Comparative genomics, phylogenomic reconstruction and divergence time analysis of 32 Delphinium species based on complete chloroplast genomes

Delphinium , characterized by distinctive spurred flowers, comprises numerous high-altitude endemic species with significant ornamental and medicinal value. However, the intrageneric phylogenetic relationships and evolutionary history of Delphinium have remained poorly resolved, primarily due to the limited variability of traditional DNA markers and complex morphological homoplasy. In this study, complete chloroplast genomes of three Delphinium spp. were sequenced and combined with those of 29 Delphinium spp. publicly available plastomes to conduct a comprehensive phylogenomic analysis and divergence time estimation. The Delphinium plastomes exhibited a highly conserved quadripartite structure, with genome sizes ranging from 153,769 to 157,339 bp. Comparative genomic analyses revealed 2,426 SSRs, and although narrow variable windows ( π > 0.02) were detected within or adjacent to five loci ( ndhA - ndhH , ycf2 , trnG - trnfM - rps14 , rrn23 , trnI ), their overall sequences remained highly conserved at the species level. Given their moderate overall divergence and reduced substitution saturation, these five loci are suitable for higher-level phylogeny, whereas conventional DNA barcodes and SSC-derived regions remain more effective for species-level discrimination. Phylogenomic reconstruction based on whole plastomes robustly supported the monophyly of Delphinium and resolved six well-supported primary clades, outperforming concatenated protein-coding genes in topological resolution. Furthermore, divergence time estimation revealed that the Delphinium crown group originated in the early Eocene (ca. 53.23 Ma; 95% HPD: 51.68-54.77 Ma). An initial major lineage divergence occurred during the Oligocene (ca. 28.53 Ma) driven by global cooling, followed by exPLoSive diversification during the Pliocene and Pleistocene (e.g., rapid radiation of core clades at 3.47 Ma). This recent rapid radiation was likely triggered by the intense uplift of the Qinghai-Tibet Plateau and recurrent Northern Hemisphere glaciations. Collectively, our study provides new insights into the plastome evolution and phylogeny of Delphinium , offering a robust evolutionary framework for understanding its diversification and facilitating future population genetic analyses.