Geographical distribution of close kin in southern right whales on feeding grounds.

This study investigated the close kinship structure of southern right whales on feeding grounds during austral summer seasons. The study was based on biopsy samples of 171 individual whales, which were genotyped with 14 microsatellite DNA loci. Kinship was investigated by using the LOD (Log Odds) score, a relatedness index for a pair of genotypes. Based on a cut-off point of LODPO > 6, which was chosen to balance false positives and negatives, a total of 28 dyads were inferred. Among these, 25 were classified as parent-offspring pairs. Additional genetic (mitochondrial DNA haplotypes) and biological (estimated body length, sex) data were used to provide additional information on the inferred close kin pairs. The elapsed time between sampling varied from 0 (close kin detected in the same austral summer season) to 17 years. All the kin pairs occurred within the Antarctic Indo sector (85°-135°E) and no pair occurred between whales within and outside of this sector. Six pairs were between individuals in high (Antarctic) and lower latitudes. Results of the present analysis on kinship are consistent with the views that whales in the Indo sector of the Antarctic are related with the breeding ground in Southwest Australia, and that whales from this population can occupy different feeding grounds. The present study has the potential to contribute to the conservation of the southern right whales through the monitoring of important population parameters such as population sizes and growth rate, in addition to assist the interpretation of stock structure derived from standard population genetic analyses.

Molecular Evidence for Relaxed Selection on the Enamel Genes of Toothed Whales (Odontoceti) with Degenerative Enamel Phenotypes.

Different species of toothed whales (Odontoceti) exhibit a variety of tooth forms and enamel types. Some odontocetes have highly prismatic enamel with Hunter-Schreger bands, whereas enamel is vestigial or entirely lacking in other species. Different tooth forms and enamel types are associated with alternate feeding strategies that range from biting and grasping prey with teeth in most oceanic and river dolphins to the suction feeding of softer prey items without the use of teeth in many beaked whales. At the molecular level, previous studies have documented inactivating mutations in the enamel-specific genes of some odontocete species that lack complex enamel. At a broader scale, however, it is unclear whether enamel complexity across the full diversity of extant Odontoceti correlates with the relative strength of purifying selection on enamel-specific genes. Here, we employ sequence alignments for seven enamel-specific genes (ACP4, AMBN, AMELX, AMTN, ENAM, KLK4, MMP20) in 62 odontocete species that are representative of all extant families. The sequences for 33 odontocete species were obtained from databases, and sequences for the remaining 29 species were newly generated for this study. We screened these alignments for inactivating mutations (e.g., frameshift indels) and provide a comprehensive catalog of these mutations in species with one or more inactivated enamel genes. Inactivating mutations are rare in Delphinidae (oceanic dolphins) and Platanistidae/Inioidea (river dolphins) that have higher enamel complexity scores. By contrast, mutations are much more numerous in clades such as Monodontidae (narwhal, beluga), Ziphiidae (beaked whales), Physeteroidea (sperm whales), and Phocoenidae (porpoises) that are characterized by simpler enamel or even enamelless teeth. Further, several higher-level taxa (e.g., Hyperoodon, Kogiidae, Monodontidae) possess shared inactivating mutations in one or more enamel genes, which suggests loss of function of these genes in the common ancestor of each clade. We also performed selection (dN/dS) analyses on a concatenation of these genes and used linear regression and Spearman's rank-order correlation to test for correlations between enamel complexity and two different measures of selection intensity (# of inactivating mutations per million years, dN/dS values). Selection analyses revealed that relaxed purifying selection is especially prominent in physeteroids, monodontids, and phocoenids. Linear regressions and correlation analyses revealed a strong negative correlation between selective pressure (dN/dS values) and enamel complexity. Stronger purifying selection (low dN/dS) is found on branches with more complex enamel and weaker purifying selection (higher dN/dS) occurs on branches with less complex enamel or enamelless teeth. As odontocetes diversified into a variety of feeding modes, in particular, the suction capture of prey, a reduced reliance on the dentition for prey capture resulted in the relaxed selection of genes that are critical to enamel development.

