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Background: Scutellaria baicalensis Georgi has been extensively used as a medicinal herb in China for over 2000 years. They may be intentionally or inadvertently substituted or blended with comparable species in the local market, threatening clinical medication safety. Molecular markers are effective tools to prevent misidentification and eliminate doping and falsification among Scutellaria plants. This study screened four highly variable regions to identify Scutellaria and its adulterants. In addition, a phylogenetic analysis was performed using the complete cp genome combined with published Scutellaria species samples. Moreover, a comparative analysis of the cp genomes was conducted to investigate the cp genome evolution of S. baicalensis. Results: The complete cp genome of five species of Scutellaria was sequenced for the first time, and four previously published Scutellaria species were re-sequenced. They all exhibited a conserved quadripartite structure in their cp genomes, including two distinct regions, namely a small and large single copy region, respectively, and two inverted repeats encompassing the majority of ribosomal RNA genes. Furthermore, the nine species exhibited high conservation from aspects of the genome structure, codon usage, repeat sequences, and gene content. Four highly variable regions (matK-rps16, ndhC-trnV-UAC, psbE-petL, and rps16-trnQ-UUG) may function as potential molecular markers for differentiating S. baicalensis from its adulterants. Additionally, the monophyly of Scutellaria was ascertained and could be reclassified into two subgenera, subgenus Anaspis and subgenus Scutellaria, as evidenced by the phylogenetic analyses on sequences of cp genome and shared protein-coding sequences. According to the molecular clock analysis, it has been inferred that the divergence of Scutellaria occurred at approximately 4.0 Mya during the Pliocene Epoch. Conclusion: Our study provides an invaluable theoretical basis for further Scutellaria species identification, phylogenetics, and evolution analysis. [ABSTRACT FROM AUTHOR]

• Chloroplast genome sequences of five endangered Paris species were de novo assembled. • Subgenera Paris sensu strict is sister to the Daiswa and Trillium. • Phylogeny of genus Paris was inferred using protein-coding genes sequences. • Divergence times of Paris were inferred based on molecular evolution. Paris L. genus has been a precious traditional herb for more than 2000 years in China. However, due to overexploitation and habitat destruction, Paris is threatened by extinction. Similar morphological features cause the classification of Paris species in dispute. The chloroplast (cp) genome approach has been used to investigate the evolution of Paris. However, some studies confirm that the cp genome may result in misleading relationships because of the length variation, gaps/indels deletion, and incorrect models of sequence evolution in concatenated datasets. Therefore, there is a high demand for a reconstructed phylogenetic relationship and developed genetic markers to conserve these species. Recent studies have demonstrated that the protein-coding genes could provide a better phylogenetic relationship in the phylogenetic investigation. In this study, the complete cp genomes of five species were characterized, and the length of five cp genomes ranges from 162,927 bp to 165,267 bp, covering 89 protein-coding genes, 38 tRNA, and eight rRNA. The analysis of the repeat sequences, codon usage, RNA-editing sites, and comparison of cp genomes shared a high degree of conservation. Based on the protein-coding genes, the phylogenetic tree confirmed Paris 's position in the order Melanthiaceae, providing maximum support for a sister relationship of the subgenera Paris sensu strict (Paris s.s.) with the Daiswa and Trillium. In addition, the molecular clock showed that subgenus Paris was inferred to have occurred at about 52.81 Mya, whereas subgenus Daiswa has originated at 24.56 Mya, which was consistent with the phylogenetic investigation. This study provided a valuable insight into the evolutionary dynamics of cp genome structure in the family Melanthiaceae, and it also contributes to the bioprospecting and conservation of Paris species. [ABSTRACT FROM AUTHOR]