Phylogenomics clarifies Balanophora evolution, plastid reduction, and obligate asexuality origins

Holoparasitic plants are non-green plants that depend entirely on their host plants for essential resources, which often results in functional reduction and gene loss. However, the timing and extent of gene loss associated with parasitism remain unclear. Although Balanaphara is known to have highly reduced plastid genomes, only five species from a few geographically restricted regions have been studied. Here, we expanded Balanaphara sampling with seven species from twelve populations across Taiwan and Japan. We assembled their plastid genomes and transcriptomes and inferred multi-gene phylogenies from diverse plastid and nuclear markers. The uncovered relationships within the genus imply independent origins of obligate apomixis in island populations of several species. All the plastid genomes are highly reduced (14-16 kbp), gene-dense, completely syntenic, strongly AT-biased (87-88%), and with the same non-canonical genetic code (TAG->Trp) as previously reported in other Balanaphara species. To further understand the functional role of these non-photosynthetic plastids, we predicted the subcellular localization of nuclear-encoded proteins based on expression data. Strikingly, over 800 Balanaphara proteins were predicted to be plastid-targeted, suggesting a retained capacity for the biosynthesis of amino acids, fatty acids, riboflavin, lipoic acid, heme, isoprenoids, and components of the glycolysis and pentose phosphate pathways. Our findings show that the extreme plastid genome reduction in Balanaphara mainly occurred before the origin of the clade and primarily erased photosynthesis-related functions. Numerous highly expressed nuclear genes of Balanaphara still retain chloroplast transit peptides, and the resulting proteins are likely targeted to the non-photosynthetic organelle that remains metabolically active. Similar to other parasitic eukaryotes with non-photosynthetic plastids, such as apicomplexans, the loss of photosynthesis led to extreme plastid genome reduction without massive elimination of other photosynthesis-unrelated functions. Balanophoraceae thus emerges as a fascinating model for reconstructing the evolutionary changes associated with photosynthesis loss in land plants.