Partial mycoheterotrophy in the arbuscular mycorrhizal Gentiana squarrosa (Gentianaceae) demonstrated by coculture assays using C3 and C4 plants

Partial mycoheterotrophy is a nutritional mode in which green plants supplement photosynthetically derived carbon with fungal carbon obtained from associated fungi. In green arbuscular mycorrhizal (AM) plants, however, detecting partial mycoheterotrophy using natural ¹³C abundance is challenging, because the δ¹³C signatures of AM fungi often resemble those of their host plants. To overcome this limitation, we exploited the large natural δ¹³C contrast between C₃ and C₄ plants as an intrinsic tracer and tested for the incorporation of donor-derived carbon into the shoots of Gentiana squarrosa (Gentianaceae). Using an AM fungus commonly associated with G. squarrosa, we conducted coculture experiments with either a C₃ or C₄ companion plant. A compartmentalized U-shaped coculture system was employed, in which the roots of the companion plant were separated from G. squarrosa seedlings by a nylon mesh that permitted only hyphal passage, thereby minimizing alternative carbon transfer pathways, such as indirect transfer via CO₂ released by donor root respiration. Shoot δ¹³C values of G. squarrosa were significantly higher when seedlings were connected to a C₄ companion than when connected to a C₃ companion. Furthermore, among individuals grown with a C₄ companion, shoot dry mass was strongly and positively correlated with shoot δ¹³C values. These results demonstrate the incorporation of donor-derived carbon into G. squarrosa via AM fungal networks, providing robust evidence of partial mycoheterotrophy and suggesting that the mycoheterotrophy contributed to growth in this AM-associated species.