Comparative analysis of the genetic structures of Kogia spp. populations in the western North Pacific.

The two Kogia species, the pygmy sperm whale (K. breviceps) and the dwarf sperm whale (K. sima), have similar morphological and biological features as well as diets. Both species are deep divers, and both have wide distributions from tropical to warm-temperate zones. Although K. breviceps is larger than K. sima, there are few reports of habitat differentiation between the two species. The distribution of K. breviceps is concentrated in higher-latitudes, and this species dives deeper than K. sima. We investigated whether these two species differ in their population structures in the western North Pacific. Using stranded specimens from Japan, we compared the population genetic patterns of the two Kogia species using mtDNA control region variation (941 bp). In total, 34 K. breviceps samples and 54 K. sima samples from stranded individuals around Japan were successfully sequenced. Thirty haplotypes were detected in K. breviceps and 34 in K. sima, indicating high genetic diversity for both. Almost all these haplotypes are unique to the western North Pacific, but did not constitute distinct phylogeographic clades within either species. We detected differences between the species in the shape of haplotype networks and in the potential time of population expansion, indicating that the western North Pacific population of the two biologically similar species could have different population demographies. This may reflect differences in evolutionary histories and in the details of their ecological niches.

Ancestry testing of "Old Tom," a killer whale central to mutualistic interactions with human whalers.

Cooperative hunting between humans and killer whales (Orcinus orca) targeting baleen whales was reported in Eden, New South Wales, Australia, for almost a century. By 1928, whaling operations had ceased, and local killer whale sightings became scarce. A killer whale from the group, known as "Old Tom," washed up dead in 1930 and his skeleton was preserved. How these killer whales from Eden relate to other populations globally and whether their genetic descendants persist today remains unknown. We extracted and sequenced DNA from Old Tom using ancient DNA techniques. Genomic sequences were then compared with a global dataset of mitochondrial and nuclear genomes. Old Tom shared a most recent common ancestor with killer whales from Australasia, the North Atlantic, and the North Pacific, having the highest genetic similarity with contemporary New Zealand killer whales. However, much of the variation found in Old Tom's genome was not shared with these widespread populations, suggesting ancestral rather than ongoing gene flow. Our genetic comparisons also failed to find any clear descendants of Tom, raising the possibility of local extinction of this group. We integrated Traditional Custodian knowledge to recapture the events in Eden and recognize that Indigenous Australians initiated the relationship with the killer whales before European colonization and the advent of commercial whaling locally. This study rectifies discrepancies in local records and provides new insight into the origins of the killer whales in Eden and the history of Australasian killer whales.

Quantification by droplet digital PCR and species identification by metabarcoding of environmental (e)DNA from Blainville's beaked whales, with assisted localization from an acoustic array.

Detection and identification of species, subspecies or stocks of whales, dolphins and porpoises at sea remain challenging, particularly for cryptic or elusive species like beaked whales (Family: Ziphiidae). Here we investigated the potential for using an acoustically assisted sampling design to collect environmental (e)DNA from beaked whales on the U.S. Navy's Atlantic Undersea Test and Evaluation Center (AUTEC) in The Bahamas. During 12 days of August 2019, we conducted 9 small-boat surveys and collected 56 samples of seawater (paired subsamples of 1L each, including controls) using both a spatial collection design in the absence of visual confirmation of whales, and a serial collection design in the proximity of whales at the surface. There were 7 sightings of whales, including 11 Blainville's beaked whales (Mesoplodon densirostris). All whales were located initially with the assistance of information from a bottom-mounted acoustic array available on the AUTEC range. Quantification by droplet digital (dd)PCR from the four spatial design collections showed no samples of eDNA above the threshold of detection and none of these 20 samples yielded amplicons for conventional or next-generation sequencing. Quantification of the 31 samples from four serial collections identified 11 likely positive detections. eDNA barcoding by conventional sequencing and eDNA metabarcoding by next-generation sequencing confirmed species identification for 9 samples from three of the four serial collections. We further resolved five intra-specific variants (i.e., haplotypes), two of which showed an exact match to previously published haplotypes and three that have not been reported previously to the international repository, GenBank. A minimum spanning network of the five eDNA haplotypes, with all other published haplotypes of Blainville's beaked whales, suggested the potential for further resolution of differences between oceanic populations.

