Changing partners in the dark: isotopic and molecular evidence of ectomycorrhizal liaisons between forest orchids and trees

In the mycorrhizal symbiosis, plants exchange photosynthates for mineral nutrients acquired by fungi from the soil. This mutualistic arrangement has been subverted by hundreds of mycorrhizal plant species that lack the ability to photosynthesize. The most numerous examples of this behaviour are found in the largest plant family, the Orchidaceae. Although these non-photosynthetic orchid species are known to be highly specialized exploiters of the ectomycorrhizal symbiosis, photosynthetic orchids are thought to use free-living saprophytic, or pathogenic, fungal lineages. However, we present evidence that putatively photosynthetic orchids from five species which grow in the understorey of forests: (i) form mycorrhizas with ectomycorrhizal fungi of forest trees; and (ii) have stable isotope signatures indicating distinctive pathways for nitrogen and carbon acquisition approaching those of non-photosynthetic orchids that associate with ectomycorrhizal fungi of forest trees. These findings represent a major shift in our understanding of both orchid ecology and evolution because they explain how orchids can thrive in low-irradiance niches and they show that a shift to exploiting ectomycorrhizal fungi precedes viable losses of photosynthetic ability in orchid lineages.     

Identity and Specificity of Rhizoctonia-Like Fungi from Different Populations of Liparis japonica (Orchidaceae) in Northeast China

Mycorrhizal association is known to be important to orchid species, and a complete understanding of the fungi that form mycorrhizas is required for orchid ecology and conservation. Liparis japonica (Orchidaceae) is a widespread terrestrial photosynthetic orchid in Northeast China. Previously, we found the genetic diversity of this species has been reduced recent years due to habitat destruction and fragmentation, but little was known about the relationship between this orchid species and the mycorrhizal fungi. The Rhizoctonia-like fungi are the commonly accepted mycorrhizal fungi associated with orchids. In this study, the distribution, diversity and specificity of culturable Rhizoctonia-like fungi associated with L. japonica species were investigated from seven populations in Northeast China. Among the 201 endophytic fungal isolates obtained, 86 Rhizoctonia-like fungi were identified based on morphological characters and molecular methods, and the ITS sequences and phylogenetic analysis revealed that all these Rhizoctonia-like fungi fell in the same main clade and were closely related to those of Tulasnella calospora species group. These findings indicated the high mycorrhizal specificity existed in L. japonica species regardless of habitats at least in Northeast China. Our results also supported the wide distribution of this fungal partner, and implied that the decline of L. japonica in Northeast China did not result from high mycorrhizal specificity. Using culture-dependent technology, these mycorrhizal fungal isolates might be important sources for the further utilizing in orchids conservation.     

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

? Premise of the study: 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. ? Methods: 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. ? Key results: 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. ? Conclusions: 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.     

Parallel evolutionary paths to mycoheterotrophy in understorey Ericaceae and Orchidaceae: ecological evidence for mixotrophy in Pyroleae

Several forest understorey achlorophyllous plants, termed mycoheterotrophs (MHs), obtain C from their mycorrhizal fungi. The latter in turn form ectomycorrhizas with trees, the ultimate C source of the entire system. A similar nutritional strategy occurs in some green forest orchids, phylogenetically close to MH species, that gain their C via a combination of MH and photosynthesis (mixotrophy). In orchid evolution, mixotrophy evolved in shaded habitats and preceded MH nutrition. By generalizing and applying this to Ericaceae, we hypothesized that green forest species phylogenetically close to MHs are mixotrophic. Using stable C isotope analysis with fungi, autotrophic, mixotrophic and MH plants as comparisons, we found the first quantitative evidence for substantial fungi-mediated mixotrophy in the Pyroleae, common ericaceous shrubs from boreal forests close to the MH Monotropoideae. Orthilia secunda, Pyrola chlorantha, Pyrola rotundifolia and Chimaphila umbellata acquired between 10.3 and 67.5% of their C from fungi. High N and ¹⁵N contents also suggest that Pyroleae nutrition partly rely on fungi. Examination of root fungal internal transcribed spacer sequences at one site revealed that 39 species of mostly endophytic or ectomycorrhizal fungi, including abundant Tricholoma spp., were associated with O. secunda, P. chlorantha and C. umbellata. These fungi, particularly ectomycorrhizal associates, could thus link mixotrophic Pyroleae spp. to surrounding trees, allowing the C flows deduced from isotopic evidence. These data suggest that we need to reconsider ecological roles of understorey plants, which could influence the dynamics and composition of forest communities.     

