Computational protocol: Generic affiliations of Canthium species placed under Pyrostria group B sensu Bridson (Vanguerieae, Rubiaceae) inferred from morphology and molecular data

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[…] The ITS and trnL-F sequences were assembled and edited using the Codon Code Aligner version 3.0.1. Novel sequences of the four Philippine Canthium from each of the markers used were incorporated with several related sequences from the work of Lantz and Bremer ([]) taken from the GenBank (Table ). Ixora coccinea L. and Mussaenda erythrophylla Schumach. & Thonn., considered as closely related to Vanguerieae were used as the outgroups. Sequences were aligned manually using Se-Al v.1.0al (Rambaut []).Bayesian inference (BI) was used to estimate phylogenetic positions of the Philippine endemic Canthium species. The analysis was carried out using the MrBayes v.3.1.2p software (Huelsenbeck and Ronquist []; Ronquist and Huelsenbeck []; Altekar et al. []). Model selection for the best-performing evolutionary models were determined under three model selection criteria: a) Akaike Information Criterion (AIC) (Akaike []), b) AICc (seconder order criterion of AIC, necessary for smaller samples) and c) the Bayesian Information Criterion (BIC) (Schwartz []). The selected models were HKY and GTR + G for the ITS and trnL-F, respectively. In analyzing single marker, the best performing model was selected and one million generation was considered with a sample frequency of 1000 and four parallel chains. For combined analyses, model selection as well as the settings is similar with that of the single-marker analysis, however there were a total of three million running generations. Clades with posterior probability (PP) exceeding 0.95 were regarded as strongly supported.Parsimony analysis was conducted using PAUP version4.0b (Swofford []). Heuristic search was carried out to determine the most parsimonious trees utilizing a tree-bisection reconnection (TBR) branch swapping using 10,000 random addition sequences, with MULTREES option on. Consistency index (Kluge and Farris []) and retention index (Farris []) were calculated to determine if the data is far from being homoplasious. Bootstrapping was determined using 10,000 replicates, MULTREES option off, TBR branch swapping, and five random addition sequences. Clades receiving greater than 90% were considered strongly supported. […]

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