Computational protocol: Molecular Systematics of Genus Atractylodes (Compositae, Cardueae): Evidence from Internal Transcribed Spacer (ITS) and trnL-F Sequences

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Protocol publication

[…] Susanna et al.[] used ITS, trnL-F, and matK to study the phylogenetic relationship of Cardueae. ITS, trnL-F, and matK sequence and analysis was also applied in the present study. However, our studies show that the substitution sites of matK in Atractylodes are very limited; thus, ITS and trnL-F were reported.The raw sequences were assembled and edited using the BioEdit software ver.5.0.9 [] and adjusted manually as necessary. The novel sequences generated from Atractylodes and the sequences downloaded from GenBank were subjected to multiple alignment using the CLUSTALW 1.83 software package with default settings []. ITS and trnL-F + ITS datasets were constructed, and all contained 49 individuals from 7 taxa.To examine the extent of conflict between the ITS and trnL-F datasets, the partition homogeneity test (also known as the incongruence length difference, ILD; []) was carried out using PAUP*. The text was implemented with 1000 partition homogeneity test replicates, using a heuristic search option with simple addition of taxa, TBR branch swapping and MaxTrees ser to 1000. The results suggest that both data sets are congruent (p-value > 0.05) and can be combined.Parsimony analysis. Maximum parsimony (MP) analyses were carried out using PAUP* v.4.0b10 []. For each analysis, maximum parsimony trees were sought using the heuristic search strategies of PAUP* (with 1000 replicate analyses, random stepwise addition of taxa, tree bisection and reconnection (TBR) branch swapping, and setting the maximum number of trees to 10,000). Bootstrap values were calculated from 1,000,000 replicate analyses using fast stepwise addition of taxa, and only those values compatible with the majority-rule consensus tree were recorded. All most parsimonious trees (MPTs) were saved and PAUP* was used to compute a strict consensus. Tree lengths, consistency index (CI) and retention index (RI) were calculated excluding uninformative characters.Bayesian Inference (BI) analysis. Datasets were conducted using MrBayes version 3.1.2 []. Prior to these analyses, the best-fit nucleotide substitution models of each partition were selected by AIC implemented in MrModeltest v.2.3 [] (GTR + G for the ITS datasets, GTR for the cpDNA datasets, GTR + I + G for the cpDNA +nrDNA datasets). Four simultaneous Markov chain Monte Carlo (MCMC) chains were run using MrBayes version 3.1.2 [], there heated and one cold, with the temperature adjusted to 0.5 in order to keep an appropriate heat range for the four chains. From a random starting tree, the Bayesian analysis was run for 20 million generations, and the trees were saved to a file every 1000 generations. Branch lengths of the trees were saved. Each analysis reached stationarity (the average standard deviation of split frequencies between runs ≤0.01) well before the end of the run. Burn-in (=5,000) trees were discarded, and the remaining trees and their parameters were saved. The 50% majority rule consensus tree was constructed. The results of the Bayesian analysis are reported as posterior probabilities [], which are equal to the percentage of trees sampled when a given clade is resolved. Only PP scores exceeding 50% are shown.Maximum likelihood (ML) analysis. ML analyses were performed using PHYML version 2.4 [] using the same model of substitution with 1000 bootstrap replicates. […]

Pipeline specifications

Software tools BioEdit, Clustal W, PAUP*, MrBayes, MrModelTest, PhyML
Application Phylogenetics