Computational protocol: Pisorisporiales, a new order of aquatic and terrestrial fungi for Achroceratosphaeria and Pisorisporium gen. nov. in the Sordariomycetes

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Protocol publication

[…] Cultures used for DNA isolations were grown as previously described by and DNA was extracted following the protocols of . Procedures for amplifying and sequencing the nuc18S, nuc28S and rpb2 were performed as described in . Sequences were edited using Sequencher v. 5.0 software (Gene Codes Corp., Ann Arbor, MI, USA).GenBank accession numbers for newly sequenced taxa and other homologous sequences of members of the Sordariomycetes and Leotiomycetes retrieved from GenBank are listed in . Sequences were manually aligned in BioEdit v. (). The nuclear ribosomal loci were aligned according to the secondary structure of Saccharomyces cerevisiae Meyen ex E.C. Hansen in order to improve the decisions on homologous characters and introduction of gaps (, , These procedures and alignment of the rpb2 sequences were performed as described in .The single-locus datasets (nuc28S: 1 923 characters and 77 sequences, nuc18S: 1 805 characters and 68 sequences, rpb2 segments 5–7: 1 213 characters and 48 sequences) were examined for topological incongruence among loci. For each individual locus, 500 bootstrap replicates were generated with RAxML-HPC v. 7.0.3 (, ) and compared visually for topological conflict between supported clades in phylogenetic trees. A conflict between two loci was assumed to occur when a clade appeared monophyletic with bootstrap support of ≥ 75 % in one tree, but was supported as non-monophyletic in another (). Individual, conflict-free alignments were concatenated to combine sequences for subsequent phylogenetic analyses. The multiple sequence alignment is deposited in TreeBASE (Study no. 16406). [...] Phylogenetic relationships of the unidentified fungus were resolved by an analysis of nuc18S, nuc28S and rpb2 sequences of representatives of 19 orders or individual families of the Sordariomycetes. We analysed the first 2/3 of the 5’ half of the nuc28S, the almost entire nuc18S, and segments 5–7 of rpb2. Bases 1–148 of the nuc18S, 1–85 of the nuc28S, and 1–58 of the rpb2 alignments at the 5’-end and 1 457–1 923 of the nuc28S alignment at the 3’-end were excluded from analyses because of incompleteness of the majority of the available sequences. The combined dataset was partitioned into several subsets of nucleotide sites, i.e. nuc28S, nuc18S, and first, second and third codon positions of rpb2. Two members of the Leotiomycetes, Leotia lubrica and Microglossum rufum were used to root the multilocus phylogeny.The program MrModeltest2 v. 2.3 () was used to infer the appropriate substitution model that would best fit the model of DNA evolution for each sequence dataset and each partition of the combined datasets. Maximum likelihood (ML) and Bayesian inference (BI) analyses were used to estimate phylogenetic relationships. ML analysis was performed with RAxML-HPC v. 7.0.3 with a GTRCAT model of evolution. Nodal support was determined by non-parametric bootstrapping (BS) with 1 000 replicates.BI analysis was performed in a likelihood framework as implemented in MrBayes v. 3.0b4 software package to reconstruct phylogenetic trees (). For the combined nuc18S, nuc28S and rpb2 dataset we used for each partition the GTR+I+G substitution model. Two Bayesian searches were performed using the default parameters. Analyses were run for 10 M generations, with trees sampled every 1 000 generations. Tracer v. 1.6.0. () was used to confirm convergence of trees and burn-in. The first 50 000 trees, which represented the burn-in phase of the analysis, were discarded. The remaining trees were used for calculating posterior probabilities (PP) of recovered branches (). […]

Pipeline specifications

Software tools Sequencher, BioEdit, RAxML, MrModelTest, MrBayes
Databases TreeBASE
Application Phylogenetics