Mycorrhizal fungal community composition in seven orchid species inhabiting Song Mountain,Beijing, China

Mycorrhizal fungi play an important role in the germination and growth of orchids essentially influencing their survival,abundance, and spatial distribution. In this study, we investigated the composition of the mycorrhizal fungal community in seven terrestrial orchid species inhabiting Song Mountain, Beijing, China, using Illumina MiSeq high-throughput sequencing. The mycorrhizal communities in the seven orchids were mainly composed of members of the Ceratobasidiaceae, Sebacinales, and Tulasnellaceae, while a number of ectomycorrhizal fungi belonging to the Russulaceae, Tricholomataceae, Thelephoraceae, and Cortinariaceae were occasionally observed. However, the dominant fungal associates and mycorrhizal community differed significantly among the orchid species as well as subhabitats. These findings confirm the previous observation that sympatric orchid species show different preferences for mycorrhizal fungi, which may drive niche partitioning and contribute to their cooccurrence.     

Continent-wide distribution in mycorrhizal fungi: implications for the biogeography of specialized orchids

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.     

Comparative study of nutritional mode and mycorrhizal fungi in green and albino variants of Goodyera velutina,an orchid mainly utilizing saprotrophic rhizoctonia

The majority of chlorophyllous orchids form mycorrhizal associations with so‐called rhizoctonia fungi, a phylogenetically heterogeneous assemblage of predominantly saprotrophic fungi in Ceratobasidiaceae, Tulasnellaceae, and Serendipitaceae. It is still a matter of debate whether adult orchids mainly associated with rhizoctonia species are partially mycoheterotrophic. Here, we investigated the nutritional modes of green and albino variants of Goodyera velutina, an orchid species considered to be mainly associated with Ceratobasidium spp., by measuring their ¹³C and ¹⁵N abundances, and by molecular barcoding of their mycorrhizal fungi. Molecular analysis revealed that both green and albino variants of G. velutina harbored a similar range of mycobionts, mainly saprotrophic Ceratobasidium spp., Tulasnella spp., and ectomycorrhizal Russula spp. In addition, stable isotope analysis revealed that albino variants were significantly enriched in ¹³C but not so greatly in ¹⁵N, suggesting that saprotrophic Ceratobasidium spp. and Tulasnella spp. are their main carbon source. However, in green variants, ¹³C levels were depleted and those of ¹⁵N were indistinguishable from the co‐occurring autotrophic plants. Therefore, we concluded that the albino G. velutina variants are fully mycoheterotrophic plants whose C derives mainly from saprotrophic rhizoctonia, while the green G. velutina variants are mainly autotrophic plants, at least at our study site, in spite of their additional associations with ectomycorrhizal fungi. This is the first report demonstrating that adult nonphotosynthetic albino variants can obtain their nutrition mainly from nonectomycorrhizal rhizoctonia.     

