Computational protocol: The largest type study of Agaricales species to date: bringing identification and nomenclature of Phlegmacium (Cortinarius) into the DNA era

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[…] DNA was extracted from a few milligrams of dried material (a piece of lamella) with the NucleoSpin Plant kit (Macherey-Nagel, Düren, Germany), or with various CTAB protocols in Brandrud’s and Frøslev’s specimens (see , ). Primers ITS 1F and ITS 4 (, ) were used to amplify ITS regions. The same primer pairs were used in direct sequencing. For problematic material the primer combinations ITS 1F/ITS 2 and ITS 3/ITS 4 were also used. PCR amplifications were performed in a 25 μL reaction mix with about 70 ng extracted DNA, 1 U Phusion High-Fidelity DNA polymerase and 1× HF buffer (Finnzymes), 200 mM of each dNTP and 0.5 μM of each primer. The PCR reactions were run on a MBS 0.2 G Thermal Cycler (Thermo Hybaid) with the following settings: denaturation for 30 s at 98 °C, followed by 35 cycles of denaturation for 10 s at 98 °C, annealing for 30 s at 50 °C and extension for 30 s at 72 °C. The PCR products were purified using an ExoSAP-IT purification kit (Amersham Biosciences). Sequencing was performed on both strands using a BigDye Terminator v. 1.1 Sequencing kit (Applied Biosystems). Reactions were performed in 10 μL with 1 μL of PCR product, 1.3 mM of primer (ITS 1F or ITS 4), 1 μL 5X sequencing buffer, and 1 μL of Terminator Ready Reaction Mix. Reactions were run for 1 min at 96 °C, followed by 30 cycles of 30 s at 96 °C, 15 s at 50 °C and 4 min at 60 °C. Unincorporated dye terminators and primers were removed by Sephadex G-50 DNA Grade Fine (Amersham Biosciences) purification system, and the reactions were analysed by ABI 3730 DNA Analyzer (Applied Biosystems) automatic sequencer. Sequences were assembled and edited with Sequencer 4.1 (Gene Codes, Ann Arbor, Michigan, USA). A total of 405 new ITS sequences were produced for this study. Collections and GenBank sequences used for the phylogenetic analysis are given in and . The alignment of 461 ITS sequences was produced with the MUSCLE program () under default settings and followed by manual adjustments in BioEdit ( The alignment is 812 nucleotides long (including gaps) and is available at TreeBASE under S14832 ( inference (BI) was performed with MrBayes v. 3.1.1 (). The best substitution model for alignment was estimated by both the Akaike information criterion and the Bayesian information criterion with jModelTest 0.1.1 (). GTR model was chosen. Two independent runs with four chains in each were performed 6 000 000 generations with sampling every 100th generation. All trees sampled before stationarity were discarded with a 25 % safety margin (burn-in of 15 000 trees, 1 500 000 generations). Sampled trees from both runs were combined in a 50 % majority rule consensus phylogram with posterior probabilities (PP). The analyses were run with computer clusters of the CSC, IT Center for Science, Espoo, Finland. […]

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