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Protocol publication

[…] All newly obtained raw sequences were assembled and edited using Geneious version 7.0 []. Boundaries of DNA regions were verified by BLAST searches in GenBank. Preliminary alignments were produced using MAFFT version 6.0 [], then adjusted manually in BioEdit version 7.0 []. Aligned matrices of four DNA regions were combined using SequenceMatrix version 1.7 []. The nrITS and concatenated plastid datasets were analyzed separately and in combination. No nucleotide positions were excluded from the analyses.Bayesian Inference (BI) and Maximum Likelihood (ML) methods were used for phylogenetic reconstruction. Partioned BI analyses were performed using MrBayes version 3.2.3 [], with DNA substitution models selected for each gene partition by the Bayesian information criterion (BIC) using jModeltest version 2 [, ]. The best-fit models used for each DNA region are listed in Table . Markov Chain Monte Carlo (MCMC) analyses were run in MrBayes for 10,000,000 generations for each dataset, with each run comprising four incrementally heated chains. The BI analyses were started with a random tree and sampled every 1000 generations. The first 20 % of the trees were discarded as burn-in, and the remaining trees were used to generate a majority-rule consensus tree. Posterior probability values (PP) ≥ 0.95 were considered as well supported [–]. ML tree searches and bootstrap estimation of clade support were conducted with RAxML []. These analyses used the GTR substitution model with gamma-distributed rate heterogeneity among sites and the proportion of invariable sites estimated from the data. For the ML analyses, bootstrap support (BS) ≥ 70 % were considered well supported []. Both BI and ML analyses, as well as jModeltest, were performed at the CIPRES Science Gateway ( incongruence between BI and ML analyses using the same dataset was visually compared using TreeGraph version 2 []. The incongruence length difference test (ILD) [] was not used to assess topological conflict between the nuclear and concatenated plastid datasets, because this analysis has been shown to be misleading [, ]. We used a conservative PP ≥ 0.95 and BS ≥ 70 % as threshold for identifying significantly incongruent clades. Due to only a weak conflict, we combined the total datasets for the additional phylogenetic analyses. [...] Ancestral states of four morphological characters (phyllotaxy, corolla tube, galea tooth, and galea beak) were inferred by mapping characters on to the BI tree of the total dataset. Outgroups were removed. Parsimony reconstructions were performed using Mesquite version 2.75 []. Following Ree [], each character was coded categorically with two states, 0 and 1. States were assigned as follows: phyllotaxy, alternate/sub-opposite (0) or opposite/whorled (1); corolla tube, short (0) or long (1); and galea teeth, absent (0) or present (1); galea beak, absent (0) or present (1). […]

Pipeline specifications

Software tools Geneious, MAFFT, BioEdit, Sequence Matrix, MrBayes, jModelTest, RAxML, CIPRES Science Gateway, TreeGraph, Mesquite
Organisms Paris verticillata
Diseases Precursor Cell Lymphoblastic Leukemia-Lymphoma