Summary Mycoheterotrophic plants depend entirely on fungal associations for organic nutrients. While most mycoheterotrophic plants are associated with the mycorrhizal partners of surrounding green plants, some mycoheterotrophs are believed to obtain carbon from decaying litter or dead wood by parasitising saprotrophic fungi, based on culture experiments and 13C and 15N isotopic signatures. The carbon age (the time since carbon was fixed from atmospheric CO2 by photosynthesis) can be estimated by measuring the concentration of 14C arising from the bomb tests of the 1950s and 1960s. Given that mycorrhizal fungi obtain photosynthate from their plant partners, and saprotrophic wood‐decaying fungi obtain carbon from older sources, radiocarbon could represent a new and powerful tool to investigate carbon sources of mycoheterotrophic plants. We showed that the Δ14C values of mycoheterotrophs exploiting ectomycorrhizal fungi were close to 0‰, similar to those of autotrophic plants. By contrast, the Δ14C values of mycoheterotrophs exploiting saprotrophic fungi ranged from 110.7‰ to 324.8‰, due to the 14C‐enriched bomb carbon from dead wood via saprotrophic fungi. Our study provides evidence supporting that some mycoheterotrophic orchids depend on forest woody debris. Our study also indicates that radiocarbon could be used to predict the trophic strategies of mycoheterotroph‐associated fungal symbionts.

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 N and 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.

Oxygyne Schltr. (Thismiaceae) is a rare and little-known genus of achlorophyllous mycoheterotrophic perennial herbs with one of the most remarkable distributions of all angiosperm plant genera globally, being disjunct between Japan and West–Central Africa. Each species is known only from a single location, and in most cases from a single specimen. This monographic study names, describes and maps two new species, Oxygyne duncanii Cheek from cloud forest in SW Region Cameroon and O. frankei Cheek from gallery forest in the Central African Republic, representing the first new Oxygyne species described from Africa in 112 years, and raising the number of described Oxygyne species from four to six. Oxygyne duncanii is remarkable for sharing more morphological characters with two of the three Japanese species (O. hyodoi C.Abe & Akasawa, O. shinzatoi (H. Ohashi) Tsukaya) than with the geographically much closer type species of the genus, O. triandra from Mt Cameroon. Based mainly on herbarium specimens and field observations made in Cameroon and Japan during a series of botanical surveys, we provide descriptions, synonymy, mapping and extinction risk assessments for each species of Oxygyne, together with keys to the genera of Thismiaceae and the species of Oxygyne. The subterranean structures of African Oxygyne are described for the first time, and found to be consistent with those of the Japanese species. We review and reject an earlier proposal that the Japanese species should be segregated from the African species as a separate genus, Saionia Hatus. The only character that separates the two disjunct species groups is now flower colour: blue or partly-blue in the Japanese species compared with orange-brown in the African species. Studies of the pollination biology and mycorrhizal partners of Oxygyne are still lacking. Two of the six species, O. triandra Schltr. and O. hyodoi, appear to be extinct, and the remaining four are assessed as Critically Endangered using the IUCN 2012 criteria. To avoid further extinction, an urgent requirement is for conservation management of the surviving species in the wild. Since few achlorophyllous mycoheterotrophs have been successfully cultivated from seed to maturity, ex situ conservation will not be viable for these species and protection in the wild is the only viable option. While natural habitat survives, further botanical surveys could yet reveal additional new species between Central Africa and Japan.

Thismia is a genus of > 80 mycoheterotrophic species characterized by a peculiar appearance and complex floral morphology. A significant proportion of the species and morphological diversity of Thismia has only been uncovered in the past two decades, and new discoveries continue to be made. Given that many new data have recently become available, and the most comprehensive taxonomic revision of the genus from 1938 addresses less than half of the currently known species, previous hypotheses for species relationships and infrageneric taxonomic classification in Thismia was in need of review. Extensive molecular phylogenetic studies of Thismia at the genus level have never been presented. We investigate the phylogenetic relationships of 41 species (and one variety) of Thismia from the Old World. Our study comprises 68 specimens (for 28 of which the data were newly generated), including outgroup taxa broadly representing Thismiaceae (= Burmanniaceae p.p. sensuAPG IV, 2016), and is based on two nuclear and one mitochondrial marker. We use maximum likelihood and Bayesian inference to infer relationships among the taxa. We also constructed a morphological dataset of 12 mostly floral characters, comparing these characters to hypotheses based on molecular evidence to identify putative synapomorphies for major clades and to discuss hypotheses regarding the evolution of structural traits in the genus. Our analyses indicate that the majority of currently accepted infrageneric taxa of Thismia are polyphyletic. We find support for the monophyly of the Old World group, in which we recognize five well-supported lineages (clades); the only New World species studied appears to be related to the Neotropical genus Tiputinia. Ancestral state reconstructions demonstrate that the evolution of most morphological characters was homoplastic, but we identify characters that provide each of the five clades of Old World Thismia with a unique morphological description. The geographical distribution of the species under study is also shown to be consistent with the major clades. Our investigation provides a phylogenetic basis for the development of a novel sectional classification of Thismia reflecting morphological and geographical traits.

Few studies have examined the reproductive ecology of mycoheterotrophic plants, but the existing literature hypothesizes that they adopt a self-pollinating strategy. Although growing evidence indicates that some rewarding mycoheterotrophic plants depend (at least partially) on an insect-mediated pollination system, it remains unclear whether some mycoheterotrophic plants can attract pollinators without nectar or other rewards. Moreover, in a broader evolutionary/ecological context, the question of whether the evolution of mycoheterotrophy induces a shift in pollination pattern is still unknown. Here I present a comparative investigation into the breeding system of two fully mycoheterotrophic orchids, Cymbidium macrorhizon and C. aberrans, and their closest extant relative, the mixotrophic C. lancifolium. Pollination experiments were conducted to determine the breeding system of these plants. In addition, flower visitors that might contribute to pollination were recorded. Flowers at different maturity stages were examined to investigate mechanisms enabling or limiting self-fertilization. While nectarless flowers of C. lancifolium and C. macrorhizon can successfully attract potential pollinator honeybees, all three Cymbidium possess an effective self-pollination system in which the rostellum that physically separates the stigma and pollinia is absent. Because mixotrophic and mycoheterotrophic Cymbidium occupy low-light niches, pollinator foraging would be negatively influenced by low-light intensity. In partial and fully mycoheterotrophic Cymbidium, autogamy would likely be favoured as a reproductive assurance to compensate for pollinator limitation due to their lack of nectar and pollinators' hostile habitat preferences.

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This is the first quantitative investigation of the host range and selectivity of hemiparasitic plants of the Santalales. The preference for Fabaceae as hosts may be linked to the greater nutrient availability in these nitrogen-fixing plants.

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