Initial Evidence for Adaptive Selection on the NADH Subunit Two of Freshwater Dolphins by Analyses of Mitochondrial Genomes

<div><p>A small number of cetaceans have adapted to an entirely freshwater environment, having colonized rivers in Asia and South America from an ancestral origin in the marine environment. This includes the ‘river dolphins’, early divergence from the odontocete lineage, and two species of true dolphins (Family Delphinidae). Successful adaptation to the freshwater environment may have required increased demands in energy involved in processes such as the mitochondrial osmotic balance. For this reason, riverine odontocetes provide a compelling natural experiment in adaptation of mammals from marine to freshwater habitats. Here we present initial evidence of positive selection in the NADH dehydrogenase subunit 2 of riverine odontocetes by analyses of full mitochondrial genomes, using tests of selection and protein structure modeling. The codon model with highest statistical support corresponds to three discrete categories for amino acid sites, those under positive, neutral, and purifying selection. With this model we found positive selection at site 297 of the NADH dehydrogenase subunit 2 (<i>d_N/d_S</i>>1.0,) leading to a substitution of an Ala or Val from the ancestral state of Thr. A phylogenetic reconstruction of 27 cetacean mitogenomes showed that an Ala substitution has evolved at least four times in cetaceans, once or more in the three ‘river dolphins’ (Families Pontoporidae, Lipotidae and Inidae), once in the riverine <i>Sotalia fluviatilis</i> (but not in its marine sister taxa), once in the riverine <i>Orcaella brevirostris</i> from the Mekong River (but not in its marine sister taxa) and once in two other related marine dolphins. We located the position of this amino acid substitution in an alpha-helix channel in the trans-membrane domain in both the <i>E</i>. <i>coli</i> structure and <i>Sotalia fluviatilis</i> model. In <i>E</i>. <i>coli</i> this position is located in a helix implicated in a proton translocation channel of respiratory complex 1 and may have a similar role in the NADH dehydrogenases of cetaceans.</p></div

Selection models tested.

<p>Selection models tested.</p

Posterior probability per site for <i>d</i>_<i>N</i><i>/d</i>_<i>S</i> >1 (in practice <i>d</i>_<i>N</i><i>/d</i>_<i>S</i> = 2.24 given the best-fitting codon model) across the <i>ND2</i> gene.

<p>The red point corresponds to site 297</p

Primers used for amplification of mitochondrial genomes and the ND2 gene of cetacean species included in this study.

<p>Primers designed for this study are shown in bold. TD refers to touchdown PCR.</p><p>Primers used for amplification of mitochondrial genomes and the ND2 gene of cetacean species included in this study.</p

Panel A. Molecular surface comparison of the ND2 truncation in dolphins over the <i>E</i>. <i>coli</i> respiratory complex I membrane domains.

<p>Molecular surface and secondary structure of the <i>E</i>. <i>coli</i> complex I membrane domains structures (PDB: 3RKO) superimposed to the ND2 model of <i>S</i>. <i>guianensis</i>. <i>E</i>. <i>coli</i> subunit surfaces are colored as follows: NuoL, purple; NuoM, blue; NuoN, yellow; NuoA, pink; NuoJ, green and NuoK, fuchsia. <i>S</i>. <i>fluviatilis</i> ND2 subunit colored in Cyan and alanine position 297 labeled. Dashed circle highlights the TM helixes of <i>E</i>. <i>coli</i> not present in <i>S</i>. <i>guianensis</i>. Panel B. Superimposed structures of the predicted ND2 model from <i>S</i>. <i>fluviatilis</i> and the NuoN subunit in <i>E</i>. <i>coli</i>. Figure shows the structural overlap between template (PDB: 3RKO) in yellow and the model in grey. Transmembrane helixes of <i>E</i>.<i>coli</i> are labelled TM1-14. <i>S</i>. <i>fluviatilis</i> TM9a, TM9b and TM10 are colour cyan and in TM10 Threonine at position 297 is highlighted in red. Panel C. Topology diagram of the antiporter-like subunits of ND2 from <i>S</i>. <i>fluviatilis</i> and NuoN subunit in <i>E</i>. <i>coli</i>. Two inverted repeats over the conserved core of ND2 in the model, represent the internal structural symmetry and are shown in green and blue. In transparent yellow, representation of N-terminal part of <i>E</i>. <i>Coli</i> not present in <i>Sotalia sp</i>. In transparent green the C-terminal of <i>E</i>. <i>Coli</i>, present but not well conserved in the <i>Sotalia sp</i>. models. Position 297 is indicated as red dot. Possible function as sodium proton antiporter displayed as Na+/ H⁺?. Panel D. Structural analysis of position 297 substitution over the ND2 model between riverine <i>S</i>. <i>fluviatilis</i> and marine S. guianensis dolphins. Predicted structure of the subunit ND2 of <i>Sotalia sp</i>, in grey; Transmembrane helixes TM4a, TM4b and TM5, in green; and TM9a, TM9b and TM10 in cyan. Position 297 is highlighted in red and the amino acids in close proximity (Met 245, Leu248 and Thr300) in blue. Left side: Over the model structure the dashed box highlights key residues in the space of close proximity to position 297. Upper-right side: <i>S</i>. <i>fluviatilis</i> model showing Ala in position 297 in red; Lower-right side: S. guianensis model showing Thr in position 297 in red; potential hydrogen bond shown as a dashed green line and possible interaction of the rotamer shown as a dash pink line.</p

Correction: The paradox of HBV evolution as revealed from a 16^th century mummy

Correction: The paradox of HBV evolution as revealed from a 16^th century mumm

Phylogenetic analysis reveals that the HBV sequence reads from NASD24 are from a subgenotype D3 virus.

<p>Maximum likelihood phylogeny of HBV sequences representative of the full genotypic diversity. Tip labels indicate viral genotype. The draft genome from HBV reads from NASD24 (Italian Mummy) and the JN315779 sequence (Korean Mummy) are colored in red. Nodes with bootstrap support above 70% are displayed. All horizontal branch lengths are scaled according to the number of nucleotide substitutions per site and the tree is mid-point rooted for clarity only.</p

Comparison of prior and posterior probability distribution estimates for the age of ancient HBV samples NASD24SEQ and JN315779.

<p>Histogram showing the probability density estimation distributions for the Bayesian analysis of NASD24SEQ and JN315779 both with and without sequence data from subset a-ii, using a normal prior approximating the radiocarbon dates for the samples and with the internal calibration scheme using the estimation of entry into the Americas from Llamas et al. 2016 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006750#ppat.1006750.ref055" target="_blank">55</a>] to calibrate the node separating genotypes F and H.</p