Comparative Analysis of Complete Chloroplast Genomes of Penstemon digitalis Nutt. ex Sims and Insights into Phylogenetic Relationships of Penstemon

Background: Penstemon, a core genus of the tribe Cheloneae in Plantaginaceae, possesses significant ecological and horticultural value. However, its interspecific phylogenetic relationships remain unclear due to morphological convergence, hybridization events, and limited genomic resources. This study aimed to characterize the complete chloroplast (cp) genome of Penstemon digitalis and explore its genomic features and phylogenetic position within Plantaginaceae. Methods: We sequenced the cp genome of P. digitalis using high-throughput sequencing, assembled and annotated the genome, and compared it with other Plantaginaceae species. Repetitive sequences and hypervariable regions were identified. Phylogenetic analyses were performed using maximum likelihood (ML) based on complete cp genomes from 46 taxa (36 Plantaginaceae, six Scrophulariaceae, and four outgroup species). Results: The cp genome is 152,993 bp with a typical quadripartite structure, containing 132 functional genes (87 protein-coding, 37 tRNA, and eight rRNA). Comparative analyses showed high gene content and structural conservation within the tribe Cheloneae. Several hypervariable regions and repeats were detected as potential markers. ML analysis strongly supported the monophyly of Penstemon among the sampled species (100% bootstrap) and placed P. digitalis in a well-supported clade within the genus; Penstemon was recovered as the sister group of Russelia equisetiformis. Conclusions: This study provides the first complete cp genome resource for P. digitalis, confirming its phylogenetic affinity and offering valuable genomic data for future studies on population genetics, species delimitation, and adaptive evolution in Penstemon. These findings will aid taxonomic revision and conservation efforts for this diverse genus.