Convergent evolution of the genomes of marine mammals

Abstract

Marine mammals from different mammalian orders share several phenotypic traits adapted to the aquatic environment and are therefore a classic example of convergent evolution. To investigate convergent evolution at the genomic level, we sequenced and de novo assembled the genomes of three species of marine mammals (the killer whale, walrus and manatee) from three mammalian orders that share independently evolved phenotypic adaptations to a marine existence. Our comparative genomic analyses found that convergent amino acid substitutions were widespread throughout the genome, and that a subset were in genes evolving under positive selection and putatively associated with a marine phenotype. However, we found higher levels of convergent amino acid substitutions in a control set of terrestrial sister taxa to the marine mammals. Our results suggest that while convergent molecular evolution is relatively common, adaptive molecular convergence linked to phenotypic convergence is comparatively rare.

While there are potentially several genomic routes to reach the same phenotypic outcome, it has been suggested that the genomic changes underlying convergent evolution may to some extent be reproducible, and that convergent phenotypic traits may commonly arise from the same genetic changes^1–3. Phenotypic convergence has indeed been connected to identical single amino acid replacements within a protein coding gene occurring independently in unrelated taxa^4,5; however, such examples are rare and, to the best of our knowledge, no previous study has conducted a genome-wide scan for such convergent substitutions. Here, we present high-coverage whole genomes of four marine mammal species: the walrus (Odobenus rosmarus), bottlenose dolphin (Tursiops truncatus), killer whale (Orcinus orca) and manatee (Trichechus manatus latirostris). These genomes provide a unique opportunity to address a key evolutionary question: the role of molecular convergence in the evolution of shared derived phenotypic adaptations to a novel environment⁶.

Mammals have evolved to inhabit the marine environment on multiple independent occasions. Cetaceans (whales, dolphins and porpoises) and sirenians (manatees and dugongs) emerged during the Eocene epoch^7–10 through diversification from the Cetartiodactyla and Afrotheria, respectively. Pinnipeds (seals, sea lions and walrus) emerged approximately 20 MY later during the Miocene from within the Carnivora^7,8. Despite their independent evolutionary origins, pinnipeds, sirenians and cetaceans share a number of phenotypic adaptations to the pathogenic, locomotory, thermal, sensory, communication and anaerobic challenges of an aquatic existence, including limbs adapted for swimming, bone density adapted to manage buoyancy and a large total oxygen store relative to body size^6–8.

We de novo sequenced and assembled the genomes of killer whale, manatee and walrus, and increased the coverage of the previous draft bottlenose dolphin genome by applying a whole genome shotgun strategy using the Roche 454 and Illumina HiSeq platforms (Supplementary Table 1). We then predicted a set of 16,878 orthologous genes for the four marine mammal genomes and six other mammalian genomes (human, alpaca, cow, dog, elephant and the opossum as an outgroup; Supplementary Table 2). Following filtering this resulted in the inclusion of 14,883 protein-coding orthologs for killer whale, 10,597 for the dolphin, 15,396 for the walrus and 14,674 for the manatee.

We investigated molecular convergence among these species at two levels: first, identifying protein coding genes evolving under positive selection in all three orders; second, identifying convergent amino acid substitutions within these protein coding genes. To identify genes evolving under positive selection we performed a series of four different likelihood ratio tests, one on the combined marine mammal branches, and one on each of the individual branches leading to manatee, walrus, and to the order containing the dolphin and the killer whale (see branches coloured red in Fig. 1). One hundred and ninety-one genes were under positive selection across the combined marine mammal branches, five after conservatively correcting for multiple testing (Supplementary Table 3). These five included the glutathione metabolism pathway gene, ANPEP. Glutathione has been experimentally demonstrated to increase the antioxidant capacity in the tissue cells of cetaceans and is thought to prevent damage by reactive oxygen species under the hypoxic conditions of long underwater dives¹¹ (Supplementary Fig. 1). There were no parallel substitutions along each of the marine mammal branches in this gene. However, another glutathione metabolism pathway gene, GCLC, was detected as evolving under positive selection across the combined marine mammal branches (prior to multiple testing correction) and contained an identical substitution along all three branches (Table 1).

Figure 1.

