Computational protocol: Taxonomic Re-evaluation of Megacollybia Species in Korea

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Protocol publication

[…] We edited raw sequences using PHYDIT 3.2 []. For phylogenetic analyses, DNA sequences were aligned using ClustalX 1.81 [] with manual adjustment using PHYDIT. We excluded ambiguously aligned regions in the phylogenetic analyses. Maximum parsimony (MP) analysis was performed using the heuristic search option in PAUP* 4.0 [] with the following settings: all characters were equally weighted, gaps were treated as missing characters, starting trees were obtained by random addition with 1,000 replicates, and tree bisection-reconnection (TBR) branch swapping algorithm was used. Maximum likelihood (ML) phylogenetic tree with bootstrap analysis was constructed using the general time-reversible (GTR) evolutionary model and the gamma distribution of rate heterogeneity. Nodal supports for MP and ML analyses were estimated using nonparametric bootstrapping with 1,000 replicates. The option of each replicate for bootstrapping analyses was chosen as a heuristic search of 100 stepwise random addition replicates and TBR branch swapping for each bootstrap replicate. For Bayesian inference (BI), MrBayes 3.1 [] was employed to infer the phylogenetic relationships. Posterior probabilities (PP) were approximated using the metropolis-coupled Markov Chain Monte Carlo method. Two parallel runs were conducted with one cold and three heated chains for 5 million generations, respectively, starting with a random tree. The trees were sampled every 100 generations. We interpreted the convergence of two independent runs had converged when the average standard deviation of the split frequencies dropped below 0.01. The trees obtained before the convergence were discarded using the burn-in command, and the remaining trees were used to calculate a 50% majority consensus tree and to estimate PP. PP values below 0.95 were not considered significant. Based on the studies of Moncalvo et al. [] and Hughes et al. [], we selected three species, Clitocybula oculus AFTOL-ID 1554, Gerronema strombodes TENN60718 and G. subclavatum DAOM194858 as outgroups. […]

Pipeline specifications

Software tools Clustal W, MrBayes
Application Phylogenetics