Evolution of in the angiosperms: sequence, splicing, and expression in a clade of early transitional mycoheterotrophic orchids.

The plastid-targeted transcription factor () has been implicated in chloroplast biogenesis, plastid genome stability, and fungal defense response, which together represent characteristics of interest for the study of autotrophic losses across the angiosperms. While gene loss in the plastid and nuclear genomes has been well studied in mycoheterotrophic plants, the evolution of the molecular mechanisms impacting genome stability is completely unknown. Here, we characterize the evolution of in four early transitional mycoheterotrophic orchid species in the genus by synthesizing the results of phylogenetic, transcriptomic, and comparative genomic analyses with genomic sequences sampled from 21 orders of angiosperms. We found an increased number of non-canonical isoforms assembled from all but the greenest species, including intron retention in some isoforms. Within , phylotranscriptomic analyses revealed the presence of tissue-specific differential expression of in only the most photosynthetically capable species and a coincident increase in the number of non-canonical isoforms assembled from fully mycoheterotrophic species. Gene- and codon-level tests of selective regimes did not infer significant signal of either relaxed selection or episodic diversifying selection in but did so for relaxed selection in the late-stage full mycoheterotrophic orchids and . Additionally, nucleotide substitutions that most likely impact the function of , such as nonsense mutations, were only observed in late-stage mycoheterotrophs. We propose that our findings suggest that splicing and expression changes may precede the selective shifts we inferred for late-stage mycoheterotrophic species, which therefore does not support a primary role for in the transition to mycoheterotrophy in the Orchidaceae. Taken together, this study provides the most comprehensive view of evolution across the angiosperms to date.