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.