μM) in MS medium supplemented with the cytokinin TDZ (6.8 μM) favoured shoot multiplication. A mean number of 27.56 ± 0.54 shoots with 3.92 ± 0.11 number of roots were produced per explant. The response of the seed derived protocorm to the different types of organic additives viz., peptone and yeast extract and coconut water was also evaluated. The addition of these organic additives to the medium containing TDZ enhanced the number of shoot regeneration. The plantlets were acclimatized in plastic pots containing sterilized vermiculite. The survival rate was 100 % when maintained in the culture room condition (25 ± 2 °C). Screening of the antibacterial, antioxidant activity and estimation of total phenolics and flavonoid content of methanolic extracts of micropropagated plants were also carried out and compared with that of the wild-grown plants. In all the tests, methanolic extract from wild-grown plants showed higher antioxidant, antimicrobial activity, total phenolics and flavonoid content than in vitro propagated plants.

재배조건과 수확시기에 따른 천마의 기능성 물질함량 변화

Gastrodia elata Blume is a achlorophyllous orchid plant that has been used in traditional medicine to treat many human illnesses, such as vertigo, hemiplegia, and epilepsy. It contains three major pharmacological substances; gastrodin, ergothioneine, and vanillyl alcohol. Here we investigated the quantitative changes in those functional components by cultivation conditions and harvest times. The content of ergothioneine was different with cultivation areas, while gastrodin accumulated the higher levels than vanillyl alcohol regardless of cultivation areas. The smaller tubers contained the higher concentrations of gastrodin and the lower concentrations of ergothioneine than did the larger tubers. The cultivation under sun shade screen increased the concentrations of both gastrodin and vanillyl alcohol but decreased the levels of ergothioneine. We also determined the contents of functional substances depending on the harvest times. The concentration of gastrodin was higher in October than in April, while the level of ergothioneine was higher in April than in October. Interestingly, the use of sexually propagated seed tubers produced 1.9 times higher levels of gastrodin compared to vegetatively propagated seed tubers. In this study, we found that three major functional substances in G. elata tubers altered depending on various cultivation practices, which might be important to increase the economic values of G. elata tubers.

ISSR 표지에 의한 천마의 유전 다양성분석 및 기능성 물질분석

Gastrodia elata, an achlorophyllous orchid plant, is rare medicinal plant. We investigated the genetic diversity in G. elata from 4 locations by using Inter-Simple Sequence Repeats (ISSR) markers. Shannon`s information Index (S.I.) indicating genetic diversity ranged from 0.255 (Pocheon) to 0.322 (Muju) with the mean of 0.29. The level of genetic diversity was lower than other plant and most genetic diversity was allocated among individuals within populations (26.81%). The UPGMA dendrogram based on genetic distance failed in showing decisive geographic relationship. In the case of gastrodin (GA), the major components in G. elata, Sangju was highest. The ergothionine (ERG) was detected a lot of contents in Muju and Pocheon. In conclusion, our results is very important information for explaining relationship of genetic variation and functional substances without the effects of environment factors and developing genetic marker by ISSR in G. elata, which may be responsible for the development of breeds with a lot of functional substance in G. elata.

Limited carbon and mineral nutrient gain from mycorrhizal fungi by adult Australian orchids

In addition to autotrophic and fully mycoheterotrophic representatives, the orchid family comprises species that at maturity obtain C and N partially from fungal sources. These partial mycoheterotrophs are often associated with fungi that simultaneously form ectomycorrhizas with trees. This study investigates mycorrhizal nutrition for orchids from the southwestern Australian biodiversity hotspot. The mycorrhizal fungi of 35 green and one achlorophyllous orchid species were analyzed using molecular methods. Nutritional mode was identified for 27 species by C and N isotope abundance analysis in comparison to non-orchids from the same habitat. As a complementary approach, (13)CO(2) pulse labeling was applied to a subset of six orchid species to measure photosynthetic capacity. Almost all orchids associated with rhizoctonia-forming fungi. Due to much higher than expected variation within the co-occurring nonorchid reference plants, the stable isotope approach proved challenging for assigning most orchids to a specialized nutritional mode; therefore, these orchids were classified as autotrophic at maturity. The (13)CO(2) pulse labeling confirmed full autotrophy for six selected species. Nonetheless, at least three orchid species (Gastrodia lacista, Prasophyllum elatum, Corybas recurvus) were identified as nutritionally distinctive from autotrophic orchids and reference plants. Despite the orchid-rich flora in southwestern Australia, partial mycoheterotrophy among these orchids is less common than in other parts of the world, most likely because most associate with saprotrophic fungi rather than ectomycorrhizal fungi.

Ergothioneine accumulation in a medicinal plant Gastrodia elata.

Ergothioneine (ERG) has long been recognized as a potent and stable antioxidant. In nature, ERG is synthesized only in fungi and mycobacteria, not in plants. The lifecycle of Gastrodia elata, an achlorophyllous orchid plant, is completely dependent on the presence of symbiotic fungi, particularly Armillaria mellea. In this study, we report for the first time that G. elata accumulated ERG whose levels were correlated with the concentrations of ERG in A. mellea. The contents of ERG in G. elata were significantly higher in actively developing tissues, such as seed capsules and newly growing corms, than in mature rhizomes. The ERG levels in rhizomes were significantly correlated with antioxidant capacities and were quantitatively comparable to the previously established major pharmacological components in G. elata. These results indicated that ERG is a newly found functional substance in G. elata, which may be responsible for the beneficial health effects of G. elata on various human diseases. Key words: Gastrodia elata, Armillaria mellea, ergothioneine, antioxidant.

Independent recruitment of saprotrophic fungi as mycorrhizal partners by tropical achlorophyllous orchids

Mycoheterotrophic orchids have adapted to shaded forest understory by shifting to achlorophylly and receiving carbon from their mycorrhizal fungi. In temperate forests, they associate in a highly specific way with fungi forming ectomycorrhizas on nearby trees, and exploiting tree photosynthates. However, many rainforests lack ectomycorrhizal fungi, and there is evidence that some tropical Asiatic species associate with saprotrophic fungi. To investigate this in different geographic and phylogenetic contexts, we identified the mycorrhizal fungi supporting two tropical mycoheterotrophic orchids from Mascarene (Indian Ocean) and Caribbean islands. We tested their possible carbon sources by measuring natural nitrogen ((15)N) and carbon ((13)C) abundances. Saprotrophic basidiomycetes were found: Gastrodia similis associates with a wood-decaying Resinicium (Hymenochaetales); Wullschlaegelia aphylla associates with both litter-decaying Gymnopus and Mycena species, whose rhizomorphs link orchid roots to leaf litter. The (15)N and (13)C abundances make plausible food chains from dead wood to G. similis and from dead leaves to W. aphylla. We propose that temperature and moisture in rainforests, but not in most temperate forests, may favour sufficient saprotrophic activity to support development of mycoheterotrophs. By enlarging the spectrum of mycorrhizal fungi and the level of specificity in mycoheterotrophic orchids, this study provides new insights on orchid and mycorrhizal biology in the tropics.