The raw material of evolution.

Estimates of whale mutation rates contribute to understanding evolutionary processes.

Wild pedigrees inform mutation rates and historic abundance in baleen whales.

Phylogeny-based estimates suggesting a low germline mutation rate (µ) in baleen whales have influenced research ranging from assessments of whaling impacts to evolutionary cancer biology. We estimated µ directly from pedigrees in four baleen whale species for both the mitochondrial control region and nuclear genome. The results suggest values higher than those obtained through phylogeny-based estimates and similar to pedigree-based values for primates and toothed whales. Applying our estimate of µ reduces previous genetic-based estimates of preexploitation whale abundance by 86% and suggests that µ cannot explain low cancer rates in gigantic mammals. Our study shows that it is feasible to estimate µ directly from pedigrees in natural populations, with wide-ranging implications for ecological and evolutionary research.

Resurrection of genus Phocanema Myers, 1959, as a genus independent from Pseudoterranova Mozgovoi, 1953, for nematode species (Anisakidae) parasitic in pinnipeds and cetaceans, respectively.

Species of the genus Pseudoterranova, infect kogiid cetaceans and pinnipeds. However, there is mounting molecular evidence that those from cetaceans and pinnipeds are not congeneric. Here, we provide further evidence of the non-monophyly of members of Pseudoterranova from phylogenetic analyses of the conserved nuclear LSU rDNA gene, entire ITS rDNA region and mtDNA cox2 gene, and identify morphological characters that may be used to distinguish the members of the two clades. We propose the resurrection of the genus Phocanema, with Ph. decipiens (sensu stricto) as the type species, to encompass Ph. decipiens, Ph. azarasi, Ph. bulbosa, Ph. cattani and Ph. krabbei, all parasites of pinnipeds. We propose to restrict the conception of genus Pseudoterranova, which now harbours two species infecting kogiid whales; Ps. kogiae (type species) and Ps. ceticola. Members of the genera Phocanema and Pseudoterranova differ by the shape and orientation of the lips, relative tail lengths, adult size, type of final host (pinniped vs. cetacean) and phylogenetic placement based on nuclear rDNA and mtDNA cox2 sequences.

Diversity of mitochondrial DNA in 3 species of great whales before and after modern whaling.

The 20th century commercial whaling industry severely reduced populations of great whales throughout the Southern Hemisphere. The effect of this exploitation on genetic diversity and population structure remains largely undescribed. Here, we compare pre- and post-whaling diversity of mitochondrial DNA (mtDNA) control region sequences for 3 great whales in the South Atlantic, such as the blue, humpback, and fin whale. Pre-whaling diversity is described from mtDNA extracted from bones collected near abandoned whaling stations, primarily from the South Atlantic island of South Georgia. These bones are known to represent the first stage of 20th century whaling and thus pre-whaling diversity of these populations. Post-whaling diversity is described from previously published studies reporting large-scale sampling of living whales in the Southern Hemisphere. Despite relatively high levels of surviving genetic diversity in the post-whaling populations, we found evidence of a probable loss of mtDNA lineages in all 3 species. This is evidenced by the detection of a large number of haplotypes found in the pre-whaling samples that are not present in the post-whaling samples. A rarefaction analysis further supports a loss of haplotypes in the South Atlantic humpback and Antarctic blue whale populations. The bones from former whaling stations in the South Atlantic represent a remarkable molecular archive for further investigation of the decline and ongoing recovery in the great whales of the Southern Hemisphere.

A genomic assessment of the marine-speciation paradox within the toothed whale superfamily Delphinoidea.

