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.     

Isotopic evidence of partial mycoheterotrophy in Burmannia coelestis (Burmanniaceae)

The Burmanniaceae contain several lineages of achlorophyllous mycoheterotrophic plants that associate with arbuscular mycorrhizal fungi (AMF). Here we investigate the isotopic profile of a green and potentially mycoheterotrophic plant in situ, Burmannia coelestis, and associated reference plants. We generated δ ¹³C and δ ¹⁵N stable isotope profiles for five populations of B. coelestis. Burmannia coelestis was significantly enriched in ¹³C relative to surrounding C₃ reference plants and significantly depleted in ¹³C relative to C₄ reference plants. No significant differences were detected in ¹⁵N enrichment between B. coelestis and reference plants. The isotopic profiles measured are suggestive of partial mycoheterotrophy in B. coelestis. Within the genus Burmannia transitions to full mycoheterotrophy have occurred numerous times, suggesting that some green Burmannia species are likely to be partially mycoheterotrophic but in many conditions this mode of nutrition may only be detectable using natural abundance stable isotopic methods, such as when associated with C₄ mycorrhizal plants.     

Partial mycoheterotrophy is common among chlorophyllous plants with Paris-type arbuscular mycorrhiza

Background and AimsAn arbuscular mycorrhiza is a mutualistic symbiosis with plants as carbon providers for fungi. However, achlorophyllous arbuscular mycorrhizal species are known to obtain carbon from fungi, i.e. they are mycoheterotrophic. These species all have the Paris type of arbuscular mycorrhiza. Recently, two chlorophyllous Paris-type species proved to be partially mycoheterotrophic. In this study, we explore the frequency of this condition and its association with Paris-type arbuscular mycorrhiza.MethodsWe searched for evidence of mycoheterotrophy in all currently published ¹³C, ²H and ¹⁵N stable isotope abundance patterns suited for calculations of enrichment factors, i.e. isotopic differences between neighbouring Paris- and Arum-type species. We found suitable data for 135 plant species classified into the two arbuscular mycorrhizal morphotypes.Key ResultsAbout half of the chlorophyllous Paris-type species tested were significantly enriched in ¹³C and often also enriched in ²H and ¹⁵N, compared with co-occurring Arum-type species. Based on a two-source linear mixing model, the carbon gain from the fungal source ranged between 7 and 93 % with ferns > horsetails > seed plants. The seed plants represented 13 families, many without a previous record of mycoheterotrophy. The ¹³C-enriched chlorophyllous Paris-type species were exclusively herbaceous perennials, with a majority of them thriving on shady forest ground.ConclusionsSignificant carbon acquisition from fungi appears quite common and widespread among Paris-type species, this arbuscular mycorrhizal morphotype probably being a pre-condition for developing varying degrees of mycoheterotrophy.     

C and N stable isotope signatures reveal constraints to nutritional modes in orchids from the Mediterranean and Macaronesia

We compared the nutritional modes and habitats of orchids (e.g., autotrophic, partially or fully mycoheterotrophic) of the Mediterranean region and adjacent islands of Macaronesia. We hypothesized that ecological factors (e.g., relative light availability, surrounding vegetation) determine the nutritional modes of orchids and thus impose restrictions upon orchid distribution. Covering habitats from dark forests to open sites, orchid samples of 35 species from 14 genera were collected from 20 locations in the Mediterranean and Macaronesia to test for mycoheterotrophy. Mycorrhizal fungi were identified via molecular analyses, and stable isotope analyses were applied to test whether organic nutrients are gained from the fungal associates. Our results show that orchids with partial or full mycoheterotrophy among the investigated species are found exclusively in Neottieae thriving in light-limited forests. Neottioid orchids are missing in Macaronesia, possibly because mycoheterotrophy is constrained by the lack of suitable ectomycorrhizal fungi. Furthermore, most adult orchids of open habitats in the Mediterranean and Macaronesia show weak or no N gains from fungi and no C gain through mycoheterotrophy. Instead isotope signatures of some of these species indicate net plant-to-fungus C transfer.     

