Evidence for partial mycoheterotrophy during fruiting in two leafless orchids, Cremastra aphylla and C. saprophytica

The evolutionary transition from autotrophy to full mycoheterotrophy is often viewed as a progressive loss of photosynthetic capacity, yet comparative studies of closely related leafless orchids that retain residual photosynthesis remain scarce. The Cremastra appendiculata species complex provides a valuable system for testing whether dependence on fungal carbon varies among leafless species. We compared the nutritional modes of two closely related leafless orchids, Cremastra aphylla and C. saprophytica, at flowering and fruiting using natural-abundance stable isotope analyses and measurements of chlorophyll concentration and fluorescence. Both species retained functional, albeit limited, photosynthetic capacity. From flowering to fruiting, chlorophyll concentrations and the maximum quantum yield of photosystem II increased, whereas δ¹³C values decreased, a pattern consistent with enhanced photosynthetic capacity during reproductive development. However, C. aphylla consistently showed greater ¹³C enrichment relative to autotrophic reference plants and had a significantly lower chlorophyll concentration during flowering than C. saprophytica, indicating greater dependence on fungal carbon. These results demonstrate that leaflessness does not necessarily imply full mycoheterotrophy and, more importantly, that dependence on fungal carbon can differ even between closely related leafless species. Together with developmental-stage-dependent changes within each species, these interspecific differences suggest that the transition from partial to full mycoheterotrophy can proceed through multiple incremental steps even after leaflessness has evolved.