Computational protocol: Discovery or Extinction of New Scleroderma Species in Amazonia?

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Protocol publication

[…] Samples for DNA extraction were excised from dry basidiomes. To avoid contamination by other fungi, pseudotissues were taken from the inner part of the basidiome. DNA extraction, amplification, and sequencing of the ITS regions including the 5.8S of the ribosomal RNA gene cluster followed the protocols mentioned by Phosri et al. []. The ITS regions were amplified with Ready-To-GoTM PCR Beads (GE healthcare Life Sciences, NJ, USA), using the primers ITS1F [] and ITS4 [], and the cycling protocol described in Martín and Winka []. Aliquots of the purified products were mixed separately with the direct and reverse primers before sending them to Macrogen (South Korea) for sequencing. Consensus sequences were assembled using Sequencher software (Gene Codes Corporation Inc, Ann Arbor, Michigan, USA). Previous to the alignment, sequences were compared with homologous sequences from the EMBL/GenBank/DDBJ [] using the BLASTn algorithm []. All new sequences have been deposited on the EMBL-EBI database and their accession numbers are presented in .Using SEQAPP software (PerkinElmer Applied Biosystems), multiple sequence alignments were performed of the consensus sequences obtained in this study and homologous sequences from the EMBL/GenBank/DDBJ, ( (Phosri et al. [], Rusevska et al. [], and Crous et al. []) shown in . The alignment was optimized visually. Alignment gaps were indicated as “-” and ambiguous nucleotides were marked as “N”.The alignment was analyzed using the programms PAUP 4.0a147 [], MrBAYES 3.2.2 [] and RAxML [] using the CIPRES portal ( []. Pisolithus arhizus FM213365 was used as outgroup, since this species is closely related to Scleroderma []. First, a parsimony analysis under a heuristic search was conducted. Gaps were treated as missing data. The tree branch robustness was estimated by bootstrap (MP-BS) analysis [] employing 10000 replicates, using the fast-step option. The starting branch lengths were obtained using the Roger-Swofford approximation method and the starting trees for branch swapping were obtained by stepwise addition. The tree bisection-reconnection (TBR) branch-swapping algorithm was used with the Multitrees options. The data were further analyzed using a Bayesian approach [,]. The posterior probabilities (PP) were approximated by sampling trees using the MCMC method. The Bayesian analysis was performed assuming the general time reversible model [] including estimation of invariant sites and assuming a discrete gamma distribution with six rate categories (GTR+I+G). A run with 2M generation starting with a random tree and employing 12 simultaneous chains was executed. Every 100th tree was saved into a file. The log-likelihood scores of sample points were plotted against the number of generations using TRACER 1.0 ( to determine that stationarity was achieved when the log-likelihood values of the sample points reached a stable equilibrium value []. The initial 1000 trees were discarded as a burn-in before calculating posterior probabilities (PP). Using the “sumt” command of MrBAYES, the majority-rule consensus tree was calculated from 19K trees sampled after reaching likelihood convergence to calculate the posterior probabilities. A third maximum likelihood bootstrapping analysis was performed with RAxML 7.2.8 [], using the default parameters as implemented on the CIPRES NSF XSEDE resource with bootstrap statistics calculated from 1000 bootstrap replicates (ML-BS) under GTR + I + G model of evolution.The phylogenetic tree was drawn with the program TreeView [] and edited in Adobe Illustrator CS3; names of clades and subclades are according to Phosri et al. [], Rusevska et al. [], and Crous et al. []. A combination of MP-BS, ML-BS, and PP was used to assess confidence for a specific node [,]. […]

Pipeline specifications

Software tools Sequencher, BLASTN, PAUP*, MrBayes, RAxML, CIPRES Science Gateway, TreeViewX, Adobe Illustrator
Databases DDBJ
Applications Miscellaneous, Phylogenetics
Organisms Homo sapiens
Diseases Scleroderma, Localized
Chemicals Sulfur