DNA extracts from each isolate were used as templates for amplification with primers VLITS1 with VLITS2 for the ITS region [74], atp6F and rnsR for the atp6-rns, and nad3F with atp9R for the nad3-atp9 mt intergenic regions [27]. All reactions were performed with Herculase polymerase (Stratagene) in a PTC-200 (MJ Research) selleck products thermocycler according to the manufacturer’s protocol,

with a minor modification involving lower annealing temperature (45°C) for all three pairs. Sequencing was performed as above. DNA sequence alignments were made using CLUSTALW [75] with the multiple alignment parameters set to default and then IWR-1 in vitro edited by visual inspection (the matrix of the concatenated dataset and its partitions

is provided in Additional File 8). Maximum parsimony (MP), Neighbor-Joining (NJ) and Bayesian inference (BI) selleck chemicals llc analyses were employed in order to create the phylogenetic trees. The PAUP* program ver. 4.0b10 [76] was used for NJ (using the Kimura-2 parameter model) and MP analyses with 1,000 and 10,000 replicates, respectively, and with random addition of taxa and tree-bisection reconnection branch swapping [76]. Reliability of nodes was assessed using 1,000 and 100 bootstrap iterations for NJ and MP analyses, respectively. The BI was performed with MrBayes ver. 3.1 [77]. A gamma distribution model of site variation was used, calculated with PAML [78]. For ITS1-5.8S-ITS2, nad3-atp9, atp6-rns and concatenated data sets, alpha (a) was 0.529, 0.966, 1.311 and 0.693, respectively. Two independent MCMCMC searches were run for each data set using different random starting points (number of generations: 1,000,000 for all datasets except

EGFR inhibitor for the concatenated set, where 2 million generations were needed) with sampling every 100 generations. Convergence was checked visually by plotting likelihood scores vs. generation for the two runs [the first 25% samples from the cold chain (relburnin = yes and burninfrac = 0.25) were discarded]. Based on this analysis, the burn-in was set to 10,000, as this was found to be clearly sufficient for the likelihood and the model parameters to reach equilibrium. Distances between trees produced by the same dataset but different method were computed with the Symmetric Difference of Robinson and Foulds [79] as implemented in program Treedist of the PHYLIP v.3.69 package [80]. Nucleotide sequence accession numbers The complete sequence of B. bassiana strain Bb147 and B. brongniartii strain IMBST 95031 have been submitted to GenBank [GenBank: EU100742 and GenBank: NC_011194, respectively].