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[…] Ninety six strains belonging to species of Penicillium, Aspergillus and related taxa were studied for their phylogenetic relationship by using their publicly available sequences of the following six loci: RPB1 and RPB2 genes coding for subunits of RNA polymerase II; Tsr1, coding for a putative ribosome biogenesis protein; Cct8, coding for the theta subunit of the TCP-1 chaperonin complex; BenA coding for the beta-tubulin protein, and CaM coding for the calcium binding protein calmodulin. The list of strains and the relevant sequences accession number used is reported in .DNA sequences of the six loci were singularly aligned with Muscle (for RPB1, RPB2, CaM, BenA, and Cct8) and ClustalW (for Tsr1) algorithms using the software MEGA7 (), manually optimized and trimmed to make sequences of equal length, and then concatenated. The alignment is deposited at TreeBASE ( Successively, the Multiple Sequence Alignment (MSA) was evaluated for quality using Transitive Consistence Score (TCS) offered by the T-Coffee web server (). The presence of rogue taxa in the set of data was evaluated through the RogueNaRok web server analysis, because the presence of these taxa can frequently have a negative impact on the results of a bootstrap analysis (e.g., the overall support in consensus trees, ). Then the sequences were manually controlled and substituted if necessary to settle the MSA. JModelTest2 (v2.1.6) () was used to find the preferred model of evolution for the concatenated dataset, PartitionFinder (v1.1.1) () was used to investigate the best-fit partitioning schemes and models of molecular evolution to be adopted in RaxML analysis of the partitioned dataset, models were selected according to Bayesian Information Criterion (BIC) for both tools. The different tools performed to infer the phylogenetic tree were as follows: a) MrBayes v3.2.6 () for posterior probabilities (Bpp) using models of evolution on concatenated dataset from JmodelTest; b) RAxML-HPC2 (v8.2.8) () for rapid bootstrap support (Rbs) using models of evolution defined by JmodelTest and PartitionFinder on concatenated and partitioned dataset, respectively; c) IQ-Tree-omp (v1.4.1) (, , ) for UFML (Ultra Fast Maximul Likelihood) support (Ibs).The CIPRES Science Gateway V 3.3 () was used to perform MrBayes analysis, setting GTR + invgamma, 107 generations, sampling every 1 000 generations with a burnin fraction of 0.25; and RaxML analyses, setting GTR + GAMMA + P-Invar, executing 1 000 rapid bootstrap inferences and thereafter a thorough ML search, for the concatenated and partitioned dataset respectively.IQ-Tree analysis were done locally, setting GTR + I + G4 for the concatenated dataset and the calculated charpartition BIC (GTR + I + G4: RPB1, RPB2, CaM, BenA, and Cct8, TPM2 + I + G4: Tsr1) for the partitioned dataset, both analyses were run with 104 ultrafast bootstrap replicates. [...] Phylogenetic analyses were conducted using nine loci (18S rDNA, 5.8S rDNA, 28S rDNA (D1-D2), RPB1, RPB2, CaM, BenA, Tsr1, Cct8) with intron regions excluded from CaM and BenA sequences. The dataset primarily consisted of publicly available sequences which are listed in . Additional Cct8, RPB1, RPB2 and Tsr1 loci of Aspergillus species were amplified and sequenced using the methods described previously by . Sequences were deposited into GenBank under the accession numbers KY006730-KY006827. All sequences were aligned by PRANK v.140603 () with default settings. Individual alignments were concatenated by using SequenceMatrix 1.8 () and the dataset was partitioned by the nine loci. An initial maximum likelihood (ML) tree was generated from the dataset by raxmlGUI 1.5b1 () using the executables of RAxML 8.2.7 () under the GTR model with gamma-distributed rate heterogeneity with 500 rapid bootstrap replicates. Sequences encoding Tsr1 are containing large number of indels therefore this initial tree was used to refine the alignment of the Tsr1 sequences by PRANK with the -F option. In the case of SSU, RPB2 and Tsr1 alignments FastGap 1.2 () was used to code the phylogenetic information of gaps as binary characters implementing the “simple indel coding” algorithm. The refined alignment of partial Tsr1 sequences and the indel matrix was incorporated in the concatenated dataset. The final ML trees and branch supports were estimated by 1 000 thorough bootstrap replicates under the GTR + Γ model with ten partitions. Bootstrap support was mapped on the ML tree using the SumTrees script of the Dendropy v4.2.0 package (). The resulted best tree and the bootstrap replicates were submitted for rogue taxon identification by the RogueNaRok (, ) web service. Bayesian analyses were performed on the partitioned dataset using MrBayes 3.2.6 () with GTR substitution model with gamma-distributed rate variation across sites for 107 generations with four chains and two replicates sampling every 1 000th generations. The burnin proportion was set to 0.25. Convergence and ESS values of the runs were examined by Tracer 1.6 ().To test the phylogenetic hypotheses of the monophyly of Aspergilli, a constraint tree was generated by Mesquite v3.04 (). Per-site log likelihoods were calculated for 20 unconstrained and 20 constrained ML searches by using RAxML. To measure the support of the two hypotheses Approximately Unbiased (AU) test was conducted by CONSEL 0.1j () with 105 replicates.Tree space visualization of ML and Bayesian analyses was carried out by using the TreeSetVis v3.01 () package for Mesquite and the RWTY v1.0.1 package for R v3.3.1 (). Sorting of the bootstrap replicates was conducted by PhySortR v1.0.7 () package in R. [...] To verify the robustness of the six and nine-genes phylogeny the branch supports of the principal nodes depicting the Aspergillus and Penicillium monophyletic topology were evaluated. Three categories of branch support (, ) were considered: parametric (Bpp, aLRT-Chi2, aBayes), nonparametric (Rbs, SH-aLRT) and hybrid (Ibs).To compute, aLRT-Chi2, SH-aLRT and aBayes branches support of the six-genes phylogeny, PhyML (v20130805) () and IQ-Tree-omp (v1.4.1) analyses were performed locally (, ). The single branch tests (SH-aLRT, aBayes) and ultrafast bootstrap approximation of the nine-genes phylogeny were also conducted by using IQ-Tree v1.4.2 in 50.000 replicates under the GTR + Γ model. […]

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