Novel mycorrhizal cheating in a green orchid: Cremastra appendiculata depends on carbon from deadwood through fungal associations.

To date, there has been no robust evidence for the exploitation of saprotrophic non-rhizoctonia fungi by green plants, although some fully mycoheterotrophic orchids are known to exploit them, and mycoheterotrophic evolution has probably occurred through intermediate mixotrophic stages. We investigated the physiological ecology of a fully mycoheterotrophic species Cremastra aphylla and its photosynthetic sister species Cremastra appendiculata, which putatively exploit saprotrophic fungi. Their mycorrhizal partners and ultimate nutritional sources were determined using molecular, stable isotopic, and radiocarbon analysis. Both Cremastra aphylla and Cremastra appendiculata were consistently associated with wood-decaying Psathyrellaceae. In addition, both species were highly enriched in carbon-13 (13 C) and, to a less degree, in nitrogen-15 (15 N). The δ13 C and δ15 N values of Cremastra appendiculata were intermediate between those of Cremastra aphylla and those of autotrophic plants. All Cremastra appendiculata samples and two Cremastra aphylla samples exhibited elevated Δ14 C values due to the acquisition of carbon fixed in wood during the past decades (14 C-enriched bomb carbon). Our multifaceted evidence indicated that both species obtained carbon from deadwood via saprotrophic fungi. Our findings strongly suggest that mixotrophic relationships associated with wood-decaying fungi represent a novel evolutionary pathway for full mycoheterotrophy in orchids.

Mycobiont diversity and first evidence of mixotrophy associated with Psathyrellaceae fungi in the chlorophyllous orchid Cremastra variabilis.

Mixotrophy (MX, also called partial mycoheterotrophy) in plants is characterized by isotopic abundances that differ from those of autotrophs. Previous studies have evaluated mycoheterotrophy in MX plants associated with fungi of similar ecological characteristics, but little is known about the differences in the relative abundances of 13C and 15N in an orchid species that associates with several different mycobionts species. Since the chlorophyllous orchid Cremastra variabilis Nakai associates with various fungi with different ecologies, we hypothesized that it may change its relative abundances of 13C and 15N depending on the associated mycobionts. We investigated mycobiont diversity in the chlorophyllous orchid C. variabilis together with the relative abundance of 13C and 15N and morphological underground differentiation (presence or absence of a mycorhizome with fungal colonization). Rhizoctonias (Tulasnellaceae, Ceratobasidiaceae, Sebacinales) were detected as the main mycobionts. High differences in δ13C values (- 34.7 to - 27.4 ‰) among individuals were found, in which the individuals associated with specific Psathyrellaceae showed significantly high relative abundance of 13C. In addition, Psathyrellaceae fungi were always detected on individuals with mycorhizomes. In the present study, MX orchid association with non-rhizoctonia saprobic fungi was confirmed, and the influence of mycobionts on morphological development and on relative abundance of 13C and 15N was discovered. Cremastra variabilis may increase opportunities to gain nutrients from diverse partners, in a bet-hedging plasticity that allows colonization of various environmental conditions.

Specialized mycorrhizal association between a partially mycoheterotrophic orchid Oreorchis indica and a Tomentella taxon.

The evolution of full mycoheterotrophy in orchids likely occurs through intermediate stages (i.e., partial mycoheterotrophy or mixotrophy), in which adult plants obtain nutrition through both autotrophy and mycoheterotrophy. However, because of its cryptic manifestation, partial mycoheterotrophy has only been confirmed in slightly more than 20 orchid species. Here, we hypothesized that Oreorchis indica is partially mycoheterotrophic, since (i) Oreorchis is closely related to leafless Corallorhiza, and (ii) it possesses clustered, multi-branched rhizomes that are often found in fully mycoheterotrophic orchids. Accordingly, we investigated the nutritional modes of O. indica in a Japanese subboreal forest by measuring the 13C and 15N abundances and by community profiling of its mycorrhizal fungi. We found that O. indica mycorrhizal samples (all 12 samples from four individuals) were predominantly colonized by a single OTU of the obligate ectomycorrhizal Tomentella (Thelephoraceae). In addition, the leaves of O. indica were highly enriched in both 13C and 15N compared with those of co-occurring autotrophic plants. It was estimated that O. indica obtained 44.4 ± 6.2% of its carbon from fungal sources. These results strongly suggest that in the Oreorchis-Corallorhiza clade, full mycoheterotrophy evolved after the establishment of partial mycoheterotrophy, rather than through direct shifts from autotrophy.