Mycoheterotrophic seedling growth of Gentiana zollingeri,a photosynthetic Gentianaceae plant species,in symbioses with arbuscular mycorrhizal fungi

Journal of Plant Research - We found mycoheterotrophic seedling growth (initial mycoheterotrophy) of Gentiana zollingeri, a spring-flowering photosynthetic species of Gentianaceae family. Small...     

Mixotrophy in orchids: insights from a comparative study of green individuals and nonphotosynthetic individuals of Cephalanthera damasonium

Some green orchids obtain carbon (C) from their mycorrhizal fungi and photosynthesis. This mixotrophy may represent an evolutionary step towards mycoheterotrophic plants fully feeding on fungal C. Here, we report on nonphotosynthetic individuals (albinos) of the green Cephalanthera damasonium that likely represent another evolutionary step. Albino and green individuals from a French population were compared for morphology and fertility, photosynthetic abilities, fungal partners (using microscopy and molecular tools), and nutrient sources (as characterized by 15N and 13C abundances). Albinos did not differ significantly from green individuals in morphology and fertility, but tended to be smaller. They harboured similar fungi, with Thelephoraceae and Cortinariaceae as mycorrhizal partners and few rhizoctonias. Albinos were nonphotosynthetic, fully mycoheterotrophic. Green individuals carried out photosynthesis at compensation point and received almost 50% of their C from fungi. Orchid fungi also colonized surrounding tree roots, likely to be the ultimate C source. Transition to mycoheterotrophy may require several simultaneous adaptations; albinos, by lacking some of them, may have reduced ecological success. This may limit the appearance of cheaters in mycorrhizal networks.     

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.     

Mycoheterotrophy evolved from mixotrophic ancestors: evidence in Cymbidium (Orchidaceae)

Background and Aims

Nutritional changes associated with the evolution of achlorophyllous, mycoheterotrophic plants have not previously been inferred with robust phylogenetic hypotheses. Variations in heterotrophy in accordance with the evolution of leaflessness were examined using a chlorophyllous–achlorophyllous species pair in Cymbidium (Orchidaceae), within a well studied phylogenetic background.

Methods

To estimate the level of mycoheterotrophy in chlorophyllous and achlorophyllous Cymbidium, natural ¹³C and ¹⁵N contents (a proxy for the level of heterotrophy) were measured in four Cymbidium species and co-existing autotrophic and mycoheterotrophic plants and ectomycorrhizal fungi from two Japanese sites.

Key Results

δ¹³C and δ¹⁵N values of the achlorophyllous C. macrorhizon and C. aberrans indicated that they are full mycoheterotrophs. δ¹³C and δ¹⁵N values of the chlorophyllous C. lancifolium and C. goeringii were intermediate between those of reference autotrophic and mycoheterotrophic plants; thus, they probably gain 30–50 % of their carbon resources from fungi. These data suggest that some chlorophyllous Cymbidium exhibit partial mycoheterotrophy (= mixotrophy).

Conclusions

It is demonstrated for the first time that mycoheterotrophy evolved after the establishment of mixotrophy rather than through direct shifts from autotrophy to mycoheterotrophy. This may be one of the principal patterns in the evolution of mycoheterotrophy. The results also suggest that the establishment of symbiosis with ectomycorrhizal fungi in the lineage leading to mixotrophic Cymbidium served as pre-adaptation to the evolution of the mycoheterotrophic species. Similar processes of nutritional innovations probably occurred in several independent orchid groups, allowing niche expansion and radiation in Orchidaceae, probably the largest plant family.     

Comparison of green and albino individuals of the partially mycoheterotrophic orchid Epipactis helleborine on molecular identities of mycorrhizal fungi,nutritional modes and gene expression in mycorrhizal roots

