Computational protocol: History of myxozoan character evolution on the basis of rDNA and EF-2 data

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Protocol publication

[…] Seven alignments of EF2, SSU and LSU data with twelve myxosporeans were constructed. The cnidarian Aurelia sp. was set as outgroup. The alignments consisted of single gene data, concatenated SSU + LSU and concatenated SSU + LSU + EF2 data. EF2 gene was aligned as nucleotide sequence (EF2nt) as well as amino acid (EF2aa) sequence data. EF2nt were analysed with all three sites in codon, with the exclusion of the third site of codon and as codons. The alignments were aligned using MAFFT program [] with E-INS-i method and gap opening penalty (--op) 5.0 and gap extension penalty (--ep) 0.0. The alignments were visualised in SEAVIEW v. 3.2 [].Maximum parsimony (MP) analyses were performed in PAUP* 4.0b10 [] using a starting tree build by heuristic search with random taxa addition, the ACCTRAN-option and the TBR swapping algorithm. Ts/Tv ratio of 1:2 was applied for rDNA data and the gaps were treated as missing data. Clade supports were estimated using 500 bootstrap-replicates with random sequence additions.Maximum likelihood (ML) analyses of EF2aa and EF2nt as codon sequence type were performed in GARLI v. 0.96 []. Amino acid analysis was done using WAG model and F3x4 method of codon frequencies was chosen for the codon based analysis. All the other ML analyses were made in PAUP* 4.0b10. The best model of evolution was determined by the likelihood ratio test (LRT) implemented in the Modeltest 3.06 []. SSU, LSU and SSU + LSU analyses were performed under GTR + Γ model. EF2nt analysis was performed under GTR + Γ + I model. Bootstrap values were calculated by 500 bootstrap re-sampling.Bayesian inference (BI) trees were constructed using MrBayes 3.0b4 []. Likelihood parameters that were set for the single gene analyses and for the data partitions of concatenated analyses correspond to the models used in ML. The number of Markov chain Monte Carlo (MCMC) generations was set to 1,000,000 with every 100th tree saved (two independent runs of four simultaneous MCMC chains). AWTY system [] was used to assess the length of MCMC run and Tracer v. 1.4.1 [] was used to ascertain the length of burn-in periods. [...] Twenty morphological and bionomical characters were chosen for the analysis (Figure ). The analysis includes (1) number of spore valves, (2) shape of spore, (3) ratio of dimensions of spore width to the thickness, (4) surface ridges and striations, (5) projections of the spore, (6) shape of suture line, (7) number of polar capsules, (8) orientation of polar capsules to a plane of suture, (9) location of polar capsules and sporoplasm, (10) shape of polar capsules, (11) position of tips of polar capsules, (12) character of polar filament, (13) number of sporoplasms, (14) mucous envelope, (15) membranaceous veil, (16) vegetative stages, (17) site of infection, (18) site specificity, (19) host, and (20) host environment. The SSU rDNA-based tree was chosen as the basis for reconstruction of ancestral states. The tree consists of 73 myxosporean species covering the myxosporean diversity of known SSU rDNA sequences and malacosporean Tetracapsuloides bryosalmonae as the outgroup. The tree was constructed using ML with GTR + Γ + I model of evolution in PAUP* based on the alignment computed by MAFFT with parameters described above. Tree branches with uncertain phylogenetic relationships - unstable positions in the tree as described in Fiala [] - were collapsed in their nodes resulting into polytomy for more accurate reconstruction of myxozoan phylogenetic relationships. History of character change was traced using the program Mesquite 2.5 []. Reconstruction of character states at ancestral nodes was done by likelihood method. We used Markov k-state 1 parameter model with the single parameter (the rate of change) []. Any particular change from one state to another is equally probable within this model. […]

Pipeline specifications

Software tools MAFFT, SeaView, GARLI, ModelTest-NG, MrBayes, PAUP*, Mesquite
Application Phylogenetics
Organisms Danio rerio