Computational protocol: Molecular Evolution of Lepidopteran Silk Proteins: Insights from the Ghost Moth, Hepialus californicus

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Protocol publication

[…] The H. californicus H- and L-fibroin transcripts were compared to lepidopteran and trichopteran sequences obtained from the NCBI database. Specifically, coding sequences for the H-fibroins of Ephestia kuehniella (accession number AY253535), Antheraea yamamai (AF410906), Bombyx mandarina (DQ459410), Bombyx mori (NM_001113262), Galleria mellonella (AF095240), Hydropsyche angustipennis (AB214507), Limnephilus decipiens (AB214509), Rhyacophila obliterata (AB354588), and Yponomeuta evonymellus (AB195979); and the L-fibroins of Bombyx mandarina (AB001820), Bombyx mori (X17291), Dendrolimus spectabilis (AB001822), Galleria mellonella (S77817), Hydropsyche angustipennis (AB214508), Limnephilus decipiens (AB214510), Papilio xuthus (AB001824), Rhyacophila obliterata (AB354590), and Yponomeuta evonymellus (AB195977) were downloaded. Sequence alignments were performed with ClustalW (Thompson et al. ) and refined by eye.Signatures of selection on the protein coding regions of L-fibroin and the carboxyl-terminal region of H-fibroin were examined through maximum likelihood analyses using the codeml package of PAML (Yang ). PAML determined ω, the ratio of non-synonymous substitutions per non-synonymous site to synonymous substitutions per synonymous site, for each codon. We used an input tree based on recent studies of higher-level lepidopteran relationships (Kjer et al. ; Whiting ; Wiegmann et al. ) and analyzed the data with four different models: M1a, neutral model; M2a, selection model; M7, beta distribution neutral model; and M8, beta distribution with selection. Models M1a versus M2a and M7 versus M8 were then compared using likelihood ratio tests (Yang et al. ).Phylogenetic analyses were conducted in PAUP* v.4.0b10 (Swofford ). In parsimony analyses, all character transformations were weighted equally. In maximum likelihood analyses, models of DNA evolution were chosen by the Akaike information criterion implemented in MODELTEST v.3.6 (Posada and Crandall ) and PAUP*. Parsimony searches utilized the branch and bound method and maximum likelihood searches were heuristic. Both parsimony and maximum likelihood searches were performed with random taxon addition and tree-bisection and reconnection. Nodal support values were assessed using bootstrap analyses (Felsenstein ). Bootstrap replicates were heuristic with tree-bisection and reconnection branch swapping. For parsimony searches, 10,000 iterations with 100 random taxon additions were done and 1,000 iterations with 10 random taxon additions were executed in maximum likelihood searches. […]

Pipeline specifications

Software tools Clustal W, PAML, PAUP*, ModelTest-NG
Application Phylogenetics
Organisms Caenorhabditis elegans
Chemicals Cysteine