Some green orchids obtain carbon from their mycorrhizal fungi, as well as from photosynthesis. These partially mycoheterotrophic orchids sometimes produce fully achlorophyllous, leaf‐bearing (albino) variants. Comparing green and albino individuals of these orchids will help to uncover the molecular mechanisms associated with mycoheterotrophy. We compared green and albino Epipactis helleborine by molecular barcoding of mycorrhizal fungi, nutrient sources based on ¹⁵N and ¹³C abundances and gene expression in their mycorrhizae by RNA‐seq and cDNA de novo assembly. Molecular identification of mycorrhizal fungi showed that green and albino E. helleborine harboured similar mycobionts, mainly Wilcoxina. Stable isotope analyses indicated that albino E. helleborine plants were fully mycoheterotrophic, whereas green individuals were partially mycoheterotrophic. Gene expression analyses showed that genes involved in antioxidant metabolism were upregulated in the albino variants, which indicates that these plants experience greater oxidative stress than the green variants, possibly due to a more frequent lysis of intracellular pelotons. It was also found that some genes involved in the transport of some metabolites, including carbon sources from plant to fungus, are higher in albino than in green variants. This result may indicate a bidirectional carbon flow even in the mycoheterotrophic symbiosis. The genes related to mycorrhizal symbiosis in autotrophic orchids and arbuscular mycorrhizal plants were also upregulated in the albino variants, indicating the existence of common molecular mechanisms among the different mycorrhizal types.     

Shifts in mycorrhizal fungi during the evolution of autotrophy to mycoheterotrophy in Cymbidium (Orchidaceae)

? Premise of the study: Mycoheterotrophic plants, which completely depend upon mycorrhizal fungi for their nutrient supply, have unusual associations with fungal partners. The processes involved in shifts in fungal associations during cladogenesis of plant partners from autotrophy to mycoheterotrophy have not been demonstrated using a robust phylogenetic framework. ? Methods: Consequences of a mycorrhizal shift were examined in Cymbidium (Orchidaceae) using achlorophyllous and sister chlorophyllous species. Fungal associates of the two achlorophyllous mycoheterotrophs (C. macrorhizon and C. aberrans), their close relatives, the chlorophyllous mixotrophs (C. goeringii and C. lancifolium) and an outgroup, the chlorophyllous autotroph C. dayanum, were identified by internal transcribed spacers of the nuclear ribosomal DNA sequences. ? Key results: Molecular identification of mycorrhizal fungi revealed: (1) the outgroup autotroph is predominantly dependent on saprobic Tulasnellaceae, (2) the mixotrophs associate with the Tulasnellaceae and ectomycorrhizal groups including the Sebacinales, Russulaceae, Thelephoraceae and Clavulinaceae, and (3) the two mycoheterotrophs are mostly specialized with ectomycorrhizal Sebacinales. ? Conclusion: Fungal partners in Cymbidium have shifted from saprobic to ectomycorrhizal fungi via a phase of coexistence of both nutritional types of fungi. These three phases correspond to the evolution from autotrophy to mycoheterotrophy via mixotrophy in Cymbidium. We demonstrate that shifts in mycorrhizal fungi correlate with the evolution of nutritional modes in plants. Furthermore, gradual shifts in fungal partners through a phase of coexistence of different types of mycobionts may play a crucial role in the evolution of mycoheterotrophic plants.     

Nitrogen and Phosphorus Nutrition in Mycorrhizal Epacridaceae of South-west Australia

Xylem transport of nitrogen and phosphorus was examined in maturemycorrhizal plants of 41 species in 15 genera of Epacridaceaein native habitat in south-west Australia. Glutamine was theprincipal nitrogenous solute of xylem of all but four species.In the latter species, arginine or asparagine predominated.Nitrate and ammonium comprised minor fractions of xylem (tracheal)sap N, except in two species in which nitrate contributed overhalf of the N. Ratios of total-N:phosphate-P in xylem sap variedwidely (mean 67±18, range 0.2–495) between speciesand habitats. Plants ofCroninia kingiana (syn.Leucopogon kingeanus)from the one habitat showed higher levels of N and P in xylemearly than late in the mycorrhizal season, but there was noconsistent evidence of higher N and P levels from upper thandeeper parts of their root systems. Study of juvenile populationsof four species of epacrids indicated that substantial fractionsof the yearly increment of N, P and dry matter was accumulatedduring the three summer months when infected mycorrhizal hairroots were absent. Glasshouse culture of mycorrhizal plantsof Epacridaceae in habitat soil enriched with decomposed andleached double (¹³C,¹⁵N)-labelled dry matter of wheat showedsubstantial labelling of shoots with¹⁵N but not with¹³C. Plantsfed similarly treated¹⁵N-labelled root residues of maize acquired¹⁵Nbut failed to generate ¹³C values different from those of controlplants. Possible avenues of mycorrhizal and non-mycorrhizalnutrition of Epacridaceae are discussed. amino acids; mycorrhizal nutrition; xylem transport; south-west Australia; Epacridaceae; nitrogen; phosphorus     

