Microsatellite fingerprinting analyses
Microsatellite dataset was edited and formatted via GeneAlEx software (Peakall & Smouse, 2006). Hardy-Weinberg equilibrium (HWE) was tested for every locus and every population with default parameters via Genepop, version 4.1.4. Linkage disequilibrium (LD) was investigated at the 5% statistical significance level among loci pairs with 1000 permutations using Arlequin, version 3.11 (Excoffier et al., 2005).
Genetic diversity of each microsatellite locus was assessed using GenAlEx, version 6.3 (Peakall & Smouse, 2006). Allelic richness (A R) was estimated with FSTAT version 1.2 (Goudet, 1995). We also estimated the genetic diversity indices of each population across all loci using GenAlEx, version 6.3 (Peakall & Smouse, 2006) and FSTAT, version 1.2 (Goudet, 1995).
Genetic differentiation between the two Cycas species was assessed in three ways. First, the Wright’s fixation index (F ST) was calculated using AMOVA implemented in Arlequin, version 3.11 (Excoffier et al., 2005) with significance tested using 1000 permutations. Secondly, a Bayesian analysis of population structure using STRUCTURE, version 2.2 (Pritchard, Stephens, & Donnelly, 2000) was conducted. In this analysis, the admixture model with the assumption of correlated allele frequencies among clusters was used. To estimate the number of clusters (K), the posterior probability of the grouping number (K = 1~15) was estimated by the Markov chain Monte Carlo (MCMC) method with 10 separate runs to evaluate the consistency of the results. Each run was estimated at 100,000 steps, with a 100,000-step burn-in. The best fit number of grouping was evaluated using ΔK in the STRUCTURE HARVESTER, version 0.6.8 tool (Earl & vonHoldt, 2012). Thirdly, an individual-based principal coordinate analysis (PCoA) was conducted in the MVSP, version 3.12 (Kovach, 1999) using the genetic distances among SSR genotypes. The result of PCoA could visualize genetic relationships among these 206 individuals from the two species with 11 populations in a two-dimensional coordinate diagram figure.
Mantel test was conducted by GenAlEx, version 6.3 (Peakall & Smouse, 2006) to examine the correlation between genetic distance,F ST/(1- F ST), and geography distance (GGD) to test whether isolation-by-distance pattern (IBD) could be supported. In addition, based on genetic distance matrices, we identified geographical locations where major genetic barriers among populations might occur with a barrier boundary analysis, using BARRIER, version 2.2 (Manni, Guerard, & Heyer, 2004).