Phylogeny of 20 eutherian mammalian genome sequences, rooted with a marsupial outgroup. Branches representing the independent evolution of marine mammal lineages, for which tests for positive selection and parallel non-synonymous amino acid substitutions were performed, are coloured red. Branches of the control set of terrestrial taxa, for which tests for positive selection and parallel non-synonymous amino acid substitutions were also performed, are coloured black. Marine mammal illustrations by Uko Gorter.

Table 1.

Positively selected genes which contain parallel substitution in all three marine mammal lineages.

Gene	Branch along which positive selection was detected (P-value)	Position	Convergent amino acid substitution
Myh7b	combined marine mammals (0.0335)	1	K→Q
Tbc1d15	combined marine mammals (0.0278)	15	N→S
Mgp	combined marine mammals (0.0014)	57	L→I
Smpx	combined marine mammals (0.0315)	49	S→L
Gclc	combined marine mammals (0.0002) and walrus (<0.0001)	220	V→M
Serpinc1	combined marine mammals (0.0241), walrus (0.0400) and cetacean (0.0009)	435	N→S
M6pr	combined marine mammals (0.0227) and cetacean (0.0242)	102	N→S
S100a9	combined marine mammals (0.0007) and manatee (0.0051)	72	A→G
Irak2	cetacean (0.0091)	481	D→E
Chrm5	cetacean (0.0449)	270	R→Q
Gpr97	manatee (0.0466)	135	S→R
Esd	manatee (0.0144)	66	D→E
Siae	manatee (0.0452)	415	I→V
Dusp27	walrus (0.0121)	850	N→S
C7orf62	walrus (0.0101)	78	S→N

Such parallel non-synonymous changes in coding genes at the same amino acid site in more than one marine mammal lineage were widespread across the genome (Fig. 2). For example, forty-four parallel non-synonymous amino acid substitutions occurred along all three marine mammal lineages; this comprised 0.05% of all non-synonymous changes. Parallel changes across any two of the marine mammal lineages occurred at an even higher rate, comprising over 1% of all changes in each combination of two marine mammal lineages (Supplementary Table 4). This pattern remained when hypermutable CpG sites were masked (Supplementary Table 5).

Figure 2.

Genome scans for convergence. Marine mammals genome revealed a large number of parallel substitutions (blue shaded bars) that occurred along the branches of at least two marine mammal lineages since they evolved from a terrestrial ancestor. Parallel substitutions that occurred in positively selected genes are shaded red.

Fifteen out of the forty-four identical non-synonymous amino acid substitutions found in all three marine mammal lineages were found in genes evolving under positive selection in at least one lineage; eight of these were inferred to have evolved under positive selection in the test including all three marine mammal lineages (Table 1; Fig. 2). This is consistent with theoretical models which demonstrate that the probability of parallel molecular evolution increases under positive selection¹². In all but three cases, these non-synonymous amino acid substitutions were due to identical nucleotide changes (Supplementary Table 6). Only two of the nucleotide changes were at hypermutable CpG sites, and in neither case was mutation due to the methylation of cytosine and the subsequent deamination of 5-methylcytosine to thymidine (the process that makes CpG sites prone to high mutation rates¹³). When the 260 non-identical amino substitutions at the same base in all three marine mammal lineages were considered, over 25% (72) were found in genes evolving under positive selection in at least one marine mammal lineage (Supplementary Table 7).

The precise phenotypic effects of the parallel substitutions cannot currently be ascertained; however, several of these fifteen positively selected genes have known functional associations that suggest a role in the convergent phenotypic evolution of the marine mammal lineages (Supplementary Table 8). For example, S100a9 and Mgp are calcium binding proteins and have a role in bone formation^14,15; Smpx plays a role in hearing and inner ear formation¹⁶; C7orf62 has known links to hyperthyroidism¹⁷; Myh7b has a role in the formation of cardiac muscle¹⁸; and Serpinc1 regulates blood coagulation¹⁹. These genes could therefore be linked to convergent phenotypic traits such as changes in bone density (S100a9, Mgp), which is high in shallow-diving species such as the manatee and the walrus to overcome neutral buoyancy, but low in the deep-diving cetacean species that collapse their lungs to overcome neutral buoyancy. Additional functional associations of these genes with shared marine phenotypes include the formation and separation of the auditory bulla from the skull in the inner ear (Smpx), the unusual periodic thyroid activity (C7orf62), cardiovascular regulation during diving (Myh7b), and the low flow rate of viscous blood particularly during diving behaviour (Serpinc1)^7,8.