Seasonal and environmental changes of mycorrhizal associations and heterotrophy levels in mixotrophic Pyrola japonica (Ericaceae) growing under different light environments

? Premise of the study: Mixotrophy is a strategy whereby plants acquire carbon both through photosynthesis and heterotrophic exploitation of mycorrhizal fungi. In Euro-American Pyroleae species studied hitherto, heterotrophy levels vary according to species, sites of study, and possibly light conditions. We investigated mycorrhizal association and mixotrophy in the Asiatic forest species Pyrola japonica, and their plasticity under different light conditions. ? Methods: Pyrola japonica was sampled bimonthly in sunny and shaded conditions from a deciduous broadleaf forest. We microscopically assessed the rate of fungal colonization and sequenced the ITS to identify the mycorrhizal fungi. We measured (13)C and (15)N isotopic abundances in P. japonica as compared with neighboring autotrophic and mycoheterotrophic plants, to evaluate P. japonica's heterotrophy level. ? Key results: Pyrola japonica formed arbutoid mycorrhizas devoid of fungal mantles, with intracellular hyphal coils and a Hartig net. It tended to be more colonized by mycorrhizal fungi in spring and summer. Most associated fungi belonged to ectomycorrhizal taxa, and 84% of identified fungi were Russula spp. Rate of mycorrhizal colonization and Russula frequency tended to be higher in shaded conditions. Both δ(13)C and δ(15)N values of P. japonica were significantly higher in autotrophic plants, showing that about half of the carbon on average was received from mycorrhizal fungi. Both isotopic values negatively correlated with light availability, suggesting higher heterotrophy levels in shaded conditions. ? Conclusions: The mixotrophic P. japonica undergoes changes in mycorrhizal symbionts and carbon nutrition according to light availability. Our results suggest that during Pyroleae evolution, a tendency to increased heterotrophy emerged in the Pyrola/Orthilia clade.     

Chlorophyllous and Achlorophyllous Specimens of <Emphasis Type="Italic">Epipactis microphylla</Emphasis> (Neottieae,Orchidaceae) Are Associated with Ectomycorrhizal Septomycetes,including Truffles

Mycoheterotrophic species (i.e., achlorophyllous plants obtaining carbon from their mycorrhizal fungi) arose many times in evolution of the Neottieae, an orchid tribe growing in forests. Moreover, chlorophyllous Neottieae species show naturally occurring achlorophyllous individuals. We investigated the fungal associates of such a member of the Neottieae, Epipactis microphylla, to understand whether their mycorrhizal fungi predispose the Neottieae to mycoheterotrophy. Root symbionts were identified by sequencing the fungal ITS of 18 individuals from three orchid populations, including achlorophyllous and young, subterranean individuals. No rhizoctonias (the usual orchid symbionts) were recovered, but 78% of investigated root pieces were colonized by Tuber spp. Other Pezizales and some Basidiomycetes were also found. Using electron microscopy, we demonstrated for the first time that ascomycetes, especially truffles, form typical orchid mycorrhizae. All identified fungi (but one) belonged to taxa forming ectomycorrhizae on tree roots, and four of them were even shown to colonize surrounding trees. This is reminiscent of mycoheterotrophic orchid species that also associate with ectomycorrhizal fungi, although with higher specificity. Subterranean and achlorophyllous E. microphylla individuals thus likely rely on tree photosynthates, and a partial mycoheterotrophy in individuals plants can be predicted. We hypothesize that replacement of rhizoctonias by ectomycorrhizal symbionts in Neottieae entails a predisposition to achlorophylly.     

