Computational protocol: Phylogeny of anaerobic fungi (phylum Neocallimastigomycota), with contributions from yak in China

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Protocol publication

[…] The novel sequences obtained for all the isolates were deposited in GenBank under the accession numbers shown in Table . The final data sets incorporated all representative sequences publicly available from pure cultures, covering each of the eight known genera (Tables , S1). In addition, our sequences were also checked against the expanded dataset of Koetschan et al. () to confirm that none of our sequences fits with any of the uncultured lineages of their phylogeny. As different numbers of reference sequences are available for the ITS1 fragment, the complete ITS region and the partial LSU respectively, the phylogenetic analyses were performed separately using three data sets. Two Monoblepharella strains, as representatives of Monoblepharidales, were used as outgroup based on its relatively close relationship to anaerobic fungi of Neocallimastigales (James et al. ). The sequence datasets were initially aligned using MAFFT v. 7 (Katoh and Standley ), and were manually optimised using MEGA v. 6 (Tamura et al. ). For the ITS data sets, the alignment was obtained with the Q-INS-i method of iterative refinement as implemented in MAFFT to consider their secondary structure due to the highly variable nature of these sequences.Phylogenetic analyses of individual locus datasets were based on Bayesian inference (BI), Maximum Likelihood (ML) and Maximum Parsimony (MP) analyses. For BI, the best evolutionary model for each partition was determined using MrModeltest2 (Nylander ) and incorporated into the analyses. A Markov Chain Monte Carlo (MCMC) algorithm was used to generate phylogenetic trees using MrBayes v. 3.2.1 (Ronquist and Huelsenbeck ; Ronquist et al. ) under the optimal criteria for each locus. The MCMC analysis lasted until the average standard deviation of split frequencies came below 0.01 with trees saved every 1000 generations. The first 25 % of saved trees were discarded as the ‘burn-in’ phase and posterior probabilities (PP) were determined from the remaining trees. The MP analysis was performed using PAUP v. 4.0b10 (Phylogenetic Analysis Using Parsimony; Swofford ). Phylogenetic relationships were estimated by heuristic searches with 1000 random addition sequences. Tree bisection-reconnection was used, with the branch swapping option set on ‘best trees’ only with all characters weighted equally and alignment gaps treated as fifth state. The tree length (TL), consistency index (CI), retention index (RI) and rescaled consistence index (RC) were calculated for the MP phylogenies and the bootstrap analysis (Hillis and Bull ) was based on 1000 replications. The ML analysis was performed under the GTR-GAMMA model of evolution using RAxML-VI-HPC v. 7.0.3 (Stamatakis ) with nonparametric bootstrapping using 1000 replicates. Trees were viewed in FigTree v. 1.1.2 (Rambaut ). The alignments used in the phylogenetic analyses, and resulting phylogenetic trees, were deposited in TreeBASE (submission ID S20001; https://treebase.org/). […]

Pipeline specifications

Software tools MAFFT, MEGA-V, MrModelTest, MrBayes, PAUP*, RAxML, FigTree
Databases TreeBASE
Applications Phylogenetics, GWAS
Organisms Bos grunniens