Adaptation of the Mitochondrial Genome in Cephalopods: Enhancing Proton Translocation Channels and the Subunit Interactions

<div><p>Mitochondrial protein-coding genes (mt genes) encode subunits forming complexes of crucial cellular pathways, including those involved in the vital process of oxidative phosphorylation (OXPHOS). Despite the vital role of the mitochondrial genome (mt genome) in the survival of organisms, little is known with respect to its adaptive implications within marine invertebrates. The molluscan Class Cephalopoda is represented by a marine group of species known to occupy contrasting environments ranging from the intertidal to the deep sea, having distinct metabolic requirements, varied body shapes and highly advanced visual and nervous systems that make them highly competitive and successful worldwide predators. Thus, cephalopods are valuable models for testing natural selection acting on their mitochondrial subunits (mt subunits). Here, we used concatenated mt genes from 17 fully sequenced mt genomes of diverse cephalopod species to generate a robust mitochondrial phylogeny for the Class Cephalopoda. We followed an integrative approach considering several branches of interest–covering cephalopods with distinct morphologies, metabolic rates and habitats–to identify sites under positive selection and localize them in the respective protein alignment and/or tridimensional structure of the mt subunits. Our results revealed significant adaptive variation in several mt subunits involved in the energy production pathway of cephalopods: ND5 and ND6 from Complex I, CYTB from Complex III, COX2 and COX3 from Complex IV, and in ATP8 from Complex V. Furthermore, we identified relevant sites involved in protein-interactions, lining proton translocation channels, as well as disease/deficiencies related sites in the aforementioned complexes. A particular case, revealed by this study, is the involvement of some positively selected sites, found in Octopoda lineage in lining proton translocation channels (site 74 from ND5) and in interactions between subunits (site 507 from ND5) of Complex I.</p></div

Negative selection reported by FEL site-by-site analyses.

<p>(A) Percentage of codons under negative selection in cephalopod mitochondrial subunits. (B) Cephalopoda ϖ estimates for each one of the mitochondrial subunits.</p

Cephalopoda mitogenomic consensus phylogenetic tree.

<p>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.</p

Foreground-branches tested for both branch-specific and branch-site selection models.

<p>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.</p

Datamonkey tab and related results.

<p>(A) ‘Datamonkey’ tab showing all the selected options, (B) ‘Common Sites’ table generated from the positive-selection analyses under various methods (SLAC, FEL, REL, MEME and FUBAR) – The sites 110, 172, 202 and 231 (highlighted in pink), were simultaneously detected by Datamonkey, PAML (Codeml) and TreeSAAP as positively-selected (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096243#pone.0096243.s001" target="_blank">Table S1</a>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096243#pone.0096243-Yang1" target="_blank">[1]</a>, (C) ‘Results & 3D’ tab showing the mapping of sites found in the ‘Common Sites’ table.</p

IMPACT_S: Integrated Multiprogram Platform to Analyze and Combine Tests of Selection

<div><p>Among the major goals of research in evolutionary biology are the identification of genes targeted by natural selection and understanding how various regimes of evolution affect the fitness of an organism. In particular, adaptive evolution enables organisms to adapt to changing ecological factors such as diet, temperature, habitat, predatory pressures and prey abundance. An integrative approach is crucial for the identification of non-synonymous mutations that introduce radical changes in protein biochemistry and thus in turn influence the structure and function of proteins. Performing such analyses manually is often a time-consuming process, due to the large number of statistical files generated from multiple approaches, especially when assessing numerous taxa and/or large datasets. We present IMPACT_S, an easy-to-use Graphical User Interface (GUI) software, which rapidly and effectively integrates, filters and combines results from three widely used programs for assessing the influence of selection: Codeml (PAML package), Datamonkey and TreeSAAP. It enables the identification and tabulation of sites detected by these programs as evolving under the influence of positive, neutral and/or negative selection in protein-coding genes. IMPACT_S further facilitates the automatic mapping of these sites onto the three-dimensional structures of proteins. Other useful tools incorporated in IMPACT_S include Jmol, Archaeopteryx, Gnuplot, PhyML, a built-in Swiss-Model interface and a PDB downloader. The relevance and functionality of IMPACT_S is shown through a case study on the toxicoferan-reptilian Cysteine-rich Secretory Proteins (CRiSPs). IMPACT_S is a platform-independent software released under GPLv3 license, freely available online from <a href="http://impact-s.sourceforge.net" target="_blank">http://impact-s.sourceforge.net</a>.</p></div

PAML tab and related results.

<p>(A) The <b>‘</b>PAML’ tab from IMPACT_S showing the results of the LRT test (M7 <i>vs.</i> M8), (B) Automatically extracted and tabulated ‘BEB results’ table, (C) Mapping of the positively selected sites onto the 3D protein-structure of CRiSPs (2GIZ-A), (D) Tabulated results of <i>ω</i> estimation under the site-specific models (M0, M1, M3, M7 and M8) and (E) The ‘lnL’ table, showing all the log-likelihood values for these models.</p

Evolutionary Pathway (Evpthwy) tab under TreeSAAP tab and related results.

<p>(A) ‘TreeSAAP’ tab showing the ‘Evpthwy’ tab with results of the CRiSP case study, (B) Gnuplot graph for the property “Average number of surrounding residues” with Z-scores in y-axis and Codon positions in x-axis, (C) ‘PBR’ table showing all ranges (‘From’ - ‘To’) retrieved from the sliding window analysis with the ‘Total’ count of properties for each range and their names [Properties (7–8) (+) (−) – names of properties under significant positive (Z-score ≥3.09) and negative (Z-score ≤3.09) selection, found in the categories 7 and 8].</p

Substitutions (Substs) tab under TreeSAAP tab and related results.

<p>(A) ‘TreeSAAP’ tab showing the ‘Substs’ tab and the results of the CRiSPs case study, (B) ‘PBS’ table showing all the significant codons (‘Codon’ column) and the total count (‘Total’ column) of properties for each codon and their respective names [‘Properties (7–8) (+)’ – names of properties under significant (Z-score ≥3.09) positive selection], (C) ‘PBS statistics’, with respect to the PBS table, showing the number of times (‘Total’ column) that the same property (‘Information’ column) is selected across the data set provided and (D) Alignment Filter option with the final view of the alignment containing only the codons from the PBS table with their original positions.</p

3D mapping and related functionalities under Results & 3D tab.

<p>(A) <b>‘</b>Results & 3D’ tab with the results of the CRiSPs case study, (B) ‘MR table’ (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096243#pone.0096243.s003" target="_blank">Table S3</a>) presenting six columns: ‘Codon’ containing the site found as positive selected (TreeSAAP – Z-score ≥3.09); ‘Datamonkey’ with the positive selected common sites positions from SLAC, FEL, REL, MEME and FUBAR; ‘PAML M8Site’ with the information from BEB results M8; ‘Common Sites’ – reporting the site position common to all the previous mentioned columns; ‘Total’ - count of the number of properties per site; ‘Properties (7–8) (+)’ with the names of properties found under categories 7 and 8 for Z-score ≥3.09, (only common sites are shown and highlighted) (C) 3D structure and ‘PDB Sequence’, mapping the sites positions from the ‘Common Sites’ column following the (D) Merged Results ‘Color Scheme’ according to a pre-defined number of properties: red – less than 2 (NP<2), green - between 2 and 5 (NP(2–5)) and blue - more than 5 (NP>5).</p