Mixotrophy of Platanthera minor, an orchid associated with ectomycorrhiza-forming Ceratobasidiaceae fungi

? We investigated the fungal symbionts and carbon nutrition of a Japanese forest photosynthetic orchid, Platanthera minor, whose ecology suggests a mixotrophic syndrome, that is, a mycorrhizal association with ectomycorrhiza (ECM)-forming fungi and partial exploitation of fungal carbon. ? We performed molecular identification of symbionts by PCR amplifications of the fungal ribosomal DNA on hyphal coils extracted from P. minor roots. We tested for a (13)C and (15)N enrichment characteristic of mixotrophic plants. We also tested the ectomycorrhizal abilities of orchid symbionts using a new protocol of direct inoculation of hyphal coils onto roots of Pinus densiflora seedlings. ? In phylogenetic analyses, most isolated fungi were close to ECM-forming Ceratobasidiaceae clades previously detected from a few fully heterotrophic orchids or environmental ectomycorrhiza surveys. The direct inoculation of fungal coils of these fungi resulted in ectomycorrhiza formation on P. densiflora seedlings. Stable isotope analyses indicated mixotrophic nutrition of P. minor, with fungal carbon contributing from 50% to 65%. ? This is the first evidence of photosynthetic orchids associated with ectomycorrhizal Ceratobasidiaceae taxa, confirming the evolution of mixotrophy in the Orchideae orchid tribe, and of ectomycorrhizal abilities in the Ceratobasidiaceae. Our new ectomycorrhiza formation technique may enhance the study of unculturable orchid mycorrhizal fungi.     

Evidence for novel and specialized mycorrhizal parasitism: the orchid Gastrodia confusa gains carbon from saprotrophic Mycena

We investigated the physiological ecology of the Asian non-photosynthetic orchid Gastrodia confusa. We revealed its mycorrhizal partners by using molecular identification and identified its ultimate nutritional source by analysing carbon and nitrogen natural stable isotope abundances. Molecular identification using internal transcribed spacer and large subunit nrDNA sequences showed that G. confusa associates with several species of litter- and wood-decomposer Mycena fungi. The carbon and nitrogen isotope signatures of G. confusa were analysed together with photosynthetic plant reference samples and samples of the ectomycorrhizal epiparasite Monotropa uniflora. We found that G. confusa was highly enriched in ¹³C but not greatly in ¹⁵N, while M. uniflora was highly enriched in both ¹³C and ¹⁵N. The ¹³C and ¹⁵N signatures of G. confusa were the closest to those of the fruit bodies of saprotrophic fungi. Our results demonstrate for the first time using molecular and mass-spectrometric approaches that myco-heterotrophic plants gain carbon through parasitism of wood or litter decaying fungi. Furthermore, we demonstrate that, several otherwise free-living non-mycorrhizal, Mycena can be mycorrhizal partners of orchids.     

Cryptostylis species (Orchidaceae) from a broad geographic and habitat range associate with a phylogenetically narrow lineage of Tulasnellaceae fungi

While many Australian terrestrial orchids have highly specialized mycorrhizal associations, we tested the hypothesis that the geographically widespread orchid genus Cryptostylis associates with a diversity of fungal species. Using fungal isolation and molecular approaches, we investigated the mycorrhizal associations of five Australian Cryptostylis species (27 sites sampled) and included limited sampling from three Asiatic Cryptostylis species (two sites). Like related orchid genera, Tulasnellaceae formed the main fungal associations of the Cryptostylis species we sampled, although some ectomycorrhizal, ericoid and saprotrophic fungi were detected infrequently. Each species of Australian Cryptostylis associated with three to seven Tulasnella Operational Taxonomic Units (OTUs), except for C. hunteriana where only one Tulasnella OTU was detected. In total, eleven Tulasnella OTUs associated with Australian Cryptostylis. The Asiatic Cryptostylis associated with four different Tulasnella OTUs belonging to the same lineage as the Australian species. While five Tulasnella OTUs (T. australiensis, T. prima, T. warcupii, T. densa, and T. punctata) were used by multiple species of Australian Cryptostylis, the most commonly used OTU differed between orchid species. The association with different Tulasnella fungi by Cryptostylis species co-occurring at the same site suggests that in any given environmental condition, Cryptostylis species may intrinsically favour different fungal OTUs.     

