Abstract: The genus Aquilegia is emerging as the new model system for plant development, ecology, and evolution studies. Previous research showed that pollinator shift might drive the diversification of North American Aquilegia species, and natural selection on the length of petal nectar spur might play a crucial role. In this genus, A. ecalcarata Maxim. is the only taxon that has lost nectar spurs. Previous phylogenetic results indicated that A. ecalcarata, A. yabeana Kitag., A. oxysepala var. kansuensis Bruhl., and A. rockii Munz comprised a monophyletic group. However, their pattern of genetic diversity remains unknown. In addition, little is known about the evolutionary relationship among the four species on the population level. We carried out a population genetics study with 21 representative populations based on 10 single-copy nuclear gene fragments and found that: (i) A. yabeana conserved the highest genetic diversity (both π_sil and θ_sil) and A. oxysepala var. kansuensis had the lowest level; (ii) A. ecalcarata split into two groups, with one population clustered with A. rockii and the other five populations clustered with A. oxysepala var. kansuensis; and (iii) the allele frequency spectrum showed an excess of low frequency alleles in all four species, implying that they may undergo the mutation-drift equilibrium. Our findings provide the first investigation of genetic diversity and evolutionary relationships in A. yabeana, A. oxysepala var. kansuensis, A. rockii, and A. ecalcarata. They lay the foundation for future evolutionary studies, such as speciation mediated by pollinators.

Key words: Aquilegia ecalcarata, A. oxysepala var. kansuensis, A. rockii, A. yabeana, evolutionary history, genetic diversity