2 | Materials and methods
2.1 | Sample collection and laboratory procedures
Eight populations roughly covering the whole distribution range ofPrimulina heterotricha were sampled, and included BW (Mt. Bawang), YJ (Mt. Yajia), EX (Mt. Erxian), YG (Mt. Yingge), QX (Mt. Qixian), WZ (Mt. Wuzhi), XA (Xian’an Stone Forest), and JF (Mt. Jianfeng) (Table 1; Figure 1). A total of 176 samples were collected in 2016 and 117 samples in 2022 (Table 1). Although we did not sample prior to 1994 when the Daguangba Reservoir on the Changhua River was completed, sampling from the eight populations before (2016) and after (2022) the construction of the two expressways (expressways 1 and 2 were completed in 2018 and 2019, respectively) would allow us to detect possible genetic and phylogeographic effects of habitat fragmentation. Distance between populations ranged from 6 to 72 km, and the elevation of collected populations varied from 600 (YG) to 1163 m (YJ) above sea level (Table 1). Considering the physical barriers posed by the two expressways and the dam, the populations can be tentatively divided into four groups: the northwest (NW) group of populations (i.e. BW, YJ, EX, and YG), the southeast (SE) group (QX and WZ), the southern (S) group (XA), and the southwest (SW) group (JF). In each population, fresh leaf samples were collected from individuals at least 10 m apart. Each sampled leaf was dried quickly in a separate plastic bag containing 20 to 30 g of silica gel and stored at -80℃ later.