Social and genetic connectivity despite ecological variation in a killer whale network.

Philopatric kin-based societies encourage a narrow breadth of conservative behaviours owing to individuals primarily learning from close kin, promoting behavioural homogeneity. However, weaker social ties beyond kin, and across a behaviourally diverse social landscape, could be sufficient to induce variation and a greater ecological niche breadth. We investigated a network of 457 photo-identified killer whales from Norway (548 encounters in 2008-2021) with diet data available (46 mixed-diet individuals feeding on both fish and mammals, and 411 exclusive fish-eaters) to quantify patterns of association within and between diet groups, and to identify underlying correlates. We genotyped a subset of 106 whales to assess patterns of genetic differentiation. Our results suggested kinship as main driver of social bonds within and among cohesive social units, while diet was most likely a consequence reflective of cultural diffusion, rather than a driver. Flexible associations within and between ecologically diverse social units led to a highly connected network, reducing social and genetic differentiation between diet groups. Our study points to a role of social connectivity, in combination with individual behavioural variation, in influencing population ecology in killer whales.

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.

"Type D" killer whale genomes reveal long-term small population size and low genetic diversity.

Genome sequences can reveal the extent of inbreeding in small populations. Here, we present the first genomic characterization of type D killer whales, a distinctive eco/morphotype with a circumpolar, subantarctic distribution. Effective population size is the lowest estimated from any killer whale genome and indicates a severe population bottleneck. Consequently, type D genomes show among the highest level of inbreeding reported for any mammalian species (FROH ≥ 0.65). Detected recombination cross-over events of different haplotypes are up to an order of magnitude rarer than in other killer whale genomes studied to date. Comparison of genomic data from a museum specimen of a type D killer whale that stranded in New Zealand in 1955, with 3 modern genomes from the Cape Horn area, reveals high covariance and identity-by-state of alleles, suggesting these genomic characteristics and demographic history are shared among geographically dispersed social groups within this morphotype. Limitations to the insights gained in this study stem from the nonindependence of the 3 closely related modern genomes, the recent coalescence time of most variation within the genomes, and the nonequilibrium population history which violates the assumptions of many model-based methods. Long-range linkage disequilibrium and extensive runs of homozygosity found in type D genomes provide the potential basis for both the distinctive morphology, and the coupling of genetic barriers to gene flow with other killer whale populations.

Inbreeding depression explains killer whale population dynamics.

Understanding the factors that cause endangered populations to either grow or decline is crucial for preserving biodiversity. Conservation efforts often address extrinsic threats, such as environmental degradation and overexploitation, that can limit the recovery of endangered populations. Genetic factors such as inbreeding depression can also affect population dynamics but these effects are rarely measured in the wild and thus often neglected in conservation efforts. Here we show that inbreeding depression strongly influences the population dynamics of an endangered killer whale population, despite genomic signatures of purging of deleterious alleles via natural selection. We find that the 'Southern Residents', which are currently endangered despite nearly 50 years of conservation efforts, exhibit strong inbreeding depression for survival. Our population models suggest that this inbreeding depression limits population growth and predict further decline if the population remains genetically isolated and typical environmental conditions continue. The Southern Residents also had more inferred homozygous deleterious alleles than three other, growing, populations, further suggesting that inbreeding depression affects population fitness. These results demonstrate that inbreeding depression can substantially limit the recovery of endangered populations. Conservation actions focused only on extrinsic threats may therefore fail to account for key intrinsic genetic factors that also limit population growth.

An example of DNA methylation as a means to quantify stress in wildlife using killer whales.

The cumulative effects of non-lethal stressors on the health of biodiversity are a primary concern for conservation, yet difficulties remain regarding their quantification. In mammals, many stressors are processed through a common stress-response pathway, and therefore epigenetic changes in genes of this pathway may provide a powerful tool for quantifying cumulative effects. As a preliminary assessment of this approach, we investigated epigenetic manifestations of stress in two killer whale populations with different levels of exposure to anthropogenic stressors. We used bisulfite amplicon sequencing to compare patterns of DNA methylation at 25 CpG sites found in three genes involved in stress response and identified large differences in the level of methylation at two sites consistent with differential stress exposure between Northern and Southern Resident killer whale populations. DNA methylation patterns could therefore represent a useful method to assess the cumulative effects of non-lethal stressors in wildlife.

Runs of homozygosity in killer whale genomes provide a global record of demographic histories.

