Abstract: Traits are basic attributes of organisms that form the basis for speciation and diversity. The floral nectar spur is a classic example of a key innovative trait. Differences in nectar spur morphology can lead to pollinator specialization and thereby promote reproductive isolation between species. Despite its importance, the nectar spur has been lost in some members of the columbine genus (Aquilegia), resulting in a new spurless trait, and the evolutionary influence of this trait has become a topic of scientific interest. Aquilegia ecalcarata is an important representative columbine species that lacks spurs. Here, we resequenced the genomes of 324 individuals from A. ecalcarata and four related species. We found that A. ecalcarata was divided into three groups based on the phylogenetic relationships and population genetic structures. Topology weighting analysis revealed that A. ecalcarata has multiple origins, and Patterson′s D statistic showed that the spurless trait may have one origin. Floral morphological analysis revealed significant differences between A. ecalcarata and its spurred sister groups, and the floral phenotypes of the three A. ecalcarata groups have identical or similar floral phenotypes. Our results confirmed that the spurless trait not only produced the phenotype of A. ecalcarata but also contributed to the emergence of the A. rockii phenotype. Moreover, the spurless trait promoted the divergence between A. ecalcarata and its close, spurred relatives. Our research shows that the loss of key innovative traits can play a very important role in speciation and species diversity.