Evolutionary histories and mycorrhizal associations of mycoheterotrophic plants dependent on saprotrophic fungi

Mycoheterotrophic plants (MHPs) are leafless, achlorophyllous, and completely dependent on mycorrhizal fungi for their carbon supply. Mycorrhizal symbiosis is a mutualistic association with fungi that is undertaken by the majority of land plants, but mycoheterotrophy represents a breakdown of this mutualism in that plants parasitize fungi. Most MHPs are associated with fungi that are mycorrhizal with autotrophic plants, such as arbuscular mycorrhizal (AM) or ectomycorrhizal (ECM) fungi. Although these MHPs gain carbon via the common mycorrhizal network that links the surrounding autotrophic plants, some mycoheterotrophic lineages are associated with saprotrophic (SAP) fungi, which are free-living and decompose leaf litter and wood materials. Such MHPs are dependent on the forest carbon cycle, which involves the decomposition of wood debris and leaf litter, and have a unique biology and evolutionary history. MHPs associated with SAP fungi (SAP-MHPs) have to date been found only in the Orchidaceae and likely evolved independently at least nine times within that family. Phylogenetically divergent SAP Basidiomycota, mostly Agaricales but also Hymenochaetales, Polyporales, and others, are involved in mycoheterotrophy. The fungal specificity of SAP-MHPs varies from a highly specific association with a single fungal species to a broad range of interactions with multiple fungal orders. Establishment of symbiotic culture systems is indispensable for understanding the mechanisms underlying plant–fungus interactions and the conservation of MHPs. Symbiotic culture systems have been established for many SAP-MHP species as a pure culture of free-living SAP fungi is easier than that of biotrophic AM or ECM fungi. Culturable SAP-MHPs are useful research materials and will contribute to the advancement of plant science.

     

Partial mycoheterotrophy in rhizoctonia-associated orchid Cheirostylis liukiuensis

Partially mycoheterotrophic plant species obtain organic carbon, via both photosynthesis and mycorrhizal symbiosis. In this study, we investigated the mycorrhizal fungi association and nutritional mode of Cheirostylis liukiuensis, which is suspected to be a partial mycoheterotrophic plant, due to its characteristic reduced underground organs, low-light growth environment, and some fully mycoheterotrophic species in the phylogenetically related genera. Molecular analysis of the dominant mycobiont and stable isotope analysis suggested that C. liukiuensis is a partial mycoheterotrophic plant predominantly associate with non-ectomycorrhizal Ceratobasidiaceae fungi. As examples of partial mycoheterotrophic orchids exploiting non-ectomycorrhizal rhizoctonia are still limited, this study provides valuable information on the nutritional modes of green orchids.     

Measuring carbon gains from fungal networks in understory plants from the tribe Pyroleae (Ericaceae): a field manipulation and stable isotope approach

Partial mycoheterotrophy, a newly discovered form of mixotrophy in plants, has been described in at least two major lineages of angiosperms, the orchids and ericaceous plants in the tribe Pyroleae. Partial mycoheterotrophy entails carbon gains both directly from photosynthesis and via symbiotic mycorrhizal fungi, but determining the degree of plant dependence on fungal carbon is challenging. The purpose of this study was to determine if two chlorophyllous species of Pyroleae, Chimaphila umbellata and Pyrola picta, were receiving carbon via mycorrhizal networks and, if so, if their proportional dependency on fungal carbon gains increased under reduced light conditions. This was accomplished by a field experiment that manipulated light and plants' access to mycorrhizal networks, and by using the stable carbon isotope composition (δ(13)C) of leaf soluble sugars as a marker for the level of mycoheterotrophy. Based on leaf soluble sugars δ(13)C values, we calculated a site-independent isotope enrichment factor as a measure of fungal contributions to plant C. We found that, under each treatment and over time, the two test species demonstrated different isotopic responses caused by their different intrinsic physiologies. Our data, along with previously published studies, suggest that Chimaphila umbellata is primarily an autotrophic understory plant, while Pyrola picta may be capable of partial mycoheterotrophy. However, in this study, a 50% decrease in light availability did not significantly change the relative dependency of P. picta on carbon gains via mycoheterotrophy.     

