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).