Computational protocol: Comparative phylogenies and host specialization in the alder ectomycorrhizal fungi Alnicola, Alpova and Lactarius (Basidiomycota) in Europe

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Protocol publication

[…] Sequences were manually edited using Sequencher 4.8. Gene Codes (Ann Arbor, MI). They were aligned using MAFFT version6 using the LINS-i method with standard settings [] and subsequently carefully refined by eye. Phylogenetic analyses were conducted on sequence datasets from individual and combined DNA regions using maximum likelihood reconstruction with PhyML [] and Bayesian inference as implemented in MrBayes 3.1.2 []. [...] Modeltest 3.7 [] was run for all data sets (individual and combined) to select a model of sequence evolution. Appropriate DNA substitution models and its parameters were determined based on the comparisons of negative log likelihood values in Modeltest 3.7 [] implemented in PAUP*4.0b10 []. Likelihood and prior settings were changed to meet with the settings corresponding to the models found for each marker. Gaps were coded as missing data. The analyses were initiated with random starting trees and one cold and three incrementally heated Markov chain Monte Carlo (MCMC) chains were run for 10,000,000 generations. Trees were sampled every 1000 generations. The previously determined model of sequence evolution to each data set was applied in the partitioned Bayesian analyses for all combined data sets. For each data set, MCMC runs were repeated twice. Stationarity of the Markov chain was ascertained by plotting likelihood values against number of generations for apparent stationarity. The first 1000 to 2000 trees were discarded as burn-in, and the remaining trees were used to calculate a 50% majority rule tree and to determine the posterior probabilities for the individual branches. [...] To estimate divergence times between lineages, the combined data set of ITS and MatK sequences was employed. Rate constancy was tested with a likelihood ratio test []. Because the molecular data departed from clock-like evolution, a Bayesian analysis implemented with BEAST ver. 1.4.7 [] was used under a log-normal relaxed molecular clock [] and a Yule pure birth model of speciation to estimate the times of divergence and their credibility intervals in the genus Alnus. Posterior distributions of parameters were approximated using two independent MCMC analyses of 10,000,000 generations with 10% burn-in. Samples from the two runs which yielded similar results were combined and convergence of the chains was checked using the program Tracer 1.3 []. Based on a recent Betulaceae phylogeny [] the divergence between Alnus and Betula was assigned with an age of 95 to 105 million year (My). […]

Pipeline specifications

Software tools Sequencher, MAFFT, PhyML, MrBayes, ModelTest-NG, PAUP*, BEAST
Application Phylogenetics
Chemicals Carbon