2.6 Population Genetics, Phylogeographic Analyses, and Demographic History
All the DNA sequences were edited by SeqMan (DNAstar package; DNAStar Inc., Madison, WI, United States) to obtain consensus sequences. The program MAFFT v.7.369b(Katoh & Standley, 2013) was used to align for subsequent manual adjustments. Haplotype diversity (Hd)(Nei & Tajima, 1981) and nucleotide diversity (π)(Nei & Li, 1979) for each population were calculated using DNAsp v6.0(Rozas et al., 2017) to verify the degrees and patterns of diversity. The haplotypes of the two gene fragment datasets and the distribution of all haplotypes on the map were then plotted separately in the software PopART using the Minimum spanning method(Bandelt, Forster, & Röhl, 1999). PERMUT was used to access the total diversity (HT), within-population diversity (HS) and population differentiation indices (GST and NST)(Pons & Petit, 1996). And a U-statistic was used to test the phylogeographic structure by comparing GST and NST. In addition, analyses of molecular variance (AMOVA) was performed using ARLEQUIN v3.5(Excoffier & Lischer, 2010) to assess the genetic differentiation within and between populations, and 1000 random permutations were conducted to test the significance of partitioning. To detect whether the populations in Notholirionexperienced a recent population expansion, a mismatch distribution analysis (MDA)(Schneider & Excoffier, 1999) was carried out using the ARLEQUIN v3.5(Excoffier & Lischer, 2010). Additionally, Neutrality test (Fu’s Fs; Tajima’s D)(Tajima, 1989) was also conducted to test whether there was potential population expansion in Notholirion using DnaSP v6.0 program. The smoothness of observed mismatch distribution was detected by calculating the Harpending’s raggedness index (Hrag)(Schneider & Excoffier, 1999) and the sum of squared deviations (SSD) between observed and expected mismatch distributions.