Orchid epiphytes do not receive organic substances from living trees through fungi.

Numerous studies of terrestrial orchids have demonstrated widespread partial mycoheterotrophy, particularly the possibility of obtaining organic matter from surrounding trees through a common fungal network. Fungi are also widespread in epiphytic orchid roots, but there have been no attempts to determine if epiphytes accept organic matter from the living stems of their phorophytes. We hypothesise that such transfer does not exist because epiphytes and phorophytes harbour different fungal communities. To test this hypothesis, we tagged three short Randia sp. trees with 13C-enriched CO2 and examined 13C transfer from the phorophyte into the epiphytic orchids Grosourdya appendiculata, Dendrobium oligophyllum and Gastrochilus sp. in Cat Tien National Park, (South Vietnam, Cat Tien National Park, plot size approx. 1 ha). The coincidence of fungal sequences in the orchid roots and in the branches on which they grew was also examined. We did not detect 13C label moving from phorophytes to epiphytes. Using Illumina sequencing, 162 fungal operational taxonomic units (OTUs) were detected. The fungal communities were significantly different between the roots of epiphytes and branches of phorophytes, although no strict fungal specificity at the species level was found in either epiphytes or phorophytes.

Mycorrhizal divergence and selection against immigrant seeds in forest and dune populations of the partially mycoheterotrophic Pyrola rotundifolia.

Plant populations occupying different habitats may diverge from each other over time and gradually accumulate genetic and morphological differences, ultimately resulting in ecotype or even species formation. In plant species that critically rely on mycorrhizal fungi, differences in mycorrhizal communities can contribute to ecological isolation by reducing or even inhibiting germination of immigrant seeds. In this study, we investigated whether the mycorrhizal communities available in the soil and associating with the roots of seedlings and adult plants of the partially mycoheterotrophic Pyrola rotundifolia differed between populations growing in sand dunes and forests. In addition, reciprocal germination experiments were performed to test whether native seeds showed higher germination than immigrant seeds. Our results showed that the mycorrhizal communities differed significantly between forest and dune populations, and that within populations seedlings and adults also associated with different mycorrhizal communities. In both forest and dune populations, mycorrhizal communities were dominated by members of the Thelephoraceae, but dune populations showed a higher incidence of members of the Inocybaceae, whereas forest populations showed a high abundance of members of the Russulaceae. Reciprocal germination experiments showed that native seeds showed a higher germination success than immigrant seeds and this effect was most pronounced in dune populations. Overall, these results demonstrate that plants of P. rotundifolia growing in dune and forest habitats associate with different mycorrhizal communities and that reduced germination of non-native seeds may contribute to reproductive isolation. We conclude that selection against immigrants may constitute an important reproductive barrier at early stages of the speciation process.

Comparative morphological analysis of two parallel mycoheterotrophic transitions reveals divergent and convergent traits in the genus Pyrola (Pyroleae, Ericaceae).

The genus Pyrola includes species with different degree of mycoheterotrophy; some species possess individuals that rely on all carbon through their associations with fungi (full mycoheterotrophy, FM), whereas some species obtain carbon through both fungi and photosynthesis by itself (partial mycoheterotrophy, PM). To investigate how plant functional traits of photosynthesis and reproduction are related to the degree of mycoheterotrophy in the initial stage of the transition from PM to FM, we determined morphological traits in FM (or nearly FM) and PM species in two independent lineages, P. picta and P. japonica complexes. We used herbarium specimens and examined leaf number, leaf area, flower number, and scape length in FM or nearly FM species (P. aphylla and P. subaphylla) and PM species (P. picta s.l. and P. japonica). We found a leaf area reduction in FM (or nearly FM) species in both lineages, suggesting that this is a convergent trait. The number of flowers was not significantly different between FM (or nearly FM) and PM species in both lineages. On the other hand, differences in the variation between FM (or nearly FM) and PM species were found in some traits between the two lineages. The FM (or nearly FM) species in one lineage only possessed rudimentary leaves, whereas that in the other linage possessed a few small, ordinary leaves in addition to those with only rudimentary leaves. The scape length of the FM (or nearly FM) species was significantly longer than that of PM species in one lineage, whereas it was shorter in the other lineage. The different and common variations are divergent and convergent traits, respectively, that could be associated with the transition to FM in Pylora. In addition, shoots of both PM species occasionally lacked ordinary leaves, possibly indicating possession of these shoots is preadaptation for the transition to FM in Pyrola.

