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To evaluate the nutritional modes of orchids associated with 'rhizoctonia' fungi, analyses of hydrogen (δ 2 H), carbon (δ 13 C), and nitrogen (δ 15 N) stable isotope ratios are usually adopted. However, previous studies have not fully accounted for exchangeable hydrogens, which could affect these evaluations. Here, we performed standard δ 13 C, δ 15 N, and δ 2 H analyses on bulk samples. Additionally, we conducted δ 2 H analysis on α-cellulose and cellulose nitrate samples to investigate whether the heterogeneity of exchangeable hydrogens among plant species influences the assessment of nutritional modes. The δ 2 H of orchids were consistently higher than those of surrounding autotrophic plants, irrespective of the three pretreatments. Although the rhizoctonia-associated orchid exhibited lower δ 13 C, its δ 2 H was higher than those of the autotrophs. Notably, among all response variables, δ 15 N and δ 2 H exhibited high abilities for discriminating the nutritional modes of rhizoctonia-associated orchids. These results indicate that a time-efficient bulk sample analysis is an effective method for evaluating plant nutritional modes, as the heterogeneity of exchangeable hydrogens does not significantly impact the estimation. Using δ 15 N and δ 2 H benefits the assessment of partial mycoheterotrophy among rhizoctonia-associated orchids., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

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 δ 13 C and δ 15 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 Δ 14 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., (© 2021 The Authors. New Phytologist © 2021 New Phytologist Foundation.)