The marine mammals included in this study belong to three taxonomically distant mammalian orders; therefore genomic convergence through standing genetic variation or localised introgression of regions of the genome^3,20 can be ruled out as probable causes. Identical de novo substitutions must therefore have occurred independently in each taxon during their evolution from a terrestrial ancestor. While most of these putatively adaptive convergent substitutions were also present in the recently published minke whale genome¹¹, the convergent substitutions in the Myh7b, S100a9 and GPR97 genes were not, suggesting they were either derived in the toothed whales (Odontoceti), or lost in the baleen whales (Mysteceti) following the divergence of the Odontoceti and Mysteceti.

Surprisingly, we found an unexpectedly high level of convergence along the combined branches of the terrestrial sister taxa (cow, dog and elephant) to the marine mammals (Supplementary Fig. S2, Supplementary Tables 4 and 5), along which there is no obvious phenotypic convergence. This suggests that the options for both adaptive and neutral substitutions in many genes may be limited, possibly because substitutions at alternative sites have pleiotropic and deleterious effects (see Supplementary Table 8).

Our comparison of the genomes of marine mammals has highlighted parallel molecular changes in genes evolving under positive selection and putatively associated with independently evolved, adaptive phenotypic convergence. It has been hypothesised that adaptive evolution may favour a biased subset of the available substitutions, to maximise phenotypic change^1–3, and this may explain some of our findings of convergent molecular evolution among the marine mammals. However, we also found widespread molecular convergence among the terrestrial sister taxa, suggesting that parallel substitutions may not commonly result in phenotypic convergence. The pleiotropic and often deleterious nature of most mutations, may result in the long-term survival of substitutions at a limited number of sites leaving a signature of molecular convergence within some coding genes. The parallel substitutions in 15 positively selected genes identified in this study likely represent a small proportion of the molecular changes underlying adaptive and convergent phenotypic evolution in marine mammals. Our data therefore indicate that while convergent phenotypic evolution can result from convergent molecular evolution, these cases are rare and evolution more frequently makes use of different molecular pathways to reach the same phenotypic outcome.

Online Methods

Sample collection and DNA extraction

DNA was collected from three species of marine mammals, a killer whale (Orcinus orca), a bottlenose dolphin (Tursiops truncatus), a Pacific walrus (Odobenus rosmarus divergens) and a Florida subspecies of the West Indian manatee (Trichechus manatus latirostris). The female killer whale, ‘Morgan’, stranded on the coast of the Netherlands and was then transferred to the Harderwijk Dolfinarium. A comparison of Morgan’s mitochondrial DNA sequence and learned vocal repertoire with a North Atlantic database indicated that she originated from the population of killer whales that forages primarily on the Norwegian spring-spawning stock of Atlantic herring Clupea harengus²¹. A 10 ml sample of whole blood was taken and immediately stored in a PAXgene Blood DNA Tube and PAXgene Blood RNA Tube for DNA and RNA extraction respectively. Additional biopsy samples from 5 killer whales feeding on Atlantic herring approximately off the coast of Norway, were collected and stored immediately in the preservative RNAlater. RNA was extracted and pooled from the homogenised skin biopsies of the 5 free-ranging killer whales using the Qiagen RNeasy mini kit and following the manufacturer’s guidelines. Blood samples were similarly taken from two walruses from Harderwijk Dolfinarium: from an Alaskan male (Igor) and immediately stored in a PAXgene Blood DNA Tube for whole genome sequencing, and from a Wrangel Island female (Natasja) and immediately stored in a PAXgene Blood RNA Tube for RNA sequencing to aid with annotation of the genome. DNA and RNA were extracted from whole blood using the PAXgene Blood DNA kit and PAXgene Blood RNA kit respectively and following the manufacturer’s guidelines. Bottlenose dolphin tissue samples were obtained at necropsy from dolphins in the United States Navy Marine Mammal Program. Spleen, Liver, Kidney and Skin samples were from female animals and muscle was from a male animal. Samples were used for cDNA sequencing prepared using standard methods. Lastly, blood samples were collected and DNA extracted following standard protocols from a female Florida manatee, ‘Lorelei’ born in captivity and sampled at the Homosassa Springs Wildlife State Park in Homosassa, FL, USA.

