Comprehensive analysis of 73 Aconitum chloroplast genomes reveals their structure, codon usage bias, and phylogenetic relationships within family Ranunculaceae.

Chloroplast genomes provide conserved yet informative sequences useful for inferring plant evolution and species identification. Genus Aconitum consists of around 300 traditional Indian and Chinese medicinal plant species, many native to mountainous regions, and known to be highly poisonous due to toxic diterpene alkaloids. Their accurate identification and classification are vital for traditional medicine systems, particularly for ensuring their safe use. A consistent quadripartite structure was identified across all chloroplast genomes, comprising the typical large single copy (LSC), small single copy (SSC), and two inverted repeats (IR) regions. Pan-plastome analysis unveiled 72 core and nine accessory genes, indicating an open pan-plastome characteristic. In-depth nucleotide-level homology analysis revealed that homologous genes of all accessory genes are present in all other genomes, implying the need for better chloroplast genome annotation tools that can identify all putative genes from such conserved genomes. Notably, the order of all core and accessory genes remained highly conserved across all analysed genomes, underscoring the overall evolutionary stability despite the diversity of accessory genes. Members of some core pathways are relatively absent on the chloroplast genome, suggesting their potential presence on the nuclear genome, which will be revealed after their nuclear genome sequencing. Our phylogenetic results largely supported the morphological classification, with distinct Lycoctonum and Aconitum subgenera clustering, further validating the gross accuracy except for A. flavum, suggesting a putative morphological classification discrepancy or inaccurate classification. This comparative analysis reveals a highly conserved chloroplast genome architecture across Aconitum while documenting measurable plastome-level variation among 73 species. The chloroplast phylogeny highlights instances of non-monophyletic clustering among conspecific accessions and unexpected placement of certain taxa, indicating complex evolutionary histories within the genus. Such patterns may reflect a combination of processes, including incomplete lineage sorting, chloroplast capture, or sample misidentification, rather than definitive phylogenetic incongruence. While chloroplast genomes alone cannot fully resolve evolutionary relationships in a group shaped by hybridization and polyploidy, the results provide valuable insights into plastome evolution and identify key lineages and taxa that warrant further investigation using nuclear genomic and integrative approaches.