Runs of homozygosity (ROH) occur when offspring inherit haplotypes that are identical by descent from each parent. Length distributions of ROH are informative about population history; specifically, the probability of inbreeding mediated by mating system and/or population demography. Here, we investigated whether variation in killer whale (Orcinus orca) demographic history is reflected in genome-wide heterozygosity and ROH length distributions, using a global data set of 26 genomes representative of geographic and ecotypic variation in this species, and two F1 admixed individuals with Pacific-Atlantic parentage. We first reconstructed demographic history for each population as changes in effective population size through time using the pairwise sequential Markovian coalescent (PSMC) method. We found a subset of populations declined in effective population size during the Late Pleistocene, while others had more stable demography. Genomes inferred to have undergone ancestral declines in effective population size, were autozygous at hundreds of short ROH (<1 Mb), reflecting high background relatedness due to coalescence of haplotypes deep within the pedigree. In contrast, longer and therefore younger ROH (>1.5 Mb) were found in low latitude populations, and populations of known conservation concern. These include a Scottish killer whale, for which 37.8% of the autosomes were comprised of ROH >1.5 Mb in length. The fate of this population, in which only two adult males have been sighted in the past five years, and zero fecundity over the last two decades, may be inextricably linked to its demographic history and consequential inbreeding depression.

Cultural specialization and genetic diversity: Killer whales and beyond.

Culturally-transmitted ecological specialization can reduce niche breadths with demographic and ecological consequences. I use agent-based models, grounded in killer whale biology, to investigate the potential consequences of cultural specialization for genetic diversity. In these models, cultural specialization typically reduces the number of mitochondrial haplotypes, mitochondrial haplotype diversity, mitochondrial nucleotide diversity, and heterozygosity at nuclear loci. The causal route of this decline is mostly indirect, being ascribed to a reduction in absolute population size resulting from cultural specialization. However, small group size exacerbates the decline in genetic diversity, presumably because of increased founder effects at the initiation of each cultural ecotype. These results are concordant with measures of low genetic diversity in the killer whale, although culturally-transmitted ecological specialization alone might not be sufficient to fully account for the species' very low mitochondrial diversity. The process may also operate in other species.

Excavating ghost footprints and tangled trees from modern genomes.

Due to pervasive gene flow and admixture, simple bifurcating trees often do not provide an accurate representation of relationships among diverging lineages, but limited resolution in the available genomic data and the spatial distribution of samples has hindered detailed insights regarding the evolutionary and demographic history of many species and populations. In this issue of Molecular Ecology, Foote et al. (2019) combine a powerful sampling design with novel analytical methods adopted from human genetics to describe previously unrecognized patterns of recurrent vicariance and admixture among lineages in the globally distributed killer whale (Orcinus orca). Based on sequence data from modern samples alone, they discover clear signatures of ancient admixture with a now extinct "ghost" lineage, providing one of the first accounts of archaic introgression in a nonhominid species. Coupling a cost-effective sequencing strategy with novel analytical approaches, their paper provides a roadmap for advancing inference of evolutionary history in other nonmodel species, promising exciting times ahead for our field.

Killer whale genomes reveal a complex history of recurrent admixture and vicariance.

Reconstruction of the demographic and evolutionary history of populations assuming a consensus tree-like relationship can mask more complex scenarios, which are prevalent in nature. An emerging genomic toolset, which has been most comprehensively harnessed in the reconstruction of human evolutionary history, enables molecular ecologists to elucidate complex population histories. Killer whales have limited extrinsic barriers to dispersal and have radiated globally, and are therefore a good candidate model for the application of such tools. Here, we analyse a global data set of killer whale genomes in a rare attempt to elucidate global population structure in a nonhuman species. We identify a pattern of genetic homogenisation at lower latitudes and the greatest differentiation at high latitudes, even between currently sympatric lineages. The processes underlying the major axis of structure include high drift at the edge of species' range, likely associated with founder effects and allelic surfing during postglacial range expansion. Divergence between Antarctic and non-Antarctic lineages is further driven by ancestry segments with up to four-fold older coalescence time than the genome-wide average; relicts of a previous vicariance during an earlier glacial cycle. Our study further underpins that episodic gene flow is ubiquitous in natural populations, and can occur across great distances and after substantial periods of isolation between populations. Thus, understanding the evolutionary history of a species requires comprehensive geographic sampling and genome-wide data to sample the variation in ancestry within individuals.

Consequences of culturally-driven ecological specialization: Killer whales and beyond.

