Assembly and Comparative Analysis of Five Complete Mitochondrial Genomes of the Aquilegia ecalcarata Complex

The Aquilegia ecalcarata (A. ecalcarata) complex is a valuable evolutionary model for studying parallel evolution, introgression, and incomplete lineage sorting. Deciphering this complexity requires evidence from multiple genomic compartments. While substantial nuclear and chloroplast resources exist, mitochondrial genome data, which capture the maternally inherited evolutionary history, have been absent for this group. Using long-read sequencing data, this study assembled and comprehensively characterized the mitochondrial genomes of five representative taxa within the A. ecalcarata complex. Comparative analyses reveal that the two spurless lineages, A. ecalcarata clade A and clade B, do not form a monophyletic group based on mitochondrial phylogeny. Instead, each clusters with a different spurred relative, consistent with the independent evolution of spur loss. This finding is further supported by collinearity patterns, gene content, and lineage-specific MTPT distributions. By providing the first mitochondrial genomic resource for this group, this study offers independent organellar evidence for the multiple origins of spurless phenotype in the A. ecalcarata complex.