Computational protocol: Phylogenetic Reconstruction of the Calosphaeriales and Togniniales Using Five Genes and Predicted RNA Secondary Structures of ITS, and Flabellascus tenuirostris gen. et sp. nov.

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Protocol publication

[…] Cultures used for DNA isolations were grown and procedures for amplifying and sequencing the internal transcribed spacer rDNA (ITS rDNA), small and large subunit nuclear ribosomal DNA (nuc18S rDNA, nuc28S rDNA), second largest subunit of RNA polymerase II (rpb2) were performed as described in []. Total nucleic acids were extracted from mycelia following the protocols of []. A fragment of the β-tubulin gene region was amplified and sequenced using the primers Bt2a/benA1 and Bt2b [, ]. Sequences were edited using Sequencher 5.0 software (Gene Codes Corp., Ann Arbor, MI, USA).GenBank accession numbers for ITS, nuc28S, nuc18S, actin, β-tubulin and rpb2 sequences determined for this study and other homologous sequences of members of the Calosphaeriales and Togniniales retrieved from GenBank and the CBS-KNAW strain collection are listed in . Sequences were manually aligned in BioEdit v.7.1.9 []. The nuc18S and nuc28S alignments were enhanced by utilising the homologous 2D 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 three protein-coding genes were performed as described in []. Predicted 2D models obtained for the ITS1 and ITS2 were used to determine the positions of homologous nucleotides in the ITS.The single-locus data sets were examined for topological incongruence among loci (ITS: 82 sequences and 662 characters, nuc18S: 23 sequences and 1776 characters, nuc28S: 35 sequences and 1980 characters, actin: 46 sequences and 295 characters, β-tubulin: 71 sequences and 890 characters, rpb2: 11 sequences and 1117 characters). For individual loci, 500 bootstrap replicates were generated with RAxML-HPC v.7.0.3 [, ] and compared visually for topological conflicts among 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 not supported as monophyletic in another []. Individual, conflict-free alignments were concatenated to combine sequences for two subsequent phylogenetic analyses. The multiple sequence alignments are deposited in TreeBASE (Study no. 18161). [...] We performed two phylogenetic analyses. Phylogenetic relationships among members of the Calosphaeriales were resolved based on analysis of ITS, β-tubulin, nuc18S, nuc28S, and rpb2 sequences of 37 isolates representing 21 species and five genera. Phaeoacremonium fraxinopennsylvanicum (T.E. Hinds) D. Gramaje, L. Mostert & Crous, P. minimum (Tul. & C. Tul) D. Gramaje, L. Mostert & Crous and P. novae-zealandiae L. Mostert, W. Gams & Crous were used to root the tree. In the second analysis we investigated relationships among 44 Phaeoacremonium species with the combined ITS, actin and β-tubulin sequences. Wuestneia molokaiensis Crous & J.D. Rogers and Gnomonia gnomon (Tode) J. Schröt. were used to root the tree.We analysed the 5’ half of the nuc28S, the entire nuc18S, ITS, the 5–7 segments of the rpb2 and coding and non-coding regions of β-tubulin (exons 2, 3, 4, 5 and partial 6) and actin (exons 1, 2 and partial 3). 145 bases of the nuc18S and 654 of the nuc28S were excluded from the analyses because of the incompleteness of the 5’- and 3’-ends of the majority of the available sequences. The combined data set was partitioned into ITS, nuc28S, nuc18S, rpb2, and coding and non-coding regions of actin and β-tubulin.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 the MrBayes v.3.0b4 software package to reconstruct phylogenetic trees []. Initially, an appropriate DNA substitution model that would best fit the model of DNA evolution for each sequence data set and each partition of the combined data sets was selected using MrModeltest2 v.2.3 []. Among the 24 models tested, the GTR+I+G substitution model was selected for the ITS, β-tubulin, nuc28S, rpb2; SYM+I for the coding region of actin, HKY+I+G for the non-coding region of actin and nuc18S. Multiple Bayesian searches using Metropolis-coupled Markov chain Monte Carlo sampling were conducted. One cold and three heated Markov chains were used in the analysis. Analyses were run for 10 million generations, with trees sampled every 1 000 generations. We used the Tracer v.1.6.0. [] for analysis of trace files from Bayesian MCMC runs to assess whether we have run the analysis long enough to reach convergence. In both BI analyses the runs were long enough to effectively sample each distribution and to reach convergence. 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 in the 50% majority rule consensus tree []. [...] Knowledge of 2D structure is essential for constructing a reliable multiple sequence alignment to compare nucleotides at homologous positions (in helices and loops) while searching for non-conserved co-evolving nucleotides that maintain base pairing. Consensus 2D structure models for the ITS1 and ITS2 were built using the PPfold program v.3.0 [], which uses an explicit evolutionary model and a probabilistic model of structures and relies on multiple sequence alignment of related RNA sequences. Final 2D models created for all members of the Calosphaeriales were further improved using the Mfold program [] and then adjusted manually if necessary, based on comparison of homologous positions in the alignment. The predicted 2D RNA structures of ITS1 and ITS2 were obtained in a dot bracket notation and were visualised and drawn using VARNA: Visualization Applet for RNA program [].To evaluate 2D RNA structures more precisely we classified the topology of three-way junction (family A, B or C) occurring in the determined ITS1 model and also predicted coaxial helical stacking arrangement in the junction. The freely available programs Junction Explorer [] and Cartaj [] were used for that purpose. These programs consider mainly length of the loop between helices, sequence content and either free-energy associated to base stacking interactions between the base pairs at the end of helices or frequency of the closing base pair types.Computational analysis of the 2D RNA structure of ITS was based on 37 sequences representing 21 species of the Calosphaeriales and 45 sequences of 41 Phaeoacremonium species. Of the 46 Phaeoacremonium species described to date, five do not have their ITS sequences available, viz. P. amygdalinum D. Gramaje, Armengol & L. Mostert, P. inconspicuum (Rehm) D. Gramaje, L. Mostert & Crous, P. krajdenii L. Mostert, Summerb. & Crous and P. leptorrhynchum (Durieu & Mont.) D. Gramaje, L. Mostert & Crous. For P. vibratile (Fr.) D. Gramaje, L. Mostert & Crous only ITS2 is available, which contains several sequencing errors and was therefore not included. We used ITS sequences of the two strains CBS 110118 and CBS 110368 of P. krajdenii, which were confirmed by Mostert et al. [] to be conspecific with the ex-type strain CBS 109479 based on actin and β-tubulin sequence data. […]

Pipeline specifications

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