Culturally-transmitted ecological specialization occurs in killer whales, as well as other species. We hypothesize that some of the remarkable demographic and ecological attributes of killer whales result from this process. We formalize and model (using agent-based stochastic models parametrized using killer whale life history) the cultural evolution of specialization by social groups, in which a narrowing of niche breadth is spread and maintained in a group through social learning. We compare the demographic and ecological results of cultural specialization to those of a similar model of specialization through natural selection. We found that specialization, through either the cultural or natural selection routes, is adaptive in the short term with specialization often increasing fitness. Generalization, in contrast, is rarely adaptive. The cultural evolution of specialization can lead to increased rates of group extirpation. Specialization has little effect on group size but tends to reduce population size and resource abundance. While the two specialization processes produce similar results, cultural specialization can be very much faster. The results are generally consistent with what we know of the formation and maintenance of specialist ecotypes in killer whales, and have implications for the persistence, nature and ecological effects of these apex predators.

Colonizing the Wild West: Low Diversity of Complete Mitochondrial Genomes in Western North Pacific Killer Whales Suggests a Founder Effect.

In the North Pacific, fish-eating R-type "resident" and mammal-eating T-type "transient" killer whales do not interbreed and differ in ecology and behavior. Full-length mitochondrial genomes (about 16.4 kbp) were sequenced and assembled for 12 R-type and 14 T-type killer whale samples from different areas of the western North Pacific. All R-type individuals had the same haplotype, previously described for R-type killer whales from both eastern and western North Pacific. However, haplotype diversity of R-type killer whales was much lower in the western North Pacific than in the Aleutian Islands and the eastern North Pacific. T-type whales had 3 different haplotypes, including one previously undescribed. Haplotype diversity of T-type killer whales in the Okhotsk Sea was also much lower than in the Aleutian Islands and the eastern North Pacific. The highest haplotype diversity for both R- and T-type killer whales was observed in the Aleutian Islands. We discuss how the environmental conditions during the last glacial period might have shaped the history of killer whale populations in the North Pacific. Our results suggest the recent colonization or re-colonization of the western North Pacific by small groups of killer whales originating from the central or eastern North Pacific, possibly due to favorable environmental changes after the Last Glacial Maximum.

Epistatic interactions influence terrestrial-marine functional shifts in cetacean rhodopsin.

Like many aquatic vertebrates, whales have blue-shifting spectral tuning substitutions in the dim-light visual pigment, rhodopsin, that are thought to increase photosensitivity in underwater environments. We have discovered that known spectral tuning substitutions also have surprising epistatic effects on another function of rhodopsin, the kinetic rates associated with light-activated intermediates. By using absorbance spectroscopy and fluorescence-based retinal release assays on heterologously expressed rhodopsin, we assessed both spectral and kinetic differences between cetaceans (killer whale) and terrestrial outgroups (hippo, bovine). Mutation experiments revealed that killer whale rhodopsin is unusually resilient to pleiotropic effects on retinal release from key blue-shifting substitutions (D83N and A292S), largely due to a surprisingly specific epistatic interaction between D83N and the background residue, S299. Ancestral sequence reconstruction indicated that S299 is an ancestral residue that predates the evolution of blue-shifting substitutions at the origins of Cetacea. Based on these results, we hypothesize that intramolecular epistasis helped to conserve rhodopsin's kinetic properties while enabling blue-shifting spectral tuning substitutions as cetaceans adapted to aquatic environments. Trade-offs between different aspects of molecular function are rarely considered in protein evolution, but in cetacean and other vertebrate rhodopsins, may underlie multiple evolutionary scenarios for the selection of specific amino acid substitutions.

Phylogenetic profiling and gene expression studies implicate a primary role of PSORS1C2 in terminal differentiation of keratinocytes.

PSORS1C2 is a gene located between coiled-coil alpha-helical rod protein 1 (CCHCR1) and corneodesmosin (CDSN) within the psoriasis susceptibility locus 1 (PSORS1). Here, we performed a comparative genomics analysis of the as-yet incompletely characterized PSORS1C2 gene and determined its expression pattern in human tissues. In contrast to CCHCR1, which is common to all vertebrates investigated, PSORS1C2 and CDSN are present exclusively in mammals, indicating that the latter genes have originated after the evolutionary divergence of mammals and reptiles. CDSN is conserved in aquatic mammals, whereas PSORS1C2 orthologs contain gene-inactivating frame shift mutations in whales and dolphins, in which the epidermal differentiation programme has degenerated. Reverse-transcription PCR screening demonstrated that, in human tissues, PSORS1C2 is expressed principally in the epidermis and weakly in the thymus. PSORS1C2 mRNA was strongly upregulated during terminal differentiation of human keratinocytes in vitro. Immunohistochemistry revealed exclusive expression of PSORS1C2 in the granular layer of the epidermis and in cornifying epithelial cells of Hassall's corpuscles of the thymus. In summary, our results identify PSORS1C2 as a keratinocyte cornification-associated protein that has originated in evolutionarily basal mammals and has undergone gene inactivation in association with the loss of the skin barrier function in aquatic mammals.

