A Comprehensive Comparative Analysis and Phylogenetic Investigation of the Chloroplast Genome Sequences in Four Astragalus Species

<i>Astragalus</i> L. (Fabaceae), the largest plant genus with significant medicinal value, faces critical endangerment of its wild resources and a scarcity of chloroplast genomic data. We sequenced and assembled the complete chloroplast (cp) genomes of four <i>Astragalus</i> species (<i>A. yunnanensis</i>, <i>A. yunnanensis</i> subsp. <i>incanus, A. polycladus</i> and <i>A. polycladus</i> var. <i>nigrescens</i>) and performed comparative analyses with five previously published chloroplast genomes. The cp genomes of the four <i>Astragalus</i> species ranged in size from 122,868 bp to 125,752 bp, all lacking one inverted repeat (IR) region, thus belonging to the inverted repeat lacking clade (IRLC). Annotation revealed that each genome contained 110 unique genes, including 76 protein-coding genes, 30 tRNA genes, and 4 rRNA genes. Nucleotide diversity (Pi) analysis identified mutation hotspots, including 5 non-coding regions and 5 coding regions, which could serve as potential molecular markers. Additionally, evidence of positive selection was detected in 11 genes, suggesting their possible roles in adaptive evolution to environmental changes. Phylogenetic analysis revealed distinct clades, with <i>Astragalus</i> forming a monophyletic group within Fabaceae. Notably, closely related species, subspecies, and varieties were observed to cluster together, forming sister taxa. However, despite the conservation in cp genomes, <i>A. yunnanensis</i> and <i>A. yunnanensis</i> subsp. <i>incanus</i> exhibit significant morphological differentiation in leaf shape, leaf indumentum, and stem color. This paradox strongly suggests a markedly higher evolutionary rate in the nuclear genome compared to the chloroplast genome. The cp genomes of <i>Astragalus</i> presented here serve as a key resource for studying the genus's genetic diversity and will aid in elucidating its intrageneric phylogeny.