While orchids germinate thanks to carbon from their symbiotic fungi, variable carbon exchanges exist between adult orchids and their mycorrhizal fungi. Although some truly autotrophic orchids reward their fungi with carbon at adulthood, some species remain achlorophyllous and fully dependent on fungal carbon (mycoheterotrophy). Others are photosynthetic but also import fungal carbon: The so-called mixotrophic (MX) orchids rely on fungi of diverse taxonomy and ecology. Here, we classify MX nutrition of orchids into three types. Type I mixotrophy associates with diverse Asco- and Basidiomycota that are either saprotrophic or ectomycorrhizal, entailing enrichment of the orchids in 2H, 13C, and 15N. The two other types associate with rhizoctonias, a polyphyletic assemblage of Basidiomycotas that is ancestrally mycorrhizal in orchids. Type II mixotrophy associates with rhizoctonias that secondarily evolved into saprotrophic or ectomycorrhizal ecology, and thus enrich the orchid in 2H, 13C, and 15N. Type III mixotrophy, which remains debated, associates with rhizoctonias that have retained their ancestral lifestyle, that is saprotrophic and/or endophytic in nonorchids, and only entail orchid enrichment in 2H and 15N. Based on a case study of achlorophyllous variants in Mediterranean Ophrys and on published data, we discuss the distinct nature and research perspectives of type III mixotrophy.

• Keywords
• isotopic enrichment, mycoheterotrophy, mycorrhiza, rhizoctonia, saprotrophy,
• MeSH
• Basidiomycota physiology   MeSH
• Phylogeny MeSH
• Heterotrophic Processes MeSH
• Mycorrhizae physiology   MeSH
• Orchidaceae * microbiology   physiology   MeSH
• Symbiosis MeSH
• Carbon metabolism   MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Carbon MeSH

The plant kingdom exhibits a diversity of nutritional strategies, extending beyond complete autotrophy. In addition to full mycoheterotrophs and holoparasites, it is now recognized that a greater number of green plants than previously assumed use partly of fungal carbon. These are termed partial mycoheterotrophs or mixotrophs. Notably, some species exhibit a dependency on fungi exclusively during early ontogenetic stages, referred to as initial mycoheterotrophy. Japonolirion osense, a rare plant thriving in serpentinite soils, emerges as a potential candidate for initial mycoheterotrophy or mixotrophy. Several factors support this hypothesis, including its diminutive sizes of shoot and and seeds, the establishment of Paris-type arbuscular mycorrhizal associations, its placement within the Petrosaviales-largely composed of fully mycoheterotrophic species-and its ability to face the challenging conditions of its environment. To explore these possibilities, our study adopts a multidisciplinary approach, encompassing stable isotope abundance analyses, in vitro experiments, anatomical analyses, and comparative plastome analyses. Our study aims to (1) determine whether J. osense relies on fungal carbon during germination, indicating initial mycoheterotrophy, (2) determine if it employs a dual carbon acquisition strategy as an adult, and (3) investigate potential genomic reductions in photosynthetic capabilities. Contrary to expectations, our comprehensive findings strongly indicate that J. osense maintains complete autotrophy throughout its life cycle. This underscores the contrasting nutritional strategies evolved by species within the Petrosaviales.

• Keywords
• Japonolirion osense, In vitro, Mixotrophy, Petrosaviales, Plastome, Stable isotopes,
• MeSH
• Autotrophic Processes * MeSH
• Heterotrophic Processes * MeSH
• Mycorrhizae physiology   MeSH
• Carbon metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Carbon MeSH

We have investigated whether mycobiont identity and environmental conditions affect morphology and physiology of the chlorophyllous orchid: Cremastra variabilis. This species grows in a broad range of environmental conditions and associates with saprotrophic rhizoctonias including Tulasnellaceae and saprotrophic non-rhizoctonian fungi from the family Psathyrellaceae. We cultured the orchid from seeds under aseptic culture conditions and subsequently inoculated the individuals with either a Tulasnellaceae or a Psathyrellaceae isolate. We observed underground organ development of the inoculated C. variabilis plants and estimated their nutritional dependency on fungi using stable isotope abundance. Coralloid rhizome development was observed in all individuals inoculated with the Psathyrellaceae isolate, and 1-5 shoots per seedling grew from the tip of the coralloid rhizome. In contrast, individuals associated with the Tulasnellaceae isolate did not develop coralloid rhizomes, and only one shoot emerged per plantlet. In darkness, δ13C enrichment was significantly higher with both fungal isolates, whereas δ15N values were only significantly higher in plants associated with the Psathyrellaceae isolate. We conclude that C. variabilis changes its nutritional dependency on fungal symbionts depending on light availability and secondly that the identity of fungal symbiont influences the morphology of underground organs.

• Keywords
• Cremastra variabilis, Coralloid rhizome, Orchidaceae, Stable isotope, Symbiotic culture,
• MeSH
• Agaricales * MeSH
• Basidiomycota * MeSH
• Humans MeSH
• Mycorrhizae * physiology   MeSH
• Orchidaceae * microbiology   MeSH
• Seedlings microbiology   MeSH
• Symbiosis MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

BACKGROUND: Mycorrhiza is a ubiquitous form of symbiosis based on the mutual, beneficial exchange of resources between roots of autotrophic (AT) plants and heterotrophic soil fungi throughout a complex network of fungal mycelium. Mycoheterotrophic (MH) and mixotrophic (MX) plants can parasitise this system, gaining all or some (respectively) required nutrients without known reciprocity to the fungus. We applied, for the first time, an ecological stoichiometry framework to test whether trophic mode of plants influences their elemental carbon (C), nitrogen (N), and phosphorus (P) composition and may provide clues about their biology and evolution within the framework of mycorrhizal network functioning. RESULTS: We analysed C:N:P stoichiometry of 24 temperate orchid species and P concentration of 135 species from 45 plant families sampled throughout temperate and intertropical zones representing the three trophic modes (AT, MX and MH). Welch's one-way ANOVA and PERMANOVA were used to compare mean nutrient values and their proportions among trophic modes, phylogeny, and climate zones. Nutrient concentration and stoichiometry significantly differentiate trophic modes in orchids. Mean foliar C:N:P stoichiometry showed a gradual increase of N and P concentration and a decrease of C: nutrients ratio along the trophic gradient AT < MX < MH, with surprisingly high P requirements of MH orchids. Although P concentration in orchids showed the trophy-dependent pattern regardless of climatic zone, P concentration was not a universal indicator of trophic modes, as shown by ericaceous MH and MX plants. CONCLUSION: The results imply that there are different evolutionary pathways of adaptation to mycoheterotrophic nutrient acquisition, and that the high nutrient requirements of MH orchids compared to MH plants from other families may represent a higher cost to the fungal partner and consequently lead to the high fungal specificity observed in MH orchids.

• Keywords
• Mycoheterotrophy, Nitrogen, Orchids, Phosphorus, Stoichiometry, Trophic modes,
• MeSH
• Acclimatization MeSH
• Analysis of Variance MeSH
• Nitrogen MeSH
• Phosphorus MeSH
• Mycorrhizae * MeSH
• Carbon * MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Nitrogen MeSH
• Phosphorus MeSH
• Carbon * MeSH