Morphological Character Investigation, Sample Collection, and Transcriptome Sequencing

To understand the morphological characters and living conditions of the karst and non-karst species, we performed in situ and ex situ field investigation in the Sichuan, Hubei and Shanxi Provinces of China. The morphological traits and habitats of each species were recorded, including the flowering time, flower and leaf traits and color variation, seeds dispersal and living environment condition. The young and mature leaves and flowers of U. rockii and U. henryi as well as Semiaquilegia adoxoides and Aquilegia ecalcarata were collected from living plants, and stored at −80°C until processed for total RNA isolation. Total RNA was extracted using Trizol (Life Technologies Corp.) according to the manufacturer’s protocols, and cleaned up using the RNeasy mini kit (Qiagen, Valencia, CA, United States). Poly (A) mRNA was purified using Oligo (dT) magnetic beads and interrupted into short fragments. The RNA sequencing libraries were constructed using the Illumina mRNA-Seq Prep Kit after assessing RNA quality. Finally, all cDNA libraries were sequenced on the Illumina HiSeq 2000 platform in paired-end form. A total of eight sequencing libraries were constructed in this study, including each flower and leaf libraries of the four species.