Computational protocol: Description of two three-gendered nematode species in the new genus Auanema (Rhabditina) that are models for reproductive mode evolution

Similar protocols

Protocol publication

[…] Near full-length SSU rDNA and LSU rDNA and part of the largest subunit of RNA polymerase II were amplified from worm lysates of A. freiburgensis n. sp. or from reverse transcribed total RNA and sequenced. The resulting sequences were aligned with published sequences of 18 representatives of Rhabditina (see Supplementary Data  for Genbank numbers).The alignment for SSU rDNA was done by hand using secondary structure predictions as reported earlier. LSU rDNA sequences were aligned using ClustalW2 via the EMBL EBI website ( Default parameters were used, but the gap-open-penalty was set to 25 for pairwise and multiple sequence alignment. The resulting alignment was then manually improved. The alignment of RNA Polymerase II sequences was unambiguous.To test the data for robustness to method of phylogenetic inference, we performed analyses with weighted maximum parsimony (wMP), maximum likelihood (ML) and Bayesian inference (BI). Robustness of the data to character representation was tested using bootstrap and jackknife analyses.The wMP jackknife analysis was performed with Paup*. A transversion was weighted twice a transition as in previous analyses of rhabditids. The jackknife analysis was run with 1000 replicates and 2 addition sequence replicates in each round.The ML analysis was run with RAxML ver. 7.2.8 (“BlackBox” version) via the CIPRES Science Gateway on the TeraGrid of NSF–. A six-parameter substitution model was used with a gamma correction for rate differences across sites (using 25 discrete categories of sites) and a correction for unvarying sites (GTR + G + I). Parameters were estimated from the data. The shape parameter for the gamma distribution of rates was α = 0. 465874. Estimated proportions of nucleotides were: π(A) = 0.262, π(C) = 0.210, π(G) = 0.267, π(T) = 0.261. Estimated rates for the GTR model were: ƒ(AC) = 0.934, ƒ(AG) = 2.431, ƒ(AT) = 1.635, ƒ(CG) = 0.559, ƒ(CT) = 4.591, relative to ƒ(GT) = 1.000.The BI analysis was run with MrBayes ver. 3.1.2, via the CIPRES portal. A six-parameter substitution model was used with a gamma correction for rate differences across sites and an estimate for the proportion of invariant sites (GTR + G + I). The analysis was run for 2,000,000 generations. Trees and parameters were sampled every 100 generations. Burnin was set to 10% of the samples to calculate the clade credibility values (posterior probabilities) and to estimate the model parameters, which were: π(A) = 0.278, π(C) = 0.217, π(G) = 0.257, π(T) = 0.258. Estimated rates for the GTR model were: ƒ(AC) = 0.737, ƒ(AG) = 1.615, ƒ(AT) = 1.170, ƒ(CG) = 0.622, ƒ(CT) = 3.013, relative to ƒ(GT) = 1.000. […]

Pipeline specifications

Software tools Clustal W, RAxML, MrBayes, CIPRES Science Gateway
Application Phylogenetics
Organisms Caenorhabditis elegans
Diseases Nematode Infections