Computational protocol: Mitochondrial genome sequence and gene order of Sipunculus nudus give additional support for an inclusion of Sipuncula into Annelida

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Protocol publication

[…] The amplified fragments were set up in 10 μl reaction volumes (2.5 μl DNA, 2.5 μl water, 1 μl primer (10 μM), 4 μl DCTS master mix) and sequencing PCR reactions were carried out according to the following procedure: 96°C for 20 sec (denaturation); primer-specific temperature for 20 sec (annealing); 60°C for 2 min (elongation). After 30 cycles the samples were sequenced with a CEQ™8000 capillary sequencer (Beckmann-Coulter) and the appropriate CEQ DCTS Quick Start kit (Beckmann-Coulter).While the first checking of the sequences was carried out with the CEQ 8000 software (Beckman-Coulter), the actual sequence assemblage was done with BioEdit, version 7.0.5 []. Protein coding and ribosomal RNA genes, encoded in the mtDNA, were identified by BLAST (blastn, tblastx) searches on NCBI databases and by aligning the different sipunculid fragments with the mt genome of the echiurid Urechis caupo. To revise the final consensus sequence of S. nudus, further mt-genome data of relatively closely related taxa were retrieved from the OGRe database []. The species used for sequence comparison were: Platynereis dumerilii (Annelida), Clymenella torquata (Annelida), Orbinia latreillii (Annelida), Lumbricus terrestris (Annelida), Terebratalia transversa (Brachiopoda), Terebratulina retusa (Brachiopoda), Laqueus rubellus (Brachiopoda), Urechis caupo (echiura), Epiperipatus biolleyi (Onychophora), and Flustrellidra hispida (Bryozoa), see Table for accession numbers. Transfer RNA genes and their putative secondary structures, were determined with the tRNAscan-SE [] and ARWEN [] and for the missing ones by eye inspection of candidate regions. The genome sequence was deposited in NCBI database [GenBank: FJ422961]. [...] The amino acid alignments of the protein-coding genes (except the two short and highly variable genes atp8 and nad4L) were concatenated. Sequence data from 74 species were included in the large analyses (see Table for all species names and accession numbers). The tree was rooted with two representatives of Cnidaria. Maximum likelihood analysis was performed with RAxML, ver. 7.00 [,]. mtREV+G+I was chosen as model for aminoacid substitutions. The complete dataset was partitioned, so that model parameters and amino acid frequencies were optimized for each single gene alignment. 100 bootstrap replicates were performed to infer the support of clades from the best tree. A second set of analyses were done with a reduced dataset of 30 species. This dataset was analyzed with RAxML as described above (model mtREV+G+I, partitioned according to the 12 single gene sequences), with 1000 bootstrap replicates. Secondly we did a Bayesian analysis with MrBayes ver. 3.1.2 []. In BI the mtREV+G+I model was used and 1.000.000 generations were run with 8 chains in parallel. Trees were sampled every 1000 generations, while the first 200 trees were discarded as burn-in (according to the likelihood plot). In addition we performed a ML analysis using the mtART+G+I model with Treefinder [] and "edge support" analysis, again with a partitioned dataset (= independently optimizing model parameters for the 12 genes).For comparison of the hypothesis that sipunculids might be closely related with molluscs and our best tree, we used a constraint for a ML-analysis (Sipuncula + Mollusca) of the sequence dataset using RaxML [] with parameters described above. We computed per-site log-likelihoods with RAxML for both topologies (best tree and constrained topology) and conducted an au-test as implemented in CONSEL []. […]

Pipeline specifications

Software tools BioEdit, BLASTN, TBLASTX, tRNAscan-SE, ARWEN, RAxML, MrBayes, CONSEL
Applications Genome annotation, Phylogenetics, Nucleotide sequence alignment
Organisms Sipunculus nudus