Computational protocol: Using a multilocus phylogeny to test morphology based classifications of Polystichum (Dryopteridaceae), one of the largest fern genera

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Protocol publication

[…] The resulting sequences were edited and assembled with Sequencher V.4.14 (GeneCodes Corporation, Ann Arbor, Michigan, USA). We manually performed the sequence alignment using Bioedit []. Gaps (insertion/deletion events) were considered as missing data. Phylogenetic relationships were reconstructed using maximum parsimony (MP), maximum likelihood (ML), and Bayesian inference (BI). Maximum parsimony jackknife (MPJK) analyses [] were conducted using PAUP* for each dataset with the removal probability set to approximately 37 %; and “jac” resampling emulated. One thousand replicates were performed, each from a different random addition sequence tree, with 100 TBR searches per replicate and a maximum of 100 trees held per TBR search. A final simultaneous MP analysis [, ] was conducted based on the combined dataset including the five molecular markers.Each DNA region of the concatenated molecular matrix was assigned a separate GTR+I+G substitution model. ML tree searches and 10,000 rapid bootstrapping (MLBS) were conducted using RAxML-HPC and default parameters, followed by a search for the best-scoring tree, in a single run [, ].jModelTest 2 [] was used to select the best-fit likelihood model for Bayesian analyses. The Akaike information criterion [] was used to select among models. The models selected were GTR+G (psb-trnH spacer), GTR+I+G (rps4-trnS spacer, the combined region trnL-trnF and rbcL gene). The selected models (Table ) were then used for tree searches from the respective data partitions in combination. BI analyses were performed using MrBayes v3 []. For each DNA partition, we used the appropriate model selected by jModelTest 2, and each molecular region has independent parameters and the overall rate is allowed to be different across partitions. Four chains (i.e., three heated and one cold) of Metropolis-coupled Markov chain Monte Carlo were performed for 50 million generations, sampling every 1000th generation. After checking the convergence of parameter traces among generations using Tracer [], we discarded the first 25 % of sampled trees as a “burn-in phase”. The remaining trees were then used to calculate Bayesian inference posterior probability (BIPP).ML and BI analyses were run on the CIPRES cluster available at []. […]

Pipeline specifications

Software tools Sequencher, BioEdit, RAxML, jModelTest, MrBayes
Application Phylogenetics