The Ashiu area of Kyoto Prefecture is renowned for its role as a refugium for relict plants that migrated from northern regions in glacial periods. Viola brevistipulata sensu lato in Ashiu represents such a rear-edge population at low latitude, yet its biogeographic and conservation importance has been obscured due to taxonomic issues. This study aimed to clarify the taxonomy of the Ashiu population of V. brevistipulata s.l. through phylogenetic analysis of three varieties occurring in Honshu, Japan. The analysis confirmed the genetic distinctness of var. acuminata from a group including var. brevistipulata and var. minor, and clearly identified the Ashiu population as var. acuminata, which prefers a subalpine climate. Surveys of the distribution of var. acuminata populations in Ashiu revealed their persistence at low altitudes that correspond to upper warm temperate to lower cool temperate climate zones. The riverside rocky habitats that exclude competing plants and the unique life history characteristics of var. acuminata appear to allow these relict populations to survive in a climatically less suitable area
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