Novel genetic code and record-setting AT-richness in the highly reduced plastid genome of the holoparasitic plant Balanophora

Significance Many groups of flowering plants have become parasites and have lost the capacity to carry out photosynthesis. The plastid genomes of these parasitic plants are often highly reduced in size and gene content and are divergent in other ways too. Here we report, to our knowledge, unprecedented levels and novel types of plastid-genome divergence in the bizarre, mushroom-like parasitic plant Balanophora. The miniaturized plastid genome in Balanophora is exceptionally streamlined, has evolved a novel genetic code, and possesses the most A+T-rich protein-coding genes known. These findings extend our understanding of the lower limits of genome complexity and offer exciting opportunities to explore the mutational and selective forces that drive radical genome evolution. Plastid genomes (plastomes) vary enormously in size and gene content among the many lineages of nonphotosynthetic plants, but key lineages remain unexplored. We therefore investigated plastome sequence and expression in the holoparasitic and morphologically bizarre Balanophoraceae. The two Balanophora plastomes examined are remarkable, exhibiting features rarely if ever seen before in plastomes or in any other genomes. At 15.5 kb in size and with only 19 genes, they are among the most reduced plastomes known. They have no tRNA genes for protein synthesis, a trait found in only three other plastid lineages, and thus Balanophora plastids must import all tRNAs needed for translation. Balanophora plastomes are exceptionally compact, with numerous overlapping genes, highly reduced spacers, loss of all cis-spliced introns, and shrunken protein genes. With A+T contents of 87.8% and 88.4%, the Balanophora genomes are the most AT-rich genomes known save for a single mitochondrial genome that is merely bloated with AT-rich spacer DNA. Most plastid protein genes in Balanophora consist of ≥90% AT, with several between 95% and 98% AT, resulting in the most biased codon usage in any genome described to date. A potential consequence of its radical compositional evolution is the novel genetic code used by Balanophora plastids, in which TAG has been reassigned from stop to tryptophan. Despite its many exceptional properties, the Balanophora plastome must be functional because all examined genes are transcribed, its only intron is correctly trans-spliced, and its protein genes, although highly divergent, are evolving under various degrees of selective constraint.