Computational protocol: Microbotryum silenes-saxifragae sp. nov. sporulating in the anthers of Silene saxifraga in southern European mountains

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Protocol publication

[…] The Microbotryum specimens examined in this study are listed in To elucidate the phylogenetic position of the Microbotryum specimens on Silene saxifraga their concatenated ITS + LSU sequences were analysed within a dataset that covered all caryophyllaceous anther smut species of which ITS and LSU sequences were available (, , , , , ) or that were sequenced in the present study (Microbotryum coronariae, ), comprising 19 of the 22 currently recognized species. For the final analysis the dataset was reduced to a maximum of two sequences per species. All sequences available in GenBank that clustered both within the Microbotryum sp. on Silene saxifraga clade and its sister clade (M. coronariae, M. silenes-inflatae) and which could not be assigned to any Microbotryum species (compare ) were kept.Sequence alignment was obtained using MAFFT 6.845b (, ) and the L-INS-I option. In the final alignment we retained conserved alignment positions using GBlocks () with the following options: ‘Minimum Number of Sequences for a Conserved Position’: 25, ‘Minimum Number of Sequences for a Flank Position’: 25, ‘Maximum Number of Contiguous Non-conserved Positions’: 8, ‘Minimum Length of a Block’: 5 and ‘Allowed Gap Positions’ to ‘With half’.The resulting alignment [new number of positions: 1276 (57% of the original 2232 positions) variable sites: 212] was used for phylogenetic analyses. Bayesian Analysis (BA) was performed using MrBayes 3.1.2 (, ) applying the same settings as in . Four incrementally heated chains were run for 10,000,000 generations, sampled every 100th generation, thereby resulting in 100,001 trees of which the first 25,001 sampled trees were discarded. Maximum Likelihood (ML) was performed using RAxML 7.2.6 () via the raxmlGUI (). We used the GTRGAMMA and rapid bootstrap option (). Trees were rooted with Microbotryum scabiosae following . […]

Pipeline specifications

Software tools MAFFT, Gblocks, MrBayes, RAxML, raxmlGUI
Application Phylogenetics