Abstract
BACKGROUND: The structure of chloroplast genomes (cpDNAs) in Fabaceae (Fab.) has undergone significant evolutionary modifications. Within the Papilionoideae (Pap.), the emergence of the Inverted Repeat-Lacking Clade (IRLC) represents a major genomic alteration. However, the molecular evolution and phylogenetic relationships within Pap. remain poorly resolved due to limited molecular data and incomplete research, highlighting the need for systematic investigation. PURPOSE: This study presents an in-depth analysis of the cpDNAs within the Pap., with the aim of unraveling the molecular evolution and phylogenetic interconnections among its species. METHODS: Complete cpDNAs of 18 Pap. species were sequenced using the Illumina Novaseq 6000 platform, followed by assembly and annotation. Comparative genomic analyses were conducted to elucidate structural variations and phylogenetic relationships. RESULTS: The research has uncovered significant differences in the structure and characteristics of the cpDNAs within the Pap.. The lengths of the cpDNAs of 18 species range from 121,190 bp to 158,539 bp, and they contain between 107 and 112 unique genes. Five species, namely Desmodium elegans and Indigofera bracteata, exhibit a typical quadripartite structure, while thirteen species from genera such as Astragalus (Ast.), Hedysarum (Hed.), and Caragana (Car.) are grouped within the Inverted Repeat-Lacking Clade (IRLC). Genetic characteristic analysis revealed a plentiful presence of SSR loci, with single-nucleotide repeats and dinucleotide (A/T) repeats being the most predominant. Notably, the cpDNAs of five species including D. elegans have experienced significant rearrangements. For example, an inversion of approximately 23 kilobase (kb) pairs was observed in Pueraria peduncularis and Sophora moorcroftiana. These species exhibit pronounced differences in their non-coding regions. Comparative genomic variations at cpDNA sites were identified. Moreover, by using D. elegans as a reference, six genes (ycf4, clpP, ycf1, trnI-GAU, accD, rpl32) displayed high nucleotide polymorphism (Pi > 0.1), and the Ka/Ks ratio for all protein-coding genes was determined to be less than 1. The topological structure of the constructed phylogenetic tree of 85 species was basically consistent with that of Pap.. Seven main clades were formed and relatively high bootstrap values were exhibited, further clarifying the evolutionary relationships among them. CONCLUSION: This study provides novel insights into the molecular evolution and phylogeny of Pap., offering a foundational resource for future taxonomic and evolutionary research.