# Mycoheterotrophy and plastid genome evolution in the early-diverging epidendroid orchid tribe Nervilieae: independent transitions in Epipogium and Stereosandra

## TL;DR

This study explores how a rare orchid species, Stereosandra javanica, evolved to become a non-photosynthetic parasite, revealing new insights into the genetic changes involved in this transition.

## Contribution

The study provides the first genetic data for Stereosandra and shows it represents an independent evolution of mycoheterotrophy in orchids.

## Key findings

- Stereosandra's plastid genome shows significant gene loss and relaxed selection, but not as extreme as in Epipogium.
- Nuclear phylogenomic analyses confirm Stereosandra's close relationship with Epipogium and Nervilia.
- Ancestral state reconstruction indicates multiple independent origins of mycoheterotrophy in the orchid tribe Nervilieae.

## Abstract

Parasitic organisms are of interest in evolutionary biology, often displaying drastic modifications in morphology, physiology, genomes, and ecology. These properties, however, make them challenging from a systematics perspective. Mycoheterotrophy, in which plants become non-photosynthetic parasites on fungi, is an excellent example, and this unique life history has evolved numerous times in the orchid family. Here, we focused on Stereosandra, a genus of mycoheterotrophic orchid comprising a single species, S. javanica, about which little is known. Stereosandra has been placed in the orchid tribe Nervilieae, along with the leafy, autotrophic Nervilia, and the leafless, mycoheterotrophic Epipogium. We characterized the first complete plastid genome for Stereosandra and used nuclear sequence capture to determine its relationships within Nervilieae. This study presents the first genetic data ever produced for Stereosandra. The plastid genome exhibits rampant gene losses, pseudogenes, and reduced size relative to Nervilia but not to the extent seen in Epipogium. There is evidence of relaxed negative selection in six genes in Stereosandra, including matK, which functions in Group IIA intron removal of seven plastid genes, four of which have been lost or pseudogenized in this species. Applying mixture models, plastid genomes provided weak support for a sister position of Stereosandra to a clade of Epipogium + Nervilia. Nuclear phylogenomic analyses provided strong support for the same relationships. Ancestral state reconstruction revealed clear evidence that mycoheterotrophy evolved multiple times in the tribe from leafy ancestors. This study provides a previously unidentified, convergent instance of the evolution of full mycoheterotrophy in plants. We discuss the results in the context of proposed models of reductive plastid genome evolution and the genomic and evolutionary consequences of radical life history shifts in heterotrophic plants.

Leafless, parasitic plants are among the most fascinating species, and about half of these occur within the orchid family. Yet, they are among the most difficult to place from a taxonomic perspective. Here we focused on Stereosandra javanica, a poorly collected and little-understood orchid native to Asia, using material sampled from herbaria and high-throughput plastid and nuclear sequencing. We found strong evidence that that Stereosandra represents an independent transition to non-photosynthetic parasitism in the orchids and conclude that the number of independent losses of photosynthesis may be underestimated. We discuss our findings considering recent models that characterize the transition from photosynthesis to photosynthetic loss from a genomic perspective in leafless plants.