Genome-wide SNP data suggest complex ancestry of sympatric North Pacific killer whale ecotypes.

Three ecotypes of killer whale occur in partial sympatry in the North Pacific. Individuals assortatively mate within the same ecotype, resulting in correlated ecological and genetic differentiation. A key question is whether this pattern of evolutionary divergence is an example of incipient sympatric speciation from a single panmictic ancestral population, or whether sympatry could have resulted from multiple colonisations of the North Pacific and secondary contact between ecotypes. Here, we infer multilocus coalescent trees from >1000 nuclear single-nucleotide polymorphisms (SNPs) and find evidence of incomplete lineage sorting so that the genealogies of SNPs do not all conform to a single topology. To disentangle whether uncertainty in the phylogenetic inference of the relationships among ecotypes could also result from ancestral admixture events we reconstructed the relationship among the ecotypes as an admixture graph and estimated f4-statistics using TreeMix. The results were consistent with episodes of admixture between two of the North Pacific ecotypes and the two outgroups (populations from the Southern Ocean and the North Atlantic). Gene flow may have occurred via unsampled 'ghost' populations rather than directly between the populations sampled here. Our results indicate that because of ancestral admixture events and incomplete lineage sorting, a single bifurcating tree does not fully describe the relationship among these populations. The data are therefore most consistent with the genomic variation among North Pacific killer whale ecotypes resulting from multiple colonisation events, and secondary contact may have facilitated evolutionary divergence. Thus, the present-day populations of North Pacific killer whale ecotypes have a complex ancestry, confounding the tree-based inference of ancestral geography.

Longitudinal evaluation of leukocyte transcripts in killer whales (Orcinus Orca).

Early identification of illness and/or presence of environmental and/or social stressors in free-ranging and domestic cetaceans is a priority for marine mammal health care professionals. Incorporation of leukocyte gene transcript analysis into the diagnostic tool kit has the potential to augment classical diagnostics based upon ease of sample storage and shipment, inducible nature and well-defined roles of transcription and associated downstream actions. Development of biomarkers that could serve to identify "insults" and potentially differentiate disease etiology would be of great diagnostic value. To this end, a modest number of peripheral blood leukocyte gene transcripts were selected for application to a domestic killer whale population with a focus on broad representation of inducible immunologically relevant genes. Normalized leukocyte transcript values, longitudinally acquired from 232 blood samples derived from 26 clinically healthy whales, were not visibly influenced temporally nor by sex or the specific Park in which they resided. Stability in leukocyte transcript number during periods of health enhances their potential use in diagnostics through identification of outliers. Transcript levels of two cytokine genes, IL-4 and IL-17, were highly variable within the group as compared to the other transcripts. IL-4 transcripts were typically absent. Analysis of transcript levels on the other genes of interest, on an individual animal basis, identified more outliers than were visible when analyzed in the context of the entire population. The majority of outliers (9 samples) were low, though elevated transcripts were identified for IL-17 from 2 animals and one each for Cox-2 and IL-10. The low number of outliers was not unexpected as sample selection was intentionally directed towards animals that were clinically healthy at the time of collection. Outliers may reflect animals experiencing subclinical disease that is transient and self-limiting. The immunologic knowledge derived from longitudinal immunologic studies in killer whales, as was the target of the present study, has the potential to improve diagnostics and health related decision making for this and other domestic and free-ranging cetacean species.

Spectral Tuning of Killer Whale (Orcinus orca) Rhodopsin: Evidence for Positive Selection and Functional Adaptation in a Cetacean Visual Pigment.

Cetaceans have undergone a remarkable evolutionary transition that was accompanied by many sensory adaptations, including modification of the visual system for underwater environments. Recent sequencing of cetacean genomes has made it possible to begin exploring the molecular basis of these adaptations. In this study we use in vitro expression methods to experimentally characterize the first step of the visual transduction cascade, the light activation of rhodopsin, for the killer whale. To investigate the spectral effects of amino acid substitutions thought to correspond with absorbance shifts relative to terrestrial mammals, we used the orca gene as a background for the first site-directed mutagenesis experiments in a cetacean rhodopsin. The S292A mutation had the largest effect, and was responsible for the majority of the spectral difference between killer whale and bovine (terrestrial) rhodopsin. Using codon-based likelihood models, we also found significant evidence for positive selection in cetacean rhodopsin sequences, including on spectral tuning sites we experimentally mutated. We then investigated patterns of ecological divergence that may be correlated with rhodopsin functional variation by using a series of clade models that partitioned the data set according to phylogeny, habitat, and foraging depth zone. Only the model partitioning according to depth was significant. This suggests that foraging dives might be a selective regime influencing cetacean rhodopsin divergence, and our experimental results indicate that spectral tuning may be playing an adaptive role in this process. Our study demonstrates that combining computational and experimental methods is crucial for gaining insight into the selection pressures underlying molecular evolution.

