Japonolirion osense, a close relative of the mycoheterotrophic genus Petrosavia, exhibits complete autotrophic capabilities.

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.