The impact of post-divergence gene flow in speciation has been documented across a range of taxa in recent years, and may have been especially widespread in highly mobile, wide-ranging marine species, such as cetaceans. Here, we studied individual genomes from nine species across the three families of the toothed whale superfamily Delphinoidea (Delphinidae, Phocoenidae and Monodontidae). To investigate the role of post-divergence gene flow in the speciation process, we used a multifaceted approach, including (i) phylogenomics, (ii) the distribution of shared derived alleles and (iii) demographic inference. We found the divergence of lineages within Delphinoidea did not follow a process of pure bifurcation, but was much more complex. Sliding-window phylogenomics reveal a high prevalence of discordant topologies within the superfamily, with further analyses indicating these discordances arose due to both incomplete lineage sorting and gene flow. D-statistics and f-branch analyses supported gene flow between members of Delphinoidea, with the vast majority of gene flow occurring as ancient interfamilial events. Demographic analyses provided evidence that introgressive gene flow has likely ceased between all species pairs tested, despite reports of contemporary interspecific hybrids. Our study provides the first steps towards resolving the large complexity of speciation within Delphinoidea; we reveal the prevalence of ancient interfamilial gene flow events prior to the diversification of each family, and suggest that contemporary hybridisation events may be disadvantageous, as hybrid individuals do not appear to contribute to the parental species' gene pools.

Historical Mitogenomic Diversity and Population Structuring of Southern Hemisphere Fin Whales.

Fin whales Balaenoptera physalus were hunted unsustainably across the globe in the 19th and 20th centuries, leading to vast reductions in population size. Whaling catch records indicate the importance of the Southern Ocean for this species; approximately 730,000 fin whales were harvested during the 20th century in the Southern Hemisphere (SH) alone, 94% of which were at high latitudes. Genetic samples from contemporary whales can provide a window to past population size changes, but the challenges of sampling in remote Antarctic waters limit the availability of data. Here, we take advantage of historical samples in the form of bones and baleen available from ex-whaling stations and museums to assess the pre-whaling diversity of this once abundant species. We sequenced 27 historical mitogenomes and 50 historical mitochondrial control region sequences of fin whales to gain insight into the population structure and genetic diversity of Southern Hemisphere fin whales (SHFWs) before and after the whaling. Our data, both independently and when combined with mitogenomes from the literature, suggest SHFWs are highly diverse and may represent a single panmictic population that is genetically differentiated from Northern Hemisphere populations. These are the first historic mitogenomes available for SHFWs, providing a unique time series of genetic data for this species.

Why are whales big? Genes behind ocean giants.

Gigantism is prevalent in animals, but it has never reached more extreme levels than in aquatic mammals such as whales, dolphins, and porpoises. A new study by Silva et al. has uncovered five genes underlying this gigantism, a phenotype with important connections to aging and cancer suppression in long-lived animals.

A genome and single-nucleus cerebral cortex transcriptome atlas of the short-finned pilot whale Globicephala macrorhynchus.

Cetaceans (dolphins, whales, and porpoises) have large and anatomically sophisticated brains. To expand our understanding of the cellular makeup of cetacean brains and the similarities and divergence between the brains of cetaceans and terrestrial mammals, we report a short-finned pilot whale (Globicephala macrorhynchus) single-nucleus transcriptome atlas. To achieve this goal, we assembled a chromosome-scale reference genome spanning 2.25 Gb on 22 chromosomes and profiled the gene expression of five major anatomical cortical regions of the short-finned pilot whale by single-nucleus RNA-sequencing (snRNA-seq). We identified six major cell lineages in the cerebral cortex (excitatory neurons, inhibitory neurons, oligodendrocytes, oligodendrocyte precursor cells, astrocytes, and endothelial cells), eight molecularly distinct subclusters of excitatory neurons, and four subclusters of inhibitory neurons. Finally, a comparison of snRNA-seq data from the short-finned pilot whale, human, and rhesus macaque revealed a broadly conserved cellular makeup of brain cell types. Our study provides genomic resources and molecular insights into cetacean brain evolution.

Comparative polyphasic characterization of Weissella strains isolated from beaked whale and rainbow trout (Oncorhynchus mykiss): confirmation of Weissella ceti sp. nov. and description of the novel Weissella tructae sp. nov. isolated from farmed rainbow trout.

