Computational protocol: New species, hyper-diversity and potential importance of Calonectria spp. from Eucalyptus in South China

Similar protocols

Protocol publication

[…] Total genomic DNA was extracted from 7-d-old cultures established from single-conidial propagules, grown on MEA at room temperature, using the UltraClean™ Microbial DNA isolation kit (Mo Bio Laboratories, Inc., California, USA) following the protocols provided by the manufacturer. Partial gene sequences were determined for β-tubulin (tub2), calmodulin (cmdA), histone H3 (his3), and the translation elongation factor 1-alpha (tef1) regions using the primers and protocols described by . To ensure the integrity of the sequences, the amplicons were sequenced in both directions using the same primers used for amplification. Consensus sequences for each locus were assembled in MEGA v. 5.1 () and compared with representative sequences from and . Subsequent alignments for each locus were generated in MAFFT v. 7.110 () and the ambiguously aligned regions of both ends were truncated.Phylogenetic analyses were based on both Bayesian inference (BI) and Maximum Parsimony (MP). For BI, the best evolutionary model for each locus was determined using MrModeltest () and incorporated into the analyses. MrBayes v. 3.2.1 () was used to generate phylogenetic trees under optimal criteria for each locus. A Markov Chain Monte Carlo (MCMC) algorithm of four chains was initiated in parallel from a random tree topology with the heating parameter set at 0.3. The MCMC analysis lasted until the average standard deviation of split frequencies was below 0.01 with trees saved every 1 000 generations. The first 25 % of saved trees were discarded as the “burn-in” phase and posterior probabilities (PP) were determined from the remaining trees.For MP, analyses were done using PAUP (Phylogenetic Analysis Using Parsimony, v. 4.0b10; ) with phylogenetic relationships estimated by heuristic searches with 1 000 random addition sequences. Tree-bisection-reconnection was used, with branch swapping option set on “best trees” only. All characters were weighted equally and alignment gaps treated as fifth state. Measures calculated for parsimony included tree length (TL), consistency index (CI), retention index (RI) and rescaled consistency index (RC). Bootstrap analyses () were based on 1 000 replications.Phylogenetic analyses were conducted on two separate sequence datasets. Datasets were separated based on morphological characteristics into the Prolate Group and Sphaero-Naviculate Group as defined by , making it possible to reduce the number of ambiguously aligned regions for the loci analysed. The dataset representing the Prolate Group of species was rooted to C. hongkongensis (CBS 114711 & CBS 114828) and the dataset representing the Sphaero-Naviculate Group was rooted to C. pauciramosa (CMW 5683 & CMW 30823). […]

Pipeline specifications

Software tools MEGA-V, MAFFT, MrModelTest, MrBayes, PAUP*
Applications Phylogenetics, GWAS
Chemicals Phosmet