Computational protocol: A morphological and phylogenetic revision of the Nectria cinnabarina species complex

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Protocol publication

[…] The forty-five cultures of N. cinnabarina used in the phylogenetic analyses () and representatives of other species of Nectria s. str. were grown in Difco™ potato dextrose broth in 6 cm diam Petri plates for about 3 wk. Mycelial mats were harvested in a laminar flow hood and dried with clean, absorbent paper towels. DNA was extracted with Ultra Clean™ Plant DNA Isolation Kit (MO BIO Laboratories Inc., Solana Beach, California, USA).Six loci were sequenced, namely a-actin (act) (), β-tubulin (tub) (), RNA polymerase II subunit one (rpb1) (), the internal transcribed spacer (ITS) (), large subunit nuclear ribosomal DNA (LSU) (), and translation elongation factor 1-a (tef1) (, ). The primers and PCR protocol information are listed in Tables and . PCR products were cleaned with ExoSAP-IT® (USB Corporation, Cleveland, Ohio, USA) following the manufacturer's instructions. Clean PCR products were sequenced at the DNA Sequencing Facility (Center for Agricultural Biotechnology, University of Maryland, College Park, Maryland, USA) and at MCLAB (Molecular Cloning Laboratories, San Francisco, California, USA). Sequences were assembled and edited with Sequencher v. 4.9 (Gene Codes, Madison, Wisconsin, USA). Sequences are deposited in GenBank (). [...] Sequences of the six genes were aligned with MAFFT v. 6 () and the alignment was visually improved with Mesquite v. 2.6 (). Maximum likelihood (ML) and Bayesian (BI) analyses were carried out with all sequences, first each locus separately, then with the combined/concatenated data sets. Representative members of the Nectriaceae, namely Cosmospora coccinea, Cyanonectria cyanostoma, and Thelonectria westlandica, were used as outgroups for inferring intrageneric relationships (). Nectria balansae, N. pseudocinnabarina, and N. pseudotrichia were used as outgroup taxa for the NCSC tree, including 45 isolates in the NCSC (). JMODELTEST () was used to calculate the models of nucleotide substitutions of each gene/partition for the ML and BI analyses. The number of substitution schemes was set to 11, base frequencies +F, rate variation +I and +G, and the base tree for likelihood calculations was set to “ML optimised”. 88 models were compared. After the likelihood scores were calculated, the models were selected according to the Akaike information criterion (AIC) (). Under the AIC settings, the AICc corrected for smaller samples was selected. After jMODELTEST was run, likelihood settings for trees of the Nectria tree and NCSC tree were set to each gene (Tables , ). For the ML and bootstrap analyses (BP), GARLI version 0.96 () was computed through the Grid computing () and The Lattice Project (), which includes clusters and desk tops in one integrated network (). In GARLI, the starting tree was made by stepwise-addition and the number of runs or search replicates was set to 50. 2000 ML BP replicates were done in GARLI with the starting tree chosen randomly. Bayesian analysis (BI) was done using MrBayes v. 3.1.2 (Huelsenbeck et al. , ). In MrBayes, data were partitioned by locus and the parameters of the nucleotide substitution models for each partition were set as described (Tables , ). For this analysis, two independent analyses of two parallel runs and four chains were carried out for 5 000 000 generations using MrBayes. Analyses were initiated from a random tree and trees sampled every 100th generation. The first 20 % of the resulting trees were eliminated (= “burn in”). A consensus tree (“sumt” option) and posterior probabilities (PP) were calculated in MrBayes, which combines the results from both parallel runs. A reciprocal 70 % BP threshold was used to detect topological incongruence among genes/partitions (, ). Fig. 1. […]

Pipeline specifications

Software tools Sequencher, MAFFT, Mesquite, jModelTest, GARLI, MrBayes
Application Phylogenetics
Organisms Nectria cinnabarina, Neoizziella asiatica