The weissellosis agent bacterium (WS08T = CBMAI 2730) was isolated from diseased rainbow trout (Oncorhynchus mykiss) in Brazil. The whole genome sequence of this strain was compared with the Mexican W-1 strain, also isolated from diseased rainbow trout, and with the Weissella ceti type strain CECT 7719 T (= 1119-1A-09 T = CCUG 59653 T), recovered from the beaked whale. Digital DNA-DNA hybridization pairwise analyses scored 98.7% between the Mexican W-1 and Brazilian WS08T but just 24.4% for both fish isolates compared to the W. ceti type strain CECT 7719 T. The 16S rRNA gene sequence comparisons with isolates of W. ceti, available at GenBank, were conducted. All rainbow trout-pathogenic isolates grouped close (97% bootstrap confirmation), but when this group was compared to the W. ceti type strain CECT 7719 T the similarity varied from 78.9 to 79.1%. Phenotypic assays were also conducted, and the W. ceti type strain diverged from WS08T and W-1 in the hydrolysis of aesculin, D-mannose, and potassium gluconate and in the hydrolysis of hippurate. Moreover, WS08T and W-1 showed weak growth at 5 °C whereas no growth was observed for W. ceti CECT 7719 T. The major fatty acids (> 10% total fatty acids) presented by WS08T and W-1 were summed feature 8 (C18:1 ω7c/C18:1 ω6c), summed feature 3 (C16:1 ω6c/C16:1ω7c), and C16:0. The results of phylogenetic and phenotypic analyses clearly differentiated the W. ceti CECT 7719 T type strain from the assessed pathogenic strains obtained from rainbow trout. Therefore, Weissella strains isolated from rainbow trout, here represented by strain WS08T (= CBMAI 2730), should be known as members of a novel species for which the name Weissella tructae sp. nov. is proposed.

Characterization of Pseudoterranova ceticola (Nematoda: Anisakidae) larvae from meso/bathypelagic fishes off Macaronesia (NW Africa waters).

The genus Pseudoterranova includes parasite species of cetaceans and pinnipeds. The third stage larva (L3) of seal-infecting species occur in second intermediate or paratenic fish hosts mainly in neritic waters. This study firstly describes a Pseudoterranova L3 from meso/bathypelagic fishes off Macaronesia. L3s were morphologically and genetically studied by light microscopy and sequencing of the mtDNA cox2 and entire ITS rDNA genes. Bayesian inferences were performed with sequences from the larvae and selected sequences from GenBank. The nematode L3s were molecularly identified as Pseudoterranova ceticola, a parasite of kogiid whales. Such larvae were collected from Bolinichthys indicus, Chauliodus danae, Eupharynx pelecanoides, Diaphus rafinesquii, D. mollis, Diretmus argenteus and Maulisia argipalla. They mainly occurred in the viscera of these fishes. Pseudoterranova ceticola L3 were small (< 12 mm) and whitish, and a prominent characteristic is a circumoral ridge extending from the ventral boring tooth which differentiate them from Pseudoterranova spp. L3 maturing in pinnipeds and Terranova sensu lato larvae that mature in poikilotherms. The shape of the tail: conical, long, pointed, ventrally curved and lacking mucron also distinguish these larvae from those of the pinniped-infecting Pseudoterranova spp. Phylogenetic analyses based on mtDNA cox2 and ITS rDNA sequences suggest that P. ceticola is closely related to Skrjabinisakis spp., and not with Pseudoterranova spp. parasitizing pinnipeds. The related species Skrjabinisakis paggiae, S. brevispiculata and S. physeteris (until recently belonging to genus Anisakis), are as P. ceticola also parasites of physeteroid cetaceans. The morphology and morphological variation of the larvae of the cetacean parasite P. ceticola is thoroughly described for the first time. These L3 can readily be morphologically distinguished from those of the pinniped-infecting Pseudoterranova spp. The parasite likely completes its life cycle in the mesopelagic and bathypelagic realm, with meso/bathypelagic fish as 2nd intermediate or paratenic hosts and kogiids as final host. Thus, Pseudoterranova from cetaceans appear to be morphologically, genetically, and ecologically differentiated to those from pinnipeds, suggesting that they are not congeneric.