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 15 N and 13 C 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.

You are what you get from your fungi: nitrogen stable isotope patterns in Epipactis species.

Background and Aims: Partially mycoheterotrophic plants are enriched in 13 C and 15 N compared to autotrophic plants. Here, it is hypothesized that the type of mycorrhizal fungi found in orchid roots is responsible for variation in 15 N enrichment of leaf tissue in partially mycoheterotrophic orchids. Methods: The genus Epipactis was used as a case study and carbon and nitrogen isotope abundances of eight Epipactis species, fungal sporocarps of four Tuber species and autotrophic references were measured. Mycorrhizal fungi were identified using molecular methods. Stable isotope data of six additional Epipactis taxa and ectomycorrhizal and saprotrophic basidiomycetes were compiled from the literature. Key Results: The 15 N enrichment of Epipactis species varied between 3·2 ± 0·8 ‰ ( E. gigantea ; rhizoctonia-associated) and 24·6 ± 1·6 ‰ ( E. neglecta ; associated with ectomycorrhizal ascomycetes). Sporocarps of ectomycorrhizal ascomycetes (10·7 ± 2·2 ‰) were significantly more enriched in 15 N than ectomycorrhizal (5·2 ± 4·0 ‰) and saprotrophic basidiomycetes (3·3 ± 2·1 ‰). Conclusions: As hypothesized, it is suggested that the observed gradient in 15 N enrichment of Epipactis species is strongly driven by 15 N abundance of their mycorrhizal fungi; i.e. ɛ 15 N in Epipactis spp. associated with rhizoctonias < ɛ 15 N in Epipactis spp. with ectomycorrhizal basidiomycetes < ɛ 15 N in Epipactis spp. with ectomycorrhizal ascomycetes and basidiomycetes < ɛ 15 N in Epipactis spp. with ectomycorrhizal ascomycetes.

Mycorrhizal Associations and Trophic Modes in Coexisting Orchids: An Ecological Continuum between Auto- and Mixotrophy.

Two distinct nutritional syndromes have been described in temperate green orchids. Most orchids form mycorrhizas with rhizoctonia fungi and are considered autotrophic. Some orchids, however, associate with fungi that simultaneously form ectomycorrhizas with surrounding trees and derive their carbon from these fungi. This evolutionarily derived condition has been called mixotrophy or partial mycoheterotrophy and is characterized by 13C enrichment and high N content. Although it has been suggested that the two major nutritional syndromes are clearly distinct and tightly linked to the composition of mycorrhizal communities, recent studies have challenged this assumption. Here, we investigated whether mycorrhizal communities and nutritional syndromes differed between seven green orchid species that co-occur under similar ecological conditions (coastal dune slacks). Our results showed that mycorrhizal communities differed significantly between orchid species. Rhizoctonia fungi dominated in Dactylorhiza sp., Herminium monorchis, and Epipactis palustris, which were autotrophic based on 13C and N content. Conversely, Liparis loeselii and Epipactis neerlandica associated primarily with ectomycorrhizal fungi but surprisingly, 13C and N content supported mixotrophy only in E. neerlandica. This, together with the finding of some ectomycorrhizal fungi in rhizoctonia-associated orchids, suggests that there exists an ecological continuum between the two syndromes. The presence of a large number of indicator species associating with individual orchid species further confirms previous findings that mycorrhizal fungi may be important factors driving niche-partitioning in terrestrial orchids and therefore contribute to orchid coexistence.