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

## TL;DR

Some leafy forms of a mostly leafless orchid still rely partly on fungi for nutrients, as shown by isotope and genetic studies.

## Contribution

First integrated isotopic and genetic evidence of partial mycoheterotrophy in leafy individuals of Cremastra aphylla.

## Key findings

- Leafy C. aphylla individuals show partial reliance on photosynthesis and fungal carbon.
- Both leafy and leafless forms share the same mycorrhizal fungus and lack genetic differentiation.
- Photosynthesis contributes about 40% of leaf carbon in leafy C. aphylla individuals.

## Abstract

The evolution of mycoheterotrophy represents a major evolutionary transition that is likely to proceed 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 are likely to contain chlorophyll. However, it remains unclear whether these forms represent phenotypic variants or an undescribed taxon distinct from true C. aphylla. Additionally, given the occasional co-occurrence of C. aphylla with the closely related, leafy Cremastra appendiculata, these individuals might have regained some photosynthetic capacity via hybridization or introgression.

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.

Both morphotypes exhibited elevated δ13C values in comparison 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 ∼40 % of leaf carbon and ∼20 % of floral carbon. Chlorophyll analyses confirmed active chlorophyll synthesis and photosystem II efficiency in leafy individuals, with values comparable to those of autotrophic plants. Fungal metabarcoding revealed that both morphotypes, each bearing coralloid rhizomes, were associated with the same Psathyrellaceae operational taxonomic unit, probably 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.

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 towards full mycoheterotrophy.