This study focuses on differentiating the endemic species Thlaspi violascens and T. densiflorum, which exhibit similar macromorphological characteristics and population appearances with utilizing molecular, anatomical, and micromorphological methods. The complete chloroplast genomes of both species have been sequenced for the first time using next-generation sequencing technologies, allowing for high-resolution analysis of their phylogenetic relationships. The isolated chloroplast genomes measure 152,693 base pairs for T. violascens and 152,718 base pairs for T. densiflorum. Phylogenetic analysis shows that species within the Thlaspi genus are monophyletically distinct from other genera. Although the average nucleotide diversity between the two species is low, whole-genome sequencing-based Skmer analysis has successfully enabled high-resolution species-level discrimination. Anatomically, the two species differ in several aspects, including the type of petiole tip, abaxial surface outline, epidermal cell size and layer count, petiole thickness, vascular bundle number, midrib lobes, mesophyll cell types, and collenchyma layer count. Micromorphological differences are also evident in the primary structures of the anther and ovary, as well as the secondary structures of the sepal and style. Overall, the study concludes that chloroplast genome data, petiolar anatomical features, and floral micromorphological traits can effectively distinguish between T. violascens and T. densiflorum.
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