Computational protocol: Molecular Species Delimitation in the Racomitrium canescens Complex (Grimmiaceae) and Implications for DNA Barcoding of Species Complexes in Mosses

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[…] DNA sequences were manually aligned in PhyDE® v0.995 . Phylogenetic reconstructions of taxon circumscriptions and relationships were performed based on the maximum parsimony (MP) principle and using two model-based approaches, maximum likelihood (ML) and Bayesian inference (BI). Separate MP analyses of the rps4-trnT-trnL versus ITS sequences were performed and the resulting tree topologies were checked for possible incongruence between the plastid and nuclear markers by visual inspection and by applying a partition homogeneity test (ILD test) , as implemented in PAUP* 4.0b10 (100 replicates). Calculations of pairwise Fst estimates and p-values based on plastid and ITS haplotypes were performed using Arlequin v3.5.1.3 , with haplotypes (including indel characters) delineated in TCS v1.2.1 .Phylogenetic analyses of the combined plastid and nuclear markers were run using MP, ML, and BI. To evaluate the employed markers for DNA barcoding, further MP analyses were performed of four partitions for which primers pairs are available, viz. rps4-trnT, trnT-trnL, ITS1, and ITS2. In addition, pairwise nucleotide distances between all sequences were calculated according to the K2P model (cf. , ) for the combined dataset and all partitions, and compared between and within the species of the R. canescens complex.Calculation of pairwise distances as well as MP and ML analyses were performed in PAUP. Heuristic searches under parsimony were implemented using random sequence addition with 1000 replicates and tree bisection-reconnection (TBR) branch swapping. All MP analyses were run with gaps (indels) either treated as missing data or coded as informative by a simple indel coding (SIC) strategy as implemented in SeqState . Heuristic bootstrap searches under parsimony were performed with 1000 replicates and 10 random addition cycles per bootstrap replicate with the same options in effect. To search the tree space for islands of more parsimonious trees, parsimony ratchet analyses were performed with PRAP2 in combination with PAUP, employing the default options (200 iterations, 25% of randomly chosen positions up-weighted to 2) and superimposed 10 random addition cycles.For the model-based approaches, model testing was performed in Modeltest 3.7 employing MrMTgui . GTR+Γ+I was indicated as best-fit model of the combined dataset according to the Akaike information criterion (AIC). Consequently, the settings basefreq = (0.3011 0.2002 0.2254), nst = 6, Rmat = (1.5235 5.1208 0.6544 2.3313 7.1000), rates = gamma, shape = 0.7849, and pinvar = 0.6263 were used for ML, and nst = 6 and rates = invgamma for BI. Bayesian posterior probabilities were calculated based on the Metropolis-coupled Markov chain Monte Carlo (MCMCMC) method, using MrBayes v3.1 . In a second set of Bayesian analyses the indels coded by SIC were included, with sequence and indel data treated as separate and unlinked partitions, employing the restriction site model (‘F81’) for the indel matrix. The a priori probabilities supplied were those specified in the default settings of the program. Four runs with four chains (106 generations each) were run simultaneously, with the temperature of the single heated chain set to 0.2. Chains were sampled every 1000 generations and the respective trees written to a tree file. Fifty percent majority rule consensus trees and posterior probabilities of clades were calculated by combining the four runs and using the trees sampled after the chains converged. Trace plots generated in Tracer v1.5 were used to check for convergence of the runs (plateaus of all runs at comparable likelihoods) and to infer the ‘burnin’, which ranged approximately between the first 100000 and 120000 generations (first 100–120 sampled trees). Consequently, the first 150 trees (15%) were deleted to be sure that only trees of the stationary phase were included. […]

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