FIG. 19 in Fungal biodiversity profiles 111-120

FIG. 19. — Maximum likelihood (ML) tree of Russula subgenus Heterophillidiae based on combined sequence data of nrLSU, rpb2 and tef1α. Bootstrap values from ML analysis ≥50 % and Posterior probabilities from Bayesian inference (BPP) ≥0.95 are shown at branch nodes. The newly generated sequences of R. ferruginea sp. nov. are highlighted in boldface and on coloured background. Data of sequences used for the tree are listed in Table 2. Protocols for DNA extraction, PCR and sequencing followed Vera et al. (2021). Sequences were edited in the BioEdit 7.2.5 sequence alignment editor (Hall 2013) and Geneious R10 (Kearse et al. 2012). Intra-individual polymorphic sites having more than one signal were marked with NC-IUPAC ambiguity codes. The datasets were aligned in MAFFT 7 using the E-INS-i strategy (Katoh & Standley 2013) and manually improved in Geneious R10 (Kearse et al. 2012). Divergent and ambiguously aligned positions in the nrLSU were removed with Gblocks (Castresana 2000) using the least stringent parameters. Intronic positions of rpb2 and tef1α were manually removed. For the BI analysis, the data set was divided into five partitions: 28S, the 1st+2nd, and 3rd codon positions of tef1, the 1st+2nd, and 3rd codon positions of rpb2. The best substitution model for each partition was computed jointly in PartitionFinder 1.1.1 (Lanfear et al. 2012). BI runs were computed twice in MrBayes 3.2.6 (Ronquist et al. 2012) with four Markov chain Monte Carlo (MCMC) chains for 10 million iterations until the standard deviation of split frequencies fell below the 0.01 threshold. Convergence of runs was visually assessed using the trace function in Tracer 1.6 (Rambaut et al. 2013). For the ML analysis, the concatenated alignment was loaded as a fasta file to CIPRES Science Gateway (Miller et al. 2010) and analyzed using RAxML-HPC2 on XSEDE (8.2.12) as a partitioned data set (as in the BI analysis) under the GTR+GAMMA model with 1000 bootstrap iterations as recommended by the RAxML user manual (Stamatakis 2014).