Computational protocol: Two new species and one new record of the genus Tylopilus (Boletaceae) from Indian Himalaya with morphological details and phylogenetic estimations

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Protocol publication

[…] The nrLSU and nrITS datasets were assembled according to recent previous studies on this genus (, ) and from BLAST (Altschul et al. 1997) searches in GenBank (Clark et al. 2016). As most Tylopilus collections in GenBank are not provided with both molecular markers, we were unable to establish a combined nrITS+nrLSU dataset and so have opted for present separate nrLSU and nrITS phylogenetic inferences. These (nrITS and nrLSU) sequences were aligned separately in MAFFT 7.305 (). For the nrLSU dataset, Xanthoconium sinense (KT990666 and KT990664) and X. purpureum (KT990663) from Boletaceae were used as outgroup taxa. Similarly, for the nrITS dataset, two sequences from Gyroporus (KX869874, GQ166901), another genus in Boletales (Gyroporaceae), were used as the outgroup. Phylip file formats were created in AliView () using default settings. Phylogenies were reconstructed using Maximum Likelihood (ML) in RAxML 7.2.6 () in GTRGAMMA substitution model. All parameters in the ML analyses used the default settings in RAxML and Maximum Likelihood bootstrap percentage (MLB) were obtained using nonparametric bootstrapping with 1000 replicates. Additionally (to generate supplementary data), nrLSU and nrITS sequences were also phylogenetically analysed using Bayesian analysis. The best-fit models of nucleotide evolution for nrLSU and nrITS datasets (TIMef and TrNef+G, respectively) were obtained in MrModeltest 3.7 (). Bayesian inferences were computed independently twice in MrBayes v.3.2.2 (), under TIMef (for nrLSU) and TrNef+G (for nrITS) models, respectively. Bayesian posterior probabilities values (BPP) were calculated in two simultaneous runs with the Markov Chain Monte Carlo (MCMC) algorithm (). Markov chains were run for 1000000 generations, saving a tree every 100th generation. These analyses were terminated when the average standard deviation of split frequencies fell below 0.01. The first 25% of trees was discarded as burn-in (). The convergence of runs was visually assessed using Trace function in Tracer version 1.6.0 (). […]

Pipeline specifications

Software tools MAFFT, PHYLIP, AliView, RAxML, MrModelTest, MrBayes
Application Phylogenetics
Diseases Contusions, Yellow Fever