Two widespread green Neottia species (Orchidaceae) show mycorrhizal preference for Sebacinales in various habitats and ontogenetic stages

Plant dependence on fungal carbon (mycoheterotrophy) evolved repeatedly. In orchids, it is connected with a mycorrhizal shift from rhizoctonia to ectomycorrhizal fungi and a high natural ¹³C and ¹⁵N abundance. Some green relatives of mycoheterotrophic species show identical trends, but most of these remain unstudied, blurring our understanding of evolution to mycoheterotrophy. We analysed mycorrhizal associations and ¹³C and ¹⁵N biomass content in two green species, Neottia ovata and N. cordata (tribe Neottieae), from a genus comprising green and nongreen (mycoheterotrophic) species. Our study covered 41 European sites, including different meadow and forest habitats and orchid developmental stages. Fungal ITS barcoding and electron microscopy showed that both Neottia species associated mainly with nonectomycorrhizal Sebacinales Clade B, a group of rhizoctonia symbionts of green orchids, regardless of the habitat or growth stage. Few additional rhizoctonias from Ceratobasidiaceae and Tulasnellaceae, and ectomycorrhizal fungi were detected. Isotope abundances did not detect carbon gain from the ectomycorrhizal fungi, suggesting a usual nutrition of rhizoctonia‐associated green orchids. Considering associations of related partially or fully mycoheterotrophic species such as Neottia camtschatea or N. nidus‐avis with ectomycorrhizal Sebacinales Clade A, we propose that the genus Neottia displays a mycorrhizal preference for Sebacinales and that the association with nonectomycorrhizal Sebacinales Clade B is likely ancestral. Such a change in preference for mycorrhizal associates differing in ecology within the same fungal taxon is rare among orchids. Moreover, the existence of rhizoctonia‐associated Neottia spp. challenges the shift to ectomycorrhizal fungi as an ancestral pre‐adaptation to mycoheterotrophy in the whole Neottieae.     

Mycorrhizal preference promotes habitat invasion by a native Australian orchid: Microtis media

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.     

Diversity of mycorrhizal fungi of terrestrial orchids: compatibility webs, brief encounters, lasting relationships and alien invasions

The diversity of mycorrhizal fungi associated with an introduced weed-like South African orchid (Disa bracteata) and a disturbance-intolerant, widespread, native West Australian orchid (Pyrorchis nigricans) were compared by molecular identification of the fungi isolated from single pelotons. Molecular identification revealed both orchids were associated with fungi from diverse groups in the Rhizoctonia complex with worldwide distribution. Symbiotic germination assays confirmed the majority of fungi isolated from pelotons were mycorrhizal and a factorial experiment uncovered complex webs of compatibility between six terrestrial orchids and 12 fungi from Australia and South Africa. Two weed-like (disturbance-tolerant rapidly spreading) orchids — D. bracteata and the indigenous Australian Microtis media, had the broadest webs of mycorrhizal fungi. In contrast, other native orchids had relatively small webs of fungi (Diuris magnifica and Thelymitra crinita), or germinated exclusively with their own fungus (Caladenia falcata and Pterostylis sanguinea). Orchids, such as D. bracteata and M. media, which form relationships with diverse webs of fungi, had apparent specificity that decreased with time, as some fungi had brief encounters with orchids that supported protocorm formation but not subsequent seedling growth. The interactions between orchid mycorrhizal fungi and their hosts are discussed.     

Relationships between orchid and fungal biodiversity: Mycorrhizal preferences in Mediterranean orchids