First flowering hybrid between autotrophic and mycoheterotrophic plant species: breakthrough in molecular biology of mycoheterotrophy

Among land plants, which generally exhibit autotrophy through photosynthesis, about 880 species are mycoheterotrophs, dependent on mycorrhizal fungi for their carbon supply. Shifts in nutritional mode from autotrophy to mycoheterotrophy are usually accompanied by evolution of various combinations of characters related to structure and physiology, e.g., loss of foliage leaves and roots, reduction in seed size, degradation of plastid genome, and changes in mycorrhizal association and pollination strategy. However, the patterns and processes involved in such alterations are generally unknown. Hybrids between autotrophic and mycoheterotrophic plants may provide a breakthrough in molecular studies on the evolution of mycoheterotrophy. We have produced the first hybrid between autotrophic and mycoheterotrophic plant species using the orchid group Cymbidium. The autotrophic Cymbidium ensifolium subsp. haematodes and mycoheterotrophic C. macrorhizon were artificially pollinated, and aseptic germination of the hybrid seeds obtained was promoted by sonication. In vitro flowering was observed five years after seed sowing. Development of foliage leaves, an important character for photosynthesis, segregated in the first generation; that is, some individuals only developed scale leaves on the rhizome and flowering stems. However, all of the flowering plants formed roots, which is identical to the maternal parent.     

Significant difference in mycorrhizal specificity between an autotrophic and its sister mycoheterotrophic plant species of Petrosaviaceae

Petrosaviaceae is a monocotyledonous plant family that comprises two genera: the autotrophic Japonolirion and the mycoheterotrophic Petrosavia. Accordingly, this plant family provides an excellent system to examine specificity differences in mycobionts between autotrophic and closely related mycoheterotrophic plant species. We investigated mycobionts of Japonolirion osense, the sole species of the monotypic genus, from all known habitats of this species by molecular identification and detected 22 arbuscular mycorrhizal (AM) fungal phylotypes in Archaesporales, Diversisporales, and Glomerales. In contrast, only one AM fungal phylotype in Glomerales was predominantly detected from the mycoheterotrophic Petrosavia sakuraii in a previous study. The high mycobiont diversity in J. osense and in an outgroup plant, Miscanthus sinensis (Poaceae), indicates that fungal specificity increased during the evolution of mycohetrotrophy in Petrosaviaceae. Furthermore, some AM fungal sequences of J. osense showed >99 % sequence similarity to the dominant fungal phylotype of P. sakuraii, and one of them was nested within a clade of P. sakuraii mycobionts. These results indicate that fungal partners are not necessarily shifted, but rather selected for in the course of the evolution of mycoheterotrophy. We also confirmed the Paris-type mycorrhiza in J. osense.     

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.     

Mycoheterotrophic germination of Pyrola asarifolia dust seeds reveals convergences with germination in orchids

Dust seeds that germinate by obtaining nutrients from symbiotic fungi have evolved independently in orchids and 11 other plant lineages. The fungi involved in this 'mycoheterotrophic' germination have been identified in some orchids and non-photosynthetic Ericaceae, and proved identical to mycorrhizal fungi of adult plants. We investigated a third lineage, the Pyroleae, chlorophyllous Ericaceae species whose partial mycoheterotrophy at adulthood has recently attracted much attention. We observed experimental Pyrola asarifolia germination at four Japanese sites and investigated the germination pattern and symbiotic fungi, which we compared to mycorrhizal fungi of adult plants. Adult P. asarifolia, like other Pyroleae, associated with diverse fungal species that were a subset of those mycorrhizal on surrounding trees. Conversely, seedlings specifically associated with a lineage of Sebacinales clade B (endophytic Basidiomycetes) revealed an intriguing evolutionary convergence with orchids, some of which also germinate with Sebacinales clade B. Congruently, seedlings clustered spatially together, but not with adults. This unexpected transition in specificity and ecology of partners could support the developmental transition from full to partial mycoheterotrophy, but probably challenges survival and distribution during development. We discuss the physiological and ecological traits that predisposed to the repeated recruitment of Sebacinales clade B for dust seed germination.