Computational protocol: Geological and climatic changes in quaternary shaped the evolutionary history of Calibrachoa heterophylla, an endemic South-Atlantic species of petunia

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[…] The sequences were aligned manually using GeneDoc []. Because poly-A/T regions and small inversions were highly variable and homoplastic in preliminary phylogenetic analyses, they were not considered for further analyses, as described previously e.g., [,]. The two cpDNA spacers were concatenated in all analyses. Contiguous insertion/deletion events longer than one base pair (bp) were treated as single mutation events [].Standard diversity indices including haplotype diversity (h), nucleotide diversity (π) [], and analysis of molecular variance (AMOVA []) were obtained with Arlequin 3.5.1.2 []. The AMOVAs were conducted using 1,000 permutations among collection sites and ΦST (pairwise differences). Mantel test between genetic and geographical distances and Spatial autocorrelation analyses (1,000 permutations) were performed with the program Alleles in Space 1.0 (AIS []). Close sites with small sample sizes were merged.Historical barriers to gene flow were identified using Spatial Analysis of Molecular Variation in SAMOVA 1.0 [] with 1,000 permutations. This method identifies geographically homogeneous groups of populations that are maximally differentiated from each other, and it attempts to maximize the proportion of total genetic variation among groups of populations (ΦCT). [...] The evolutionary relationships between haplotypes were estimated using the Median-Joining method (ϵ = 0 []) implemented in the Network 4.6 program (available at http://www.fluxus-engineering.com). Sequences of Calibrachoa paranensis (Dusén) Wijsman, C. serrulata (L. B. Sm. & Downs) Stehmann & Semir, and C. elegans (Miers) Stehmann & Semir that belong to the same subgenus of C. heterophylla[] were also included in the analysis (Additional file ).Phylogeny and divergence times among haplotypes were estimated using a Bayesian approach with BEAST 1.6.1 []. Two runs of 108 chains were conducted, sampling every 1,000 generations. The settings used were the Yule tree prior, the HKY substitution model with four gamma categories and the strict molecular clock. The substitution rate used was 2.8 × 10-9 (± 5.4 × 10-11) substitutions per site per year, or 0.56% per million years. This rate was chosen because it was previously estimated for Petunia for the same loci studied here [] and it is similar to those found in other plant groups (0.22–0.58% per Myr []; 0.26–0.92% per Myr []). The best-fit model of sequence evolution was identified in Modeltest 3.7 [] using the Akaike Information Criterion []. Tracer 1.5 (available at http://beast.bio.ed.ac.uk/Tracer) was used to check for convergence of the Markov chains and adequate effective sample sizes (>200) after the first 2 × 107 chains were deleted as “burn-in”. A maximum clade credibility tree was obtained using TreeAnnotator, part of the BEAST software package. Statistical support was determined by assessing the Bayesian posterior probabilities. […]

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