Orchidaceae is one of the most species-rich angiosperm families, and all orchids are fully dependent on fungi for their seed germination and their life cycle. The level of specificity of the association between orchid species and fungi can be related to the number of co-occurring orchid species. To investigate orchid mycorrhizal associations in adult-photosynthetic orchids, 16 Mediterranean orchid species belonging to 4 genera (Anacamptis, Ophrys, Orchis, and Serapias) at 11 different sites were subjected to DNA-based analysis. Eighteen operational taxonomic units representing two fungal families, Tulasnellaceae and Ceratobasidiaceae, were identified. All examined orchid species associated with different mycorrhizal fungi. Interestingly, there was a positive correlation between number of orchid species and number of mycorrhizal. Monospecific populations showed a lower number of fungi, while sympatric populations had a higher number of mycorrhizal fungi. Our results showed that Mediterranean orchid species associated with a higher number of mycorrhizal fungi confirming as photosynthetic orchids are typically generalists toward mycorrhizal fungi. Thus, photosynthetic orchids exhibit low specificity for fungal symbionts showing the potential for opportunistic associations with diverse fungi reducing competition for nutrient. We suggest that these characteristics could confer symbiotic assurance particularly in habitat with resource limitations or prone to stressful conditions.     

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.     

Addition of fungal inoculum increases germination of orchid seeds in restored grasslands

Grasslands restored on arable land often retain high residual nutrients, modified soil biota, and lower plant species diversity. Establishment of rare plant species with complex multitrophic interactions, typical of undisturbed nutrient-poor environments, may be hindered by the absence of interacting organisms. We hypothesised that the addition of a mycorrhizal symbiont improves the seed germination of orchids that crucially depend on fungi. We focused on grasslands restored on arable land 1–15 years ago featuring residual mineral nutrients and low organic matter contents compared to semi-natural grasslands and on four orchid species differing in the level of mycorrhizal specificity: high – Anacamptis pyramidalis and Orchis mascula – and low – Platanthera bifolia and Gymnadenia conopsea. Five fungal isolates obtained from non-green underground mycorrhizal orchid seedlings (protocorms) or adults' roots were tested for orchid-fungus compatibility under conditions in vitro. Orchid seeds inserted in retrievable seed packets were subsequently co-introduced with selected fungal isolates grown either on agar or sterilized hay into the soil of nine restored grasslands and incubated for twelve months. The identity of mycorrhizal fungi in retrieved protocorms was verified by molecular methods. The isolates that supported protocorm establishment in vitro enabled also protocorm formation in situ, but success rates differed among orchid species. While mycorrhizal specialists produced most protocorms after inoculation, the mycorrhizal generalists took advantage of naturally occurring fungi and produced some protocorms both in inoculated and uninoculated treatments. We showed that the addition of mycorrhizal fungi enhanced protocorm formation regardless of the modified soil environment, especially in mycorrhizal specialist orchids. This method may help to restore populations of native orchid species in their former distribution ranges, including farming-altered habitats.     

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.     

Evidence for mycorrhizal races in a cheating orchid

Disruptive selection on habitat or host-specificity has contributed to the diversification of several animal groups, especially plant-feeding insects. Photosynthetic plants typically associate with a broad range of mycorrhizal fungi, while non-photosynthetic plants that capture energy from mycorrhizal fungi ('mycoheterotrophs') are often specialized towards particular taxa. Sister myco-heterotroph species are often specialized towards different fungal taxa, suggesting rapid evolutionary shifts in specificity. Within-species variation in specificity has not been explored. Here, we tested whether genetic variation for mycorrhizal specificity occurs within the myco-heterotrophic orchid Corallorhiza maculata. Variation across three single-nucleotide polymorphisms revealed six multilocus genotypes across 122 orchids from 30 sites. These orchids were associated with 22 different fungal species distributed across the Russulaceae (ectomycorrhizal basidiomycetes) according to internal-transcribed-spacer sequence analysis. The fungi associated with four out of the six orchid genotypes fell predominantly within distinct subclades of the Russulaceae. This result was supported by Monte Carlo simulation and analyses of molecular variance of fungal sequence diversity. Different orchid genotypes were often found growing in close proximity, but maintained their distinct fungal associations. Similar patterns are characteristic of insect populations diversifying onto multiple hosts. We suggest that diversification and specialization of mycorrhizal associations have contributed to the rapid radiation of the Orchidaceae.     