DNA and RNA Sequencing and Assembly

Whole genome shotgun sequences were generated using an Illumina HiSeq platform, from DNA libraries of the killer whale, walrus, manatee and bottlenose dolphin. The dolphin had previously been Sanger sequenced at 2× coverage and library and sequencing protocols have been previous described²². The dolphin assembly was produced by assembling the ~2.5× Sanger data with ~ 3.5× Roche 454 FLX fragment data and ~30× Illumina HiSeq data. The Sanger and 454 data were combined with the Atlas assembler and then Atlas-Link²³ and Atlas-GapFill²⁴ were used to add the Illumina data and improve the scaffolds and fill intra-scaffold gaps.

The de-novo assemblies were produced using methods similar to those used in the Assemblathon II comparison. An initial assembly was generated using AllPath-LG with default parameters and MIN_CONTIG=300 and all sequence data except the 500 bp insert data. The assembled scaffolds from the initial assembly were further extended using Atlas-Link based upon the linking information provided from the 3 kb and 8 kb libraries. Atlas-GapFill was then used to fill gaps within scaffolds by locally assembling the reads associated with each gap. For the killer whale and walrus respectively, these reads were assembled into draft genomes with contig N50 sizes of 70.3 kb and 90.0 kb, and scaffold N50 sizes of 12.7 Mb and 2.6 Mb (Supplementary Table 1). The assemblies of 2,249 Mb and 2,300 Mb cover approximately 85% and 95% of the estimated 2,373 Mb killer whale and 2,400 Mb walrus genomes respectively. The improved dolphin assembly contig N50 is 11.9 kb and the scaffold N50 is 115 kb. The total assembled size of the genome is 2.33 Gb (2.55 Gb with gaps) and covers ~95.3% of the genome.

Sequencing and assembly of the manatee varied slightly from the other marine mammals: the manatee’s DNA was sequenced to 90× total coverage by Illumina sequencing technology comprising 45× coverage of 180 bp fragment libraries, 42× coverage of 3 kb sheared jumping libraries, 2× coverage of 6–14 kb sheared jumping libraries, and 1× coverage of Fosill jumping libraries (PMID: 22800726). The sequence was then assembled using ALLPATHS-LG (PMID: 21187386). The draft assembly is 3.10 Gb in size and is composed of 2.77 Gb of sequence plus gaps between contigs. The manatee genome assembly has a contig N50 size of 37.8 kb, a scaffold N50 size of 14.4 Mb, and quality metrics comparable to other Illumina genome assemblies.

Annotation

The NCBI eukaryotic genome annotation pipeline was used. The first step is repeat identification and masking using WindowMasker²⁵. Second, proteins, transcripts generated from the RNA-seq experiments and ESTs, including previously identified sequences from the study organisms or closely related organisms from RefSeq²⁶ were aligned to the genome assembly using BLAST. This included a ‘polishing’ stage using the spice-site-aware algorithm, Splign²⁷ to improve information about splice sites and exon boundaries. Protein and transcript alignments are passed to Gnomon²⁸, which uses a Hidden Markov model (HMM) tool based on Genscan²⁹ to extend predictions missing a start or stop codon or internal exon(s). Gnomon additionally creates ab initio gene predictions for regions with no evidence alignment. The final set of annotated features comprised in order of preference, RefSeq transcripts or genomic sequences and secondly Gnomon-predicted models. Each genome was additionally masked for repetitive elements using RepeatMasker³⁰. The proportion of repetitive elements constituting each is shown in Supplementary Fig. 3.

Ortholog identification and alignment

The latest human (hg19), macaque (rheMac2), marmoset (calJac3), mouse (mm9), rat (rn4), alpaca (vicPac2), cow (bosTau7), dog (canFam2), elephant (loxAfr3), baboon (papAnu2) and opossum as an outgroup (monDom5) genome assemblies were obtained from the University of California, Santa Cruz (UCSC) Genome Browser. Human-referenced whole-genome alignments were constructed from syntenic pairwise alignments with human (the “syntenic nets”) or reciprocal-best alignments with human, depending on the quality of the assembly, using the UCSC/MULTIZ alignment pipeline^31,32.

