5 CONCLUSIONS
Two main conclusions follow from our results that 1) the evolution of spur length in A. buergeriana var. buergeriana has occurred independently in different mountain regions, and 2) the few red-flowered phenotypes that occur within the species has not led to genetic differentiation. First, given that the independent evolution of floral size in different mountain regions has also recently been reported in L. album var. barbatum (Toji et al., 2021), the independent evolution of floral size among mountain regions may be a generalized event that occurs commonly in different taxa. The approach used here to test for independent evolution among mountain regions is applicable to any taxon and a variety of traits. In particular, morphological analyses combined with MIG-seq (Suyama & Matsuki, 2015), which can be used to obtain genome-wide SNPs from non-model organisms, constitute a powerful method for elucidating patterns of morphological and genetic diversification within species. Second, we found no relationship between flower color and the degree of genetic differentiation, despite the fact that pollinator isolation caused by differences in flower color has been reported in two closely related species of Aquilegia (Fulton & Hodges, 1999; Noutsos et al., 2014). We infer that in the mountainous region of Japan, where bumblebees are locally abundant large pollinators, shifts to different pollinator taxa are unlikely to occur, and the polymorphism in A. buergeriana var. buergeriana flower color is likely maintained by random genetic drift. Thus, our results are an important exception to diversification in genus Aquilegia , which is well known to have occurred by both flower-color and pollinator shifts (Whittall & Hodges, 2007).
Acknowledgements. We thank S. Duhon for English editing. We thank the Chubu District Forest Office (Forestry Agency), the Chubu Regional Office for Nature Conservation (Ministry of the Environment), and the Matsumoto Regional Office (Nagano Prefectural Government) for permission to work in the area. This study was supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT) (19H03300, 19J22443) and by the Environment Research and Technology Development Funds (4-2001) of the Environmental Restoration and Conservation Agency of Japan.