Computational protocol: Inferring Multiple Refugia and Phylogeographical Patterns in Pinus massoniana Based on Nucleotide Sequence Variation and DNA Fingerprinting

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Protocol publication

[…] Nucleotide sequences were aligned with the program BlastAlign . After the alignment, the indels were coded as binary characters. Maximum-likelihood (ML) analyses were performed using PHYLIP v. 3.67 on each locus. The general time reversible HKY model was determined by Modeltest v3.6 to be the most suitable model and was used for the subsequent nucleotide analyses. Confidence in the reconstructed lineages was tested by bootstrapping with 1000 replicates using unweighted characters.A network analysis complementary to conventional cladistics provides the power to discriminate between phylogeographical patterns that result from restricted gene flow and those that arise from historical events at the population level . The number of mutations between DNA sequence haplotypes in pairwise comparisons were estimated from the data using the Molecular Evolutionary Genetics Analysis program (MEGA, version 5.0) , then used to construct a minimum spanning network . Networks were produced with the program MINSPNET . [...] Summary statistics such as the number of haplotypes (h) and minimum number of recombinations (Rm) were determined. The level of genetic diversity within populations was quantified by measures of nucleotide diversity θ using DnaSP Version 5.1 . To make inferences about demographic changes of P. massoniana, we employed both mismatch distributions and statistical tests of neutrality. We calculated Tajima's D , and Fu and Li's D* statistic in the noncoding DNA fragments as indicators of demographic expansion in DnaSP.SAMOVA (spatial analysis of molecular variance) was applied to identify groups of geographically adjacent populations that were maximally differentiated based on sequence data . We performed the analyses based on 100 simulated annealing steps and examined maximum indicators of differentiation (FCT values) when the program was assigned to identify K = 2–6 partitions of populations.We also investigated historical demographics of populations by plotting mismatch distributions and comparing them to Poisson distributions. The parameters of demographic expansion were estimated using the methods of Schneider and Excoffier . [...] For microsatellite loci, genetic diversity within populations was assessed by calculating allele number (A), observed (Ho), and expected (He) heterozygosities by using the Arlequin program version 3.1 . The program is also performed to calculate population differentiation level (Fst), to analyze molecular variance (AMOVA), and to test Hardy-Weinberg equilibrium (HWE). SAMOVA (spatial analysis of molecular variance) was performed to identify groups of geographically adjacent populations based on microsatellite data . A NJ tree with bootstrapping values from 1000 replicates was constructed in comparison for eight populations using the PHYLIP Package v. 3.67 . STRUCTURE version 2.1 , applies a Bayesian method to infer the number of clusters (K) without using prior information of individual sampling locations. This program distributes individuals among K clusters based on their allelic frequencies and estimates the posterior probability of the data given each particular K. STRUCTURE was run for K = 1 to K = 9 clusters. Each run was pursued for 1 000,000 MCMC interactions, with an initial burn-in of 100,000 and an ancestry model that allowed for admixture, with the same alpha for all populations. To assess stability, 10 independent simulations were run for each K. The final posterior probability of K, Pr(X|K), was computed, as suggested by Pritchard et al. , using the runs with highest probability for each K. However, as indicated in the STRUCTURE documentation and Evanno et al. , Pr(X|K) usually plateaus or increases slightly after the ‘right’ K is reached. Thus, following Evanno et al. , ΔK, where the modal value of the distribution is located at the real K, was calculated. […]

Pipeline specifications

Software tools BlastAlign, PHYLIP, ModelTest-NG, MEGA, DnaSP, SAMOVA, Arlequin
Applications Phylogenetics, Population genetic analysis, Nucleotide sequence alignment
Organisms Pinus massoniana
Chemicals Nucleotides