A starting gene set was composed from of the human RefSeq, UCSC Known Genes³³, and VEGA³⁴ annotations (downloaded from UCSC on 29 July 2013). Transcripts that lacked annotated coding regions (CDSs), that had CDSs of <100 bp, or that had CDSs whose lengths were not multiples of three were discarded. These transcripts were grouped by same-stranded CDS overlap into genes (transcript clusters). All transcripts were mapped from human to each of the other mammalian species via the syntenic alignments, then subjected to a series of filters designed to minimize the impact of annotation errors, sequence quality, and changes in gene structure on subsequent analyses. Briefly, each human transcript was required (1) to map to the non-human genome via a single chain of sequence alignments including ≥80% of its CDS; (2) after mapping to a non-human species, to have ≤10% of its CDS in sequencing gaps or low quality sequence; (3) to have no frame-shift indels, unless they were compensated for within 15 bases; (4) to have no in-frame stop codons and to have all splice sites conserved. To allow for genes that are mostly conserved, but whose start or stop codons have shifted, incomplete transcripts with ~10% of bases removed from the 5´ and 3´ ends of the CDS were also considered. The final collection of ortholog sets was obtained by selecting, for each gene, the (complete or incomplete) transcript that successfully mapped to the largest number of marine mammals, with the number of other species used as a secondary criterion. In the case of a tie, the transcript with the greatest total CDS length was selected. This procedure resulted in 16,878 genes with at least two non-human orthologs, averaging ~3.3 marine species and 4.8 other species (including human) per gene.

Testing for positive selection

To find genes under positive selection, we applied four different branch-site likelihood ratio tests³⁵: cetacean clade and branch leading to cetaceans, walrus lineage, manatee lineage, and a single test for all branches involving the four marine mammals. In all tests we have used reduced parameterization introduced by (Kosiol et al.³⁶). The P-values were estimated assuming a null-distribution that is a 50:50 mixture of a chisquared distribution and a point mass at zero, leading to conservative P-value estimates³⁷. Benjamini and Hochberg method³⁸ was used to correct for multiple testing, and cutoff of false discovery rate of 0.1 was used. A comprehensive table of all genes in the study together with the list of species where orthologs were found, and with the information indicating for which tests these ortholog groups were used and the resulting P-values as well as indication whether these genes were FDR significant after multiple testing correction is available to download, see URLs. Genes found to be evolving under positive selection are listed in Supplementary Tables 3, 9–11.

GO categories were assigned to orthologous groups according to the human genome reference. Each gene was also assigned to all parental categories in the ontology. We have used two different statistical tests to detect categories with overrepresentation of positively selected genes. First, Fisher's exact test (FET; we have considered all genes with P-value < 0.05 as positives) measures enrichment of a particular GO category for positives (Supplementary Table 12). A disadvantage of this test is that results will be highly dependent on the cutoff value for positively selected genes. Second, Mann-Whitney U-test (MWU) measures shifts towards higher P-values in a particular GO category (Supplementary Table 13). Thus, MWU test does not depend on P-value cutoff, however, its results may also be affected by relaxation of constraint instead of positive selection. Holm method³⁹ was used to correct for multiple testing.

Testing for genomic convergence

Ancestral sequence reconstruction was conducted for 16,833 mammalian orthologs using the codeml program in PAMLv4.4⁴⁰. For each of the three marine mammal groups—cetaceans, manatee, and walrus—at each position the extant sequences were compared to the ancestral sequence at the most recent ancestral node. For the two cetaceans, this most recent ancestral node is the one shared with cow; for the walrus, this most recent ancestral node is the one shared with dog; and for the manatee the most recent ancestral node is shared with the elephant. The ancestral nodes are indicated as those at the root of the red branches in Fig. 1. We identified amino acid positions for which changes were inferred to have occurred, and further examined those positions that changed in more than one marine mammal group. These changes could have been shared by all three groups, or shared by any two of the three groups. Changes were further classified as parallel if they resulted in an identical amino acid state in the present day species, and common if they resulted in non-identical amino acid states in the present day species. Common changes were hypothesised to be possible indicators of convergent evolution if adaptation to an aquatic lifestyle can be accomplished via multiple different amino acids at the same position. Genes with common and parallel changes were then compared to genes found to be under positive selection, and any overlapping genes between these two sets were inferred to have undergone convergent evolution. The positions of parallel non-synonymous amino acid substitutions that were found in positively selected genes are shown in Supplementary Table 14.