Phylogeny and evolutionary dynamics of the Rubia genus based on the chloroplast genome of Rubia tibetica.

Rubia tibetica, a well-known medicinal plant, holds significant medicinal value. In this study, we sequenced and assembled the complete chloroplast genome of R. tibetica and conducted comparative analyses with four other Rubia species to provide insights into genome structure, selective pressure, and phylogenetic relationships. The chloroplast genome of R. tibetica was 154,901 bp in length and exhibited a typical quadripartite circular structure. Comparative analysis revealed a highly conserved genome structure among Rubia species, with genome sizes ranging from 153,555 bp to 155,108 bp and a GC content of approximately 37%. Codon usage analysis indicated a preference for A/U-ending codons. Selective pressure analysis identified relaxed selection in genes such as rps15 and petA, suggesting species-specific adaptive strategies. Two hypervariable regions, rps16-trnQ and psaJ-rps18, were experimentally validated as potential molecular markers through PCR amplification and sequencing. However, sequencing results of the rps16-trnQ region differed from the reference chloroplast genome, requiring further investigation. Phylogenetic analysis was performed using maximum likelihood (ML) based on the complete chloroplast genome sequences of five Rubia species and confirmed the monophyly of the Rubia genus, with R. tibetica occupying a basal position. This suggesting the retention of ancestral traits and adaptation to the unique environment of the Qinghai-Tibet Plateau. Molecular dating analysis further revealed divergence times consistent with geological and climatic events in the region. These findings provide critical insights into the evolutionary dynamics of Rubia species and offer valuable resources for their classification, conservation, and medicinal application.