Genomics reveal population structure, evolutionary history, and signatures of selection in the northern bottlenose whale, Hyperoodon ampullatus.

Information on wildlife population structure, demographic history, and adaptations are fundamental to understanding species evolution and informing conservation strategies. To study this ecological context for a cetacean of conservation concern, we conducted the first genomic assessment of the northern bottlenose whale, Hyperoodon ampullatus, using whole-genome resequencing data (n = 37) from five regions across the North Atlantic Ocean. We found a range-wide pattern of isolation-by-distance with a genetic subdivision distinguishing three subgroups: the Scotian Shelf, western North Atlantic, and Jan Mayen regions. Signals of elevated levels of inbreeding in the Endangered Scotian Shelf population indicate this population may be more vulnerable than the other two subgroups. In addition to signatures of inbreeding, evidence of local adaptation in the Scotian Shelf was detected across the genome. We found a long-term decline in effective population size for the species, which poses risks to their genetic diversity and may be exacerbated by the isolating effects of population subdivision. Protecting important habitat and migratory corridors should be prioritized to rebuild population sizes that were diminished by commercial whaling, strengthen gene flow, and ensure animals can move across regions in response to environmental changes.

Ancient whale rhodopsin reconstructs dim-light vision over a major evolutionary transition: Implications for ancestral diving behavior.

Cetaceans are fully aquatic mammals that descended from terrestrial ancestors, an iconic evolutionary transition characterized by adaptations for underwater foraging via breath-hold diving. Although the evolutionary history of this specialized behavior is challenging to reconstruct, coevolving sensory systems may offer valuable clues. The dim-light visual pigment, rhodopsin, which initiates phototransduction in the rod photoreceptors of the eye, has provided insight into the visual ecology of depth in several aquatic vertebrate lineages. Here, we use ancestral sequence reconstruction and protein resurrection experiments to quantify light-activation metrics in rhodopsin pigments from ancestors bracketing the cetacean terrestrial-to-aquatic transition. By comparing multiple reconstruction methods on a broadly sampled cetartiodactyl species tree, we generated highly robust ancestral sequence estimates. Our experimental results provide direct support for a blue-shift in spectral sensitivity along the branch separating cetaceans from terrestrial relatives. This blue-shift was 14 nm, resulting in a deep-sea signature (λmax = 486 nm) similar to many mesopelagic-dwelling fish. We also discovered that the decay rates of light-activated rhodopsin increased in ancestral cetaceans, which may indicate an accelerated dark adaptation response typical of deeper-diving mammals. Because slow decay rates are thought to help sequester cytotoxic photoproducts, this surprising result could reflect an ecological trade-off between rod photoprotection and dark adaptation. Taken together, these ancestral shifts in rhodopsin function suggest that some of the first fully aquatic cetaceans could dive into the mesopelagic zone (>200 m). Moreover, our reconstructions indicate that this behavior arose before the divergence of toothed and baleen whales.

Differential MC5R loss in whales and manatees reveals convergent evolution to the marine environment.

Melanocortin 5 receptor (MC5R), which is expressed in the terminally differentiated sebaceous gland, is a G protein-coupled receptor (GPCR). MC5R exists mostly in mammals but is completely lost in whales; only the relic of MC5R can be detected in manatees, and phenotypically, they have lost sebaceous glands. Interestingly, whales and manatees are both aquatic mammals but have no immediate common ancestors. The loss of MC5R and sebaceous glands in whales and manatees is likely to be a result of convergent evolution. Here, we find that MC5R in whales and manatees are lost by two different mechanisms. Homologous recombination of MC5R in manatees and the insertion of reverse transcriptase in whales lead to the gene loss, respectively. On one hand, in manatees, there are two "TTATC" sequences flanking MC5R, and homologous recombination of the segments between the two "TTATC" sequences resulted in the partial loss of the sequence of MC5R. On the other hand, in whales, reverse transcriptase inserts between MC2R and RNMT on the chromosome led to the loss of MC5R. Based on these two different mechanisms for gene loss in whales and manatees, we finally concluded that MC5R loss might be the result of convergent evolution to the marine environment, and we explored the impact on biological function that is significant to environmental adaptation.