Evaluating the Adequacy of Molecular Clock Models Using Posterior Predictive Simulations.

Molecular clock models are commonly used to estimate evolutionary rates and timescales from nucleotide sequences. The goal of these models is to account for rate variation among lineages, such that they are assumed to be adequate descriptions of the processes that generated the data. A common approach for selecting a clock model for a data set of interest is to examine a set of candidates and to select the model that provides the best statistical fit. However, this can lead to unreliable estimates if all the candidate models are actually inadequate. For this reason, a method of evaluating absolute model performance is critical. We describe a method that uses posterior predictive simulations to assess the adequacy of clock models. We test the power of this approach using simulated data and find that the method is sensitive to bias in the estimates of branch lengths, which tends to occur when using underparameterized clock models. We also compare the performance of the multinomial test statistic, originally developed to assess the adequacy of substitution models, but find that it has low power in identifying the adequacy of clock models. We illustrate the performance of our method using empirical data sets from coronaviruses, simian immunodeficiency virus, killer whales, and marine turtles. Our results indicate that methods of investigating model adequacy, including the one proposed here, should be routinely used in combination with traditional model selection in evolutionary studies. This will reveal whether a broader range of clock models to be considered in phylogenetic analysis.

Geographic and temporal dynamics of a global radiation and diversification in the killer whale.

Global climate change during the Late Pleistocene periodically encroached and then released habitat during the glacial cycles, causing range expansions and contractions in some species. These dynamics have played a major role in geographic radiations, diversification and speciation. We investigate these dynamics in the most widely distributed of marine mammals, the killer whale (Orcinus orca), using a global data set of over 450 samples. This marine top predator inhabits coastal and pelagic ecosystems ranging from the ice edge to the tropics, often exhibiting ecological, behavioural and morphological variation suggestive of local adaptation accompanied by reproductive isolation. Results suggest a rapid global radiation occurred over the last 350 000 years. Based on habitat models, we estimated there was only a 15% global contraction of core suitable habitat during the last glacial maximum, and the resources appeared to sustain a constant global effective female population size throughout the Late Pleistocene. Reconstruction of the ancestral phylogeography highlighted the high mobility of this species, identifying 22 strongly supported long-range dispersal events including interoceanic and interhemispheric movement. Despite this propensity for geographic dispersal, the increased sampling of this study uncovered very few potential examples of ancestral dispersal among ecotypes. Concordance of nuclear and mitochondrial data further confirms genetic cohesiveness, with little or no current gene flow among sympatric ecotypes. Taken as a whole, our data suggest that the glacial cycles influenced local populations in different ways, with no clear global pattern, but with secondary contact among lineages following long-range dispersal as a potential mechanism driving ecological diversification.

An agent-based model of dialect evolution in killer whales.

The killer whale is one of the few animal species with vocal dialects that arise from socially learned group-specific call repertoires. We describe a new agent-based model of killer whale populations and test a set of vocal-learning rules to assess which mechanisms may lead to the formation of dialect groupings observed in the wild. We tested a null model with genetic transmission and no learning, and ten models with learning rules that differ by template source (mother or matriline), variation type (random errors or innovations) and type of call change (no divergence from kin vs. divergence from kin). The null model without vocal learning did not produce the pattern of group-specific call repertoires we observe in nature. Learning from either mother alone or the entire matriline with calls changing by random errors produced a graded distribution of the call phenotype, without the discrete call types observed in nature. Introducing occasional innovation or random error proportional to matriline variance yielded more or less discrete and stable call types. A tendency to diverge from the calls of related matrilines provided fast divergence of loose call clusters. A pattern resembling the dialect diversity observed in the wild arose only when rules were applied in combinations and similar outputs could arise from different learning rules and their combinations. Our results emphasize the lack of information on quantitative features of wild killer whale dialects and reveal a set of testable questions that can draw insights into the cultural evolution of killer whale dialects.