Comprehensive chloroplast genome analysis of 73 genus Aconitum members of family Ranunculaceae reveals insights into genome structure, codon usage polymorphism, and phylogenetic relationships

Background: Understanding the chloroplast genome is pivotal to unravel evolutionary relationships within plant species and facilitate accurate species identification by utilizing conserved yet diverse sequences. Genus Aconitum consists of around 300 traditional Indian and Chinese medicinal plant species, many native to mountainous regions. Despite their medicinal value, several species are known to be highly poisonous due to the presence of toxic diterpene alkaloids. Therefore, accurate identification and classification of these species is vital for traditional medicine systems especially for their safe usage. Results: Our investigation revealed a consistent quadripartite structure across all chloroplast genomes, comprising the typical large single copy (LSC), small single copy (SSC), and two inverted repeats (IR) regions. Using the available annotations, pangenome analysis unveiled 72 core and nine accessory genes, indicating an open pangenome characteristic. In-depth nucleotide-level homology analysis revealed that homologous genes of all accessory genes are present in all other genomes, implying the requisite 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 overall evolutionary stability with the diversity of accessory genes. Members of some core pathways are relatively absent on the chloroplast genome, suggesting its potential presence on the nuclear genome, which will be revealed after their nuclear genome sequencing. Furthermore, codon usage analysis demonstrated a preference for A/T ending codons over G/C ending codons, consistent with chloroplast genomes across species. Our phylogenetic results largely supported the morphological classification, with distinct Lycoctonum and Aconitum subgenera clustering. This validated the gross accuracy except for A. tanguticum and A. flavum , which clustered in wrong subgenus clades, suggesting discrepancy in morphological classification of the species or inaccurate classification. Conclusion: This comprehensive comparative analysis of 73 Aconitum chloroplast genomes elucidated their diversity at gene and genome architecture levels along with showcasing their evolutionary relationships with each other. Leveraging morphological classifications, we investigated the concordance between traditional taxonomy and molecular data through core gene-based and whole-genome phylogeny. The observed phylogenetic incongruences, such as non-monophyly of conspecific accessions and unexpected clustering patterns, likely reflect the combined effects of incomplete lineage sorting and historical hybridization events, both of which appear to be prominent evolutionary forces shaping the genomic architecture of Aconitum .