The primary data and results for selection and phylogenetic analysis

In the manuscript, we have made a selection and phylogenetic analysis for MHC genes(DQB and DRB) in the Endangered Indo-Pacific Humpback Dolphin (Sousa chinensis).FUBAR software and CODENL program were used to test for the evidence of positive selection, and phylogenetic trees were reconstructed using MrBayes 3.1.2. The best-fit evolution model for MHC was selected on the basis of the Akaike Information Criterion(AIC) using MODELTEST 3.7. All the data underlying these analyses was submitted in a zip file(Data for Dryad)

<i>De novo</i> Assembly of the Indo-Pacific Humpback Dolphin Leucocyte Transcriptome to Identify Putative Genes Involved in the Aquatic Adaptation and Immune Response

<div><p>Background</p><p>The Indo-Pacific humpback dolphin (<i>Sousa chinensis</i>), a marine mammal species inhabited in the waters of Southeast Asia, South Africa and Australia, has attracted much attention because of the dramatic decline in population size in the past decades, which raises the concern of extinction. So far, this species is poorly characterized at molecular level due to little sequence information available in public databases. Recent advances in large-scale RNA sequencing provide an efficient approach to generate abundant sequences for functional genomic analyses in the species with un-sequenced genomes.</p><p>Principal Findings</p><p>We performed a <i>de novo</i> assembly of the Indo-Pacific humpback dolphin leucocyte transcriptome by Illumina sequencing. 108,751 high quality sequences from 47,840,388 paired-end reads were generated, and 48,868 and 46,587 unigenes were functionally annotated by BLAST search against the NCBI non-redundant and Swiss-Prot protein databases (E-value<10⁻⁵), respectively. In total, 16,467 unigenes were clustered into 25 functional categories by searching against the COG database, and BLAST2GO search assigned 37,976 unigenes to 61 GO terms. In addition, 36,345 unigenes were grouped into 258 KEGG pathways. We also identified 9,906 simple sequence repeats and 3,681 putative single nucleotide polymorphisms as potential molecular markers in our assembled sequences. A large number of unigenes were predicted to be involved in immune response, and many genes were predicted to be relevant to adaptive evolution and cetacean-specific traits.</p><p>Conclusion</p><p>This study represented the first transcriptome analysis of the Indo-Pacific humpback dolphin, an endangered species. The <i>de novo</i> transcriptome analysis of the unique transcripts will provide valuable sequence information for discovery of new genes, characterization of gene expression, investigation of various pathways and adaptive evolution, as well as identification of genetic markers.</p></div

Summary of the unigene hits in public protein databases.

<p>Summary of the unigene hits in public protein databases.</p

GO assignment for assembled unigenes.

<p>The results are summarized in three main categories: biological process, cellular component and molecular function. In total, 37,976 unigenes were assigned to GO. Classified gene objects are depicted as absolute numbers of the total number of gene objects with GO assignments.</p

Overview of the Indo-Pacific humpback dolphin leucocytes transcriptome assembly.

<p>(A) The size distribution of the contigs obtained from <i>de novo</i> assembly of high-quality clean reads. (B) The size distribution of the unigenes produced from further assembly of contigs. (C) The size distribution of the CDS produced by searching unigene sequences against various protein databases (NR, Swiss-Prot, KEGG and COG, in order) using BLASTX (E-value<10⁻⁵). (D) Size distributions of the ESTs obtained from the ESTScan results. For unigene CDS that had no hits in the databases (NR, Swiss-Prot, KEGG and COG), the BLAST results were subjected to ESTScans and then converted into peptide sequences.</p

Characterization of the assembled unigenes against NR protein databases.

<p>(<b>A</b>) E-value distribution of BLAST hits for the assembled unigenes with a cutoff of 1E-5. (<b>B</b>) Similarity distribution of the top BLAST hits for the assembled unigenes with a cutoff of 1E-5. (<b>C</b>) Species distribution of the top BLAST hits for the assembled unigenes with a cutoff of 1E-5.</p

Mercury and Selenium in Stranded Indo-Pacific Humpback Dolphins and Implications for Their Trophic Transfer in Food Chains

<div><p>As top predators in the Pearl River Estuary (PRE) of China, Indo-Pacific humpback dolphins (<i>Sousa chinensis</i>) are bioindicators for examining regional trends of environmental contaminants in the PRE. We examined samples from stranded <i>S. chinensis</i> in the PRE, collected since 2004, to study the distribution and fate of total mercury (THg), methylmercury (MeHg) and selenium (Se) in the major tissues, in individuals at different ages and their prey fishes from the PRE. This study also investigated the potential protective effects of Se against the toxicities of accumulated THg. Dolphin livers contained the highest concentrations of THg (32.34±58.98 µg g⁻¹ dw) and Se (15.16±3.66 µg g⁻¹ dw), which were significantly different from those found in kidneys and muscles, whereas the highest residue of MeHg (1.02±1.11 µg g⁻¹ dw) was found in dolphin muscles. Concentrations of both THg and MeHg in the liver, kidney and muscle of dolphins showed a significantly positive correlation with age. The biomagnification factors (BMFs) of inorganic mercury (Hg_inorg) in dolphin livers (350×) and MeHg in muscles (18.7×) through the prey fishes were the highest among all three dolphin tissues, whereas the BMFs of Se were much lower in all dolphin tissues. The lower proportion of MeHg in THg and higher Se/THg ratios in tissues were demonstrated. Our studies suggested that <i>S. chinensis</i> might have the potential to detoxify Hg via the demethylation of MeHg and the formation of tiemannite (HgSe) in the liver and kidney. The lower threshold of hepatic THg concentrations for the equimolar accumulation of Se and Hg in <i>S. chinensis</i> suggests that this species has a greater sensitivity to THg concentrations than is found in striped dolphins and Dall’s porpoises.</p></div

The mean concentrations and standard deviations (SD), in µg g⁻¹ dry weight, of total mercury (THg), selenium (Se) and methyl mercury (MeHg); the molar ratio (%) of Se to THg; and the percentage (%) of MeHg/THg in liver, kidney and muscle tissue of Indo-Pacific humpback dolphins from the Pearl River Estuary (PRE), China.

<p>JM: juvenile male (<12 years); JF: juvenile female (<9 years); AM: adult male (>12 years); AF: adult female (>9 years).</p><p>n.s.: not significant.</p><p>The mean concentrations and standard deviations (SD), in µg g⁻¹ dry weight, of total mercury (THg), selenium (Se) and methyl mercury (MeHg); the molar ratio (%) of Se to THg; and the percentage (%) of MeHg/THg in liver, kidney and muscle tissue of Indo-Pacific humpback dolphins from the Pearl River Estuary (PRE), China.</p

Clusters of orthologous group (COG) classification.

<p>In total, 16,467 of the 48,868 sequences with NR hits were grouped into 25 COG classifications.</p

Regression analysis of Se with THg, and of log₁₀ (Se/Hg) with log₁₀ MeHg in the liver and kidney of Indo-Pacific humpback dolphins (n = 28) stranded in the Pearl River Estuary (PRE) region.

<p>Regression analysis of Se with THg, and of log₁₀ (Se/Hg) with log₁₀ MeHg in the liver and kidney of Indo-Pacific humpback dolphins (n = 28) stranded in the Pearl River Estuary (PRE) region.</p