Computational protocol: Diversification, Biogeographic Pattern, and Demographic History of Taiwanese Scutellaria Species Inferred from Nuclear and Chloroplast DNA

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Protocol publication

[…] Polymerase chain reaction (PCR) was performed with 10–100 ng template DNA, 0.5–1 U Taq (Bernardo Scientific Corp., Taipei), 100 µM deoxyribonucleotide triphosphate, 0.2 µM each primer, and 0.1 µg/µL bovine serum albumin in a MultiGene thermal cycler (Labnet International, Inc.). The PCR program was set to 94°C for 3 min for enzyme activation, followed by 35 cycles of 94°C for 40 s, melting temperature for 40 s, and 72°C for 90 s, with a 5-min final extension at 72°C. PCR amplifications of five primer sets including three chloroplast regions (matK, ndhF-rpl32, and rpl32-trnL) and two low-copy nuclear regions (CHS and CAD) were performed. Optimal annealing temperatures were set at 47°C for chloroplast regions, 53°C for CHS, and 49°C for CAD regions. Within-population variation of all PCR products was screened with single-strand conformation polymorphism. Each PCR product was denatured for 10 min at 95°C and quickly moved into a −20°C cool box. Denatured products were separated by pre-cooling 10% polyacrylamide gel (acrylamide:bisacrylamide = 45∶1). PCR products with different fragment patterns were then sequenced directly in both directions using an ABI BigDye 3.1 Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). All sequence polymorphisms were visually rechecked from chromatograms with an ABI PRISM®3730XL DNA Sequencer (Perkin-Elmer, Foster City, CA, USA). PCR products were cloned with a yT&A cloning kit (Yeastern Biotech, Taipei, Taiwan) when they contained ambiguous nucleotides, and three to five clones were sequenced with the M13F and M13R primers to generate consensus sequences. The two sequences of a heterozygote were separated by comparing the sequences of the PCR product and the cloned sequence. Chromatograms were inspected by SeqMan implemented in DNASTAR ver. 7.0 (Lasergene, Germany). Gene confirmation and exon-intron junctions of each sequence were queried in the Nucleotide collection database at the National Center for Biotechnology Information website using the Nucleotide Basic Local Alignment Search Tool program and NetPlantGene server at the Center for Biological Sequence Analysis website ( All sequences were deposited in the NCBI nucleotide sequence database under the following accession numbers: JX981343∼JX981446 and JX985445∼JX985457. [...] In addition to collecting sequences, we also downloaded the matK sequences of S. minor (HM850804.1), S. scordifolia (HQ839713.1), S. hirta (HQ911383.1), S. sieberi (HQ911384.1), S. viscidula (HQ676587.1), S. rehderiana (HQ676588.1), T. gracilis (HQ911386.1, the outgroup used for matK only), and the CHS sequence of S. viscidula (EU386767.1) from GenBank for phylogenetic analyses. Sequence alignments were performed with Clustal X and manually edited using BioEdit ver. . Phylogenetic relationships were reconstructed using individual and combined sequences of five loci with the neighbor joining and Bayesian approaches implemented in Molecular Evolutionary Genetics Analysis v. 5.05 and MrBayes ver. 3.1.2 , respectively. In the neighbor-joining analysis, the maximum composite likelihood model for substitutions and pairwise deletions for the treatment of gaps were set with 1000 bootstrap replications to measure the supporting values for grouping. In the Bayesian analysis, substitution models were set according to the evaluation of Akaike information criterion (AIC) and Bayesian information criterion (BIC) scores (), and two parallel runs in Markov chain Monte Carlo (MCMC) searches were performed for 10 million generations with sampling every 1000 generations for a total of 10,000 trees in each run. The first 10% of the generations were discarded (burn in). Bayesian posterior probabilities were estimated as the proportion of trees containing each node over the trees sampled during runs.The species tree was also reconstructed using five loci with the assistance of BEAST ver. 1.6.1 . Substitution models of each of loci were set according to the evaluation of AIC and BIC scores (see ). Because we lacked fossil records for dating, we adopted the strict molecular clock in BEAST ver. 1.6.1 using a substitution rate of 2×10−9 per site per year for chloroplast genome DNA (cpDNA) loci and 1.5×10−8 per site per year for CHS and CAD with the Yule tree prior . Three independent pre-runs of 10 million generations of the length of the MCMC searches were performed to obtain better parameter priors for the next five independent 10 million generations of the MCMC process. Genealogies were sampled every 1000 generations, with the first 10% discarded as burn in. All the statistics of the output values were summarized using TRACER ver. 1.5 and both log and tree files of the last five runs were combined using LogCombiner ver. 1.6.1 . TreeAnnotator ver. 1.6.1 and FigTree ver. 1.3.1 were used for summarizing and displaying the sampled trees, respectively. [...] Given that the phylogenetic analyses indicated that the Taiwanese Scutellaria species are not monophyletic (see Results; ), topological tests were performed to examine the hypothesis that the Taiwanese species are descendants of a single common ancestor colonizing Taiwan (). The approximately unbiased (AU) test , the Kishino-Hasegawa (KH) test , and the Shimodaira-Hasegawa (SH) test , which are used to compare tree topologies with a null hypothesis, were performed using CONSEL , . […]

Pipeline specifications

Software tools BLASTN, NetPlantGene, Clustal W, BioEdit, MEGA, MrBayes, BEAST, FigTree, CONSEL
Applications Phylogenetics, WGS analysis
Organisms Sillago indica