Genomic Impact of Whaling in North Atlantic Fin Whales.

It is generally recognized that large-scale whaling in the 19th and 20th century led to a substantial reduction of the size of many cetacean populations, particularly those of the baleen whales (Mysticeti). The impact of these operations on genomic diversity of one of the most hunted whales, the fin whale (Balaenoptera physalus), has remained largely unaddressed because of the paucity of adequate samples and the limitation of applicable techniques. Here, we have examined the effect of whaling on the North Atlantic fin whale based on genomes of 51 individuals from Icelandic waters, representing three temporally separated intervals, 1989, 2009 and 2018 and provide a reference genome for the species. Demographic models suggest a noticeable drop of the effective population size of the North Atlantic fin whale around a century ago. The present results suggest that the genome-wide heterozygosity is not markedly reduced and has remained comparable with other baleen whale species. Similarly, there are no signs of apparent inbreeding, as measured by the proportion of long runs of homozygosity, or of a distinctively increased mutational load, as measured by the amount of putative deleterious mutations. Compared with other baleen whales, the North Atlantic fin whale appears to be less affected by anthropogenic influences than other whales such as the North Atlantic right whale, consistent with the presence of long runs of homozygosity and higher levels of mutational load in an otherwise more heterozygous genome. Thus, genome-wide assessments of other species and populations are essential for future, more specific, conservation efforts.

Molecular evolutionary analyses of tooth genes support sequential loss of enamel and teeth in baleen whales (Mysticeti).

The loss of teeth and evolution of baleen racks in Mysticeti was a profound transformation that permitted baleen whales to radiate and diversify into a previously underutilized ecological niche of bulk filter-feeding on zooplankton and other small prey. Ancestral state reconstructions suggest that postnatal teeth were lost in the common ancestor of crown Mysticeti. Genomic studies provide some support for this hypothesis and suggest that the genetic toolkit for enamel production was inactivated in the common ancestor of living baleen whales. However, molecular studies to date have not provided direct evidence for the complete loss of teeth, including their dentin component, on the stem mysticete branch. Given these results, several questions remain unanswered: (1) Were teeth lost in a single step or did enamel loss precede dentin loss? (2) Was enamel lost early or late on the stem mysticete branch? (3) If enamel and dentin/tooth loss were decoupled in the ancestry of baleen whales, did dentin loss occur on the stem mysticete branch or independently in different crown mysticete lineages? To address these outstanding questions, we compiled and analyzed complete protein-coding sequences for nine tooth-related genes from cetaceans with available genome data. Seven of these genes are associated with enamel formation (ACP4, AMBN, AMELX, AMTN, ENAM, KLK4, MMP20) whereas two other genes are either dentin-specific (DSPP) or tooth-specific (ODAPH) but not enamel-specific. Molecular evolutionary analyses indicate that all seven enamel-specific genes have inactivating mutations that are scattered across branches of the mysticete tree. Three of the enamel genes (ACP4, KLK4, MMP20) have inactivating mutations that are shared by all mysticetes. The two genes that are dentin-specific (DSPP) or tooth-specific (ODAPH) do not have any inactivating mutations that are shared by all mysticetes, but there are shared mutations in Balaenidae as well as in Plicogulae (Neobalaenidae + Balaenopteroidea). These shared mutations suggest that teeth were lost at most two times. Shared inactivating mutations and dN/dS analyses, in combination with cetacean divergence times, were used to estimate inactivation times of genes and by proxy enamel and tooth phenotypes at ancestral nodes. The results of these analyses are most compatible with a two-step model for the loss of teeth in the ancestry of living baleen whales: enamel was lost very early on the stem Mysticeti branch followed by the independent loss of dentin (and teeth) in the common ancestors of Balaenidae and Plicogulae, respectively. These results imply that some stem mysticetes, and even early crown mysticetes, may have had vestigial teeth comprised of dentin with no enamel. Our results also demonstrate that all odontocete species (in our study) with absent or degenerative enamel have inactivating mutations in one or more of their enamel genes.