Assembly of the Delphinium densiflorum Chloroplast Genome and Comparative Genomics Within Delphinium

Background/Objectives: Chloroplast genomes are essential for understanding the systematics and adaptive evolution of alpine plants, yet genomic data for high-altitude Delphinium species remain scarce. Delphinium densiflorum, a medicinal plant endemic to the Qinghai-Tibet Plateau, exhibits notable high-altitude adaptations, but its plastome features and evolutionary position are still unclear. This study aims to assemble and characterize its complete chloroplast genome and clarify its phylogenetic placement within Delphinium. Methods: Using Illumina NovaSeq data, we de novo assembled the D. densiflorum plastome, annotated it with CPGAVAS2, and compared it with 12 published Ranunculaceae plastomes. We analyzed IR-boundary dynamics, genome-wide sequence variation, and codon-usage bias and constructed a maximum-likelihood phylogeny based on 69 shared protein-coding genes. Results: The plastome is 154,161 bp (GC 38.24%) with a canonical quadripartite structure, encoding 131 genes (87 CDS, 8 rRNA, 37 tRNA). An IR expansion into the SSC region yields the shortest SSC reported among the compared Delphinium species and produces unique structural variants. Photosynthetic genes are extremely conserved (nucleotide diversity Pi ≤ 0.01), whereas several loci (e.g., ycf1 and psaC) are highly divergent (Pi ≥ 0.05). Codon usage shows a strong bias toward AU-ending triplets. Phylogenetically, D. densiflorum forms a 100%-bootstrap clade with other high-altitude congeners, supporting the non-monophyly of Delphinium. Conclusions: This study delineates the plastome architecture and putative adaptive signatures of D. densiflorum, identifies robust candidate loci for DNA barcoding, and provides molecular evidence for taxonomic revision and conservation strategies in Delphinium.