Isotopic and genetic evidence of partial mycoheterotrophy in leafy variants of Cremastra aphylla, a predominantly leafless orchid

Abstract Background and Aims The evolution of mycoheterotrophy represents a major evolutionary transition that likely proceeds through intermediate stages such as initial and partial mycoheterotrophy. Cremastra aphylla, a predominantly leafless, fully mycoheterotrophic orchid, occasionally produces individuals with well-developed green leaves that likely contain chlorophyll. However, it remains unclear whether these forms represent phenotypic variants or distinct taxa with independent evolutionary origins. Additionally, given the occasional co-occurrence of C. aphylla with the closely related, leafy C. appendiculata, these individuals may have regained some photosynthetic capacity via hybridization or introgression. Methods We integrated chlorophyll concentration and fluorescence measurements, stable isotope (δ13C and δ15N) analysis, MIG-seq-based phylogenomics, and metabarcoding of mycorrhizal partners to investigate the physiological ecology and evolutionary background of both leafless and leafy C. aphylla individuals. Key Results Both morphotypes exhibited elevated δ13C values compared to co-occurring autotrophic species. However, leafy individuals showed significantly lower values, indicating the presence of 13C-depleted photosynthates. A two-source mixing model based on 13C enrichment indicated that photosynthesis contributed approximately 40% of leaf carbon and 20% of floral carbon. Chlorophyll analyses confirmed active chlorophyll synthesis and photosystem II efficiency in leafy individuals, comparable to those of autotrophic plants. Fungal metabarcoding revealed that both morphotypes, each bearing coralloid rhizomes, associated with the same Psathyrellaceae OTU, likely Coprinellus magnoliae. MIG-seq analysis detected no introgression with C. appendiculata and no genetic differentiation between the morphotypes, ruling out the possibility that the leafy form represents either an introgressed lineage or an undescribed taxon. Conclusions These findings provide the first integrated isotopic and genetic evidence for partial mycoheterotrophy in leafy C. aphylla individuals, although they remain highly dependent on fungal carbon. Our results refine the current understanding of the nutritional continuum in partially mycoheterotrophic orchids and highlight C. aphylla as a valuable model for investigating the evolutionary transition toward full mycoheterotrophy.