The medicinally and ornamentally valuable genus Thunbergia faces taxonomic uncertainty, while certain Acanthaceae species are threatened by habitat loss, underscoring the need for chloroplast genome studies to support conservation efforts. The chloroplast genome of Thunbergia grandiflora was sequenced and assembled. Additionally, 28 Acanthaceae species with significant medicinal value were selected for comparative genomic analysis. Based on the chloroplast genome data of Acanthaceae species, this study conducted phylogenetic and comparative evolutionary analyses. The results preliminarily support a systematic framework that divides Acanthaceae into eight tribes within five subfamilies. Concurrently, the study revealed significant inverted repeat (IR) region structural variations. A clear correspondence was observed between the contraction of IR length and the topological structure of the phylogenetic tree. In particular, species within the genus Strobilanthes exhibited significant contraction in their IR regions, which corresponded consistently with their tendency to cluster into an independent clade in the phylogenetic tree. This suggests that structural variation in the IR regions may be closely associated with the evolutionary divergence of this group. SSR analysis revealed a prevalent mononucleotide A/T repeat dominant pattern across Acanthaceae species. Furthermore, selection pressure analysis detected positive selection acting on multiple key genes, including rbcL, rps3, rps12, cemA, and ycf4, suggesting that these genes may play important roles in the adaptive evolution of Acanthaceae. This study reveals that the chloroplast genomes of Acanthaceae exhibit distinctive characteristics in phylogenetic architecture, dynamic variations in IR regions, and adaptive evolution of key genes, providing important molecular insights for understanding the mechanisms underlying species diversity and for the conservation of medicinal resources within this family.
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