Fungal specificity bottlenecks during orchid germination and development

Fungus-subsidized growth through the seedling stage is the most critical feature of the life history for the thousands of mycorrhizal plant species that propagate by means of 'dust seeds.' We investigated the extent of specificity towards fungi shown by orchids in the genera Cephalanthera and Epipactis at three stages of their life cycle: (i) initiation of germination, (ii) during seedling development, and (iii) in the mature photosynthetic plant. It is known that in the mature phase, plants of these genera can be mycorrhizal with a number of fungi that are simultaneously ectomycorrhizal with the roots of neighbouring forest trees. The extent to which earlier developmental stages use the same or a distinctive suite of fungi was unclear. To address this question, a total of 1500 packets containing orchid seeds were buried for up to 3 years in diverse European forest sites which either supported or lacked populations of helleborine orchids. After harvest, the fungi associated with the three developmental stages, and with tree roots, were identified via cultivation-independent molecular methods. While our results show that most fungal symbionts are ectomycorrhizal, differences were observed between orchids in the representation of fungi at the three life stages. In Cephalanthera damasonium and C. longifolia , the fungi detected in seedlings were only a subset of the wider range seen in germinating seeds and mature plants. In Epipactis atrorubens , the fungi detected were similar at all three life stages, but different fungal lineages produced a difference in seedling germination performance. Our results demonstrate that there can be a narrow checkpoint for mycorrhizal range during seedling growth relative to the more promiscuous germination and mature stages of these plants' life cycle.     

Saprotrophic fungal mycorrhizal symbionts in achlorophyllous orchids: Finding treasures among the ‘molecular scraps’?

Mycoheterotrophic plants are achlorophyllous plants that obtain carbon from their mycorrhizal fungi. They are usually considered to associate with fungi that are (1) specific of each mycoheterotrophic species and (2) mycorrhizal on surrounding green plants, which are the ultimate carbon source of the entire system. Here we review recent works revealing that some mycoheterotrophic plants are not fungal-specific, and that some mycoheterotrophic orchids associate with saprophytic fungi. A re-examination of earlier data suggests that lower specificity may be less rare than supposed in mycoheterotrophic plants. Association between mycoheterotrophic orchids and saprophytic fungi arose several times in the evolution of the two partners. We speculate that this indirectly illustrates why transition from saprotrophy to mycorrhizal status is common in fungal evolution. Moreover, some unexpected fungi occasionally encountered in plant roots should not be discounted as ‘molecular scraps’, since these facultatively biotrophic encounters may evolve into mycorrhizal symbionts in some other plants.Key words: endophytic fungi, evolution of mycorrhizae, mycoheterophy, mycorrhizae, saprophytic fungi, specificityConsiderable advances were recently made in the ecology of achlorophyllous, heterotrophic plants that obtain carbon from their mycorrhizal fungi (Fig. 1). Most plants have contact with soil through mycorrhizal symbioses, in which roots associate with a suitable fungal partner. Fungi utilize soil mineral nutrients, and while sharing them with host plants, they generally receive carbon as a reward. In contrast, some achlorophyllous plants living in the shaded forest understorey have reversed the process. They receive carbon from their mycorrhizal fungi exclusively, hence the designation ‘mycoheterotrophic’ (MH) plants.¹ Mycoheterotrophy has appeared several times during the evolution of land plants, and more than 20 times among orchids that encompass half of all MH species.² In the last decade, the development of molecular tools has enabled researchers to identify many fungal symbionts, which are often uncultivable. The fungi occurring in the densely colonized roots of MH species often produce a stronger PCR signal than any fungal contaminant, making molecular tools very effective for this field of study.Open in a separate windowFigure 1Wullschlaegelia aphylla, a mycoheterotrophic orchid unspecifically associated with saprotrophic Mycena and Gymnopus species. (A) Whole plant at flowering time, with reduced, tuberoid root system at that period. (B) Section of mycorrhizal root showing intracellular hyphal pelotons at early stage (p), or late stage (undergoing lysis, lp); among orchids, the colonization of dead cortical cell (cc) is a unique feature to some saprotrophic fungi (picture by A. Faccio, University of Torino).