Do exchangeable hydrogens affect the evaluation of partial mycoheterotrophy in orchids? Insights from δ²H analysis in...

To evaluate the nutritional modes of orchids associated with 'rhizoctonia' fungi, analyses of hydrogen (δ²H), carbon (δ¹³C), and nitrogen (δ¹⁵N) 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 δ¹³C, δ¹⁵N, and δ²H analyses on bulk samples. Additionally, we conducted δ²H 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 δ²H of orchids were consistently higher than those of surrounding autotrophic plants, irrespective of the three pretreatments. Although the rhizoctonia-associated orchid exhibited lower δ¹³C, its δ²H was higher than those of the autotrophs. Notably, among all response variables, δ¹⁵N and δ²H 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 δ¹⁵N and δ²H benefits the assessment of partial mycoheterotrophy among rhizoctonia-associated orchids.