Fig 1.
Cephalopoda mitogenomic consensus phylogenetic tree.
The topology shown corresponds to the Maximum likelihood (ML) tree. The bootstrap probabilities (%) supporting each node were estimated with ML analyses using PHYML software version 3.0 (1000 bootstrap replicates, GTR+G+I model and best of NNI&SPR branch search algorithm) and are shown by the first and second values next to the branches. The RY symbol indicates that these values resulted from the RY coded alignments. The posterior probability supporting each node was estimated from Bayesian analyses using MRBAYES (GTR+G+I model; 10 000 000 generations; a sample frequency of 100 and a burn-in corresponding to 25% of the sampled trees) and corresponds to the third value next to the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Superorders–Decapodiformes (10 arms) and Octopodiformes (8 arms). The species are designated by their scientific names followed by the respective accession numbers. On the right, are indicated the names of the major taxonomic groups of cephalopods.
Fig 2.
Negative selection reported by FEL site-by-site analyses.
(A) Percentage of codons under negative selection in cephalopod mitochondrial subunits. (B) Cephalopoda ϖ estimates for each one of the mitochondrial subunits.
Fig 3.
Foreground-branches tested for both branch-specific and branch-site selection models.
Identification of the Maximum likelihood tree branches to test the adaptive evolution for each one of the 13-mitochondrial genes in 17 representative species from the molluscan Class Cephalopoda. The letters indicate the branches of interest (foreground-branches: named A to H). We performed 8 tests, where only one of the branches pointed by the letters was considered at a time; all other branches are corresponding to background-lineages for the analyzed gene. The sites represented correspond to positively selected sites by the employed approaches (CODEML and MEME). The numbers in bold, with an asterisk mark, represent sites that obtained posterior probabilities ≥ 99% (p-value < 0.05) and the other numbers, posterior probabilities ≥ 95% (p-value < 0.05), respectively.
Fig 4.
Amino acid variation in functional and structural sites of Cephalopoda ND5 subunit.
(A) Amino acid alignment for sites (i) under positive selection—3, 17, 38, 74, 159, 239, 289, 327, 475, 497 (CODEML branch-site model test) and 415 (MEME), (ii) sites involved in interactions between subunits—41, 132, 161, 162, 507, 516, 524, 525, 550, 553 (iii) sites lining proton translocation channels—74, 93, 94, 118, 125, 147, 218, 281, 392, 455 and (i) described mutations—108, 142, 143, 199 and 427. All the sites have as reference number, the site position from the Octopus vulgaris ND5 protein-sequence, highlighted with a magenta rectangle shape. Sites highlighted by: (i) an orange shape—identified by CODEML branch-site model test and employing TREESAAP analysis; (ii) cyan shape—site lining proton translocation channel and reported by CODEML branch-site model test; (iii) blue shape—site lining proton translocation channel and positively selected by TREESAAP; (iv) green shape—site involved in subunit interactions (ND5-ND4) and also positively selected by TREESAAP. Background amino acid colors represent a hydrophobicity scheme from the most hydrophobic (red) to the most hydrophilic (blue). (B) Superimposition of 3D structures of ND5 from Octopus vulgaris (magenta) and NuoL from Escherichia coli (white), viewed from inner mitochondrial membrane—map of all the previously mentioned sites. At the left are projected the sites (cyan sticks) lining the proton translocation channels (blue arrow): H+—putative channel and? H+—suggested channel at the interfaces of ND5 and ND4 subunits. At the right, the projected sites are involved in interactions between subunits (green sticks), described mutations (red sticks) and the sites under positive selection (CODEML—orange sticks; MEME—blue sticks). (C) View from the intermembrane space. TM—transmembrane helices: helices that are probably involved in conformational changes are shown in red (TM6—discontinuous helix), orange (TM11—discontinuous helix) and yellow (TM7—discontinuous helix). Connecting elements: β—β-hairpins and HL—HL helix (hot pink).
Fig 5.
Amino acid variation in functional and structural sites of Cephalopoda ND6 subunit.
Amino acid alignment for sites: (i) under positive selection—6, 32, 113 and 119 (MEME); (ii) at the surface—24 and 72; (iii) lining proton translocation channels—33, 34, 53, 58, 62, 70, 139 and 142. All the sites have as reference number the site position from the Octopus vulgaris ND6 protein-sequence. The sites lining proton translocation channel also identified by TREESAAP are highlighted with a cyan shape and with a green shape the sites positively selected by MEME and also by TREESAAP. Background amino acid colors represent a hydrophobicity scheme from red—the most hydrophobic to blue—the most hydrophilic. TM—transmembrane helices. TM and loops—according to the topology TMHMM prediction.
Fig 6.
Amino acid variation in functional and structural sites of Cephalopoda CYTB subunit.
(A) Amino acid alignment for sites (i) under positive selection—orange shape (CODEML branch-site model test) and blue shape (MEME), (ii) mutations related with human exercise intolerance—maroon sites, (iii) Qo site binding sites—orange sites, (iv) Qi site binding sites—yellow sites, (v) bL binding sites—blue sites and (vi) bH binding sites—black sites. The amino acid substitutions in some of the mentioned sites are marked with arrows. (B) Superimposition of 3D structures with the positively selected sites mapped as sticks (orange for CODEML and blue for MEME). All the sites have as reference number, the site position from the Octopus vulgaris.
Fig 7.
Amino acid variation in functional and structural sites of Cephalopoda COX2 and COX3 subunits.
(A) COX2: Amino acid alignment for (i) sites under positive selection—blue shape (MEME), (ii) mutations related with human exercise intolerance—red sites, (iii) CuA metal binding sites—black sites, (iv) sites involved in proton coupling mechanism—cyan sites and (v) metal binding site for Mg2+—green site. COX3: Amino acid alignment for (i) sites under positive selection—orange shape (CODEML branch-site model test) and blue shape (MEME), (ii) sites in the proximity of the entrance of D-channel—green sites, (iii) residues that protect the entrance of the D-channel from direct solvent exposure—cyan sites and (iv) sites related with diseases in humans—red sites. The amino acid substitutions in some of the mentioned sites are marked with arrows. (B) Superimposition of 3D structures with the sites mapped as sticks (colors according to Fig 7A). All the sites have as reference number, the site position from the Octopus vulgaris.