2.4 Population structure analysis
To explore the phylogeographic structure of ribotypes/chlorotypes, gene
diversity within populations (hS ), gene diversity
(hT ), index of gene diversity of total
populations (NST ) and index of genetic
differentiation between populations (GST ) were
calculated using the HAPLONST (Pons & Petit, 1996). This software was
also used to compute the test statistic U that compares the
values of NST and GST ,
which indicates the presence of phylogeographic structure ifNST is higher than GST(Pons & Petit, 1996).
Since the cpDNA is maternally inherited in Primulina , there is no
recombination among loci and structure analysis makes no sense for such
dataset. Population structure of nrDNA sequences was inferred using the
Bayesian clustering procedure implemented in the software STRUCTURE
v.2.3.4 (Evanno et al. , 2005), that identifies the most probable
number (K ) of genetic clusters of origin of the sampled
individuals, and assigns individuals to clusters. We used Markov Chain
Monte Carlo (MCMC) iterations as implemented in the software to explore
the parameter space considering individual memberships to the Kclusters, ranging from K = 1 (null hypothesis of panmixia) toK = 10 (the total number of populations sampled). Three
independent runs were performed with an admixture model at
105 MCMC iterations and a 105burn-in period. The most likely number of population groups (K ,
indicating the number of true clusters in the data) and the model values
(△K ) according to the second-order rate of Change of clusterK that best fit the data was calculated in Structure Harvester
(Earl & vonHoldt, 2012). The graphical representation of results was
accomplished in the CLUMPAK server (http://clumpak.tau.ac.il/index.
html).
The genetic differentiation (Fst ) and gene flow (Nm )
between populations and among different regions for nrDNA and cpDNA
sequences separately. An Analysis of Molecular Variance (AMOVA) was
conducted on nrDNA and cpDNA sequences separately to test genetic
differentiation within populations, among regions and among populations
within regions using GENALEX v. 6.503 (Peakall & Smouse, 2012). To test
whether there was local genetic variation attributable to isolation by
distance (IBD), Neiʼs genetic distances (Nei, 1973) calculated with by
MEGA v. 6.5 and geographic distances (in km) between all pair-wise
combinations of the ten populations sampled were subjected to a Mantel
test (Mantel, 1967) in GENALEX v. 6.503. The genealogical relationships
between ribotypes/chlorotypes were inferred from the Median-Joining
network (MJ) of NETWORK v4.6.1.0 (http://www.fluxus-Engineering.com/).
In order to identify and quantify potential genetic discontinuities and
biogeographic boundaries between populations from both nrITS and cpDNA
datasets, we calculated the Monmonierʼs maximum-difference algorithm in
Barrier v.2.2 (Manni et al., 2004). The robustness of these barriers was
assessed by bootstrap, as in the Barrier v.2.2.