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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.

The impact of human-mediated environmental change on the evolutionary trajectories of wild organisms is poorly understood. In particular, species’ capacities to adapt rapidly (in hundreds of generations or less), reproducibly and predictably to extreme environmental change is unclear. Silene uniflora is predominantly a coastal species, but it has also colonised isolated, disused mines with phytotoxic, zinc-contaminated soils. To test whether rapid, parallel adaptation to anthropogenic pollution has taken place, we used reduced representation sequencing (ddRAD) to reconstruct the evolutionary history of geographically proximate mine and coastal population pairs and found largely independent colonisation of mines from different coastal sites. Furthermore, our results show that parallel evolution of zinc tolerance has occurred without gene flow spreading adaptive alleles between mine populations. In genomic regions where signatures of selection were detected across multiple mine-coast pairs, we identified genes with functions linked to physiological differences between the putative ecotypes, although genetic differentiation at specific loci is only partially shared between mine populations. Our results are consistent with a complex, polygenic genetic architecture underpinning rapid adaptation. This shows that even under a scenario of strong selection and rapid adaptation, evolutionary responses to human activities (and other environmental challenges) may be idiosyncratic at the genetic level and, therefore, difficult to predict from genomic data. © The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Genomes of high latitude killer whales harbour signatures of post-glacial founding and expansion. Here, we investigate whether reduced efficacy of selection increased mutation load in founder populations, or whether recessive deleterious mutations exposed to selection in homozygous genotypes were purged. Comparing the accumulation of synonymous and non-synonymous mutations across pairs of globally sampled genomes reveals that the most significant outliers are high latitude North Atlantic genomes, which have accumulated significantly fewer non-synonymous mutations than all other populations. Comparisons with the genome of a 7.5-Kyr-old North Atlantic killer whale, inferred to be closely related to the population directly ancestral to present-day Icelandic and Norwegian populations, calibrates the timing of the action of selection on non-synonymous mutations predominantly to the mid-late Holocene. Non-synonymous mutations purged in modern Norwegian killer whale genomes are found as globally shared standing variation in heterozygote genotypes more often than expected, suggesting overdominance. Taken together, our findings are consistent with purging of recessive non-synonymous mutations exposed to selection in founder-associated homozygous genotypes.

International audience; 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 (1.5 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.

Description, Old genetic variants were key to the ability of bottlenose dolphins to repeatedly adapt to coastal waters across the world., Studying repeated adaptation can provide insights into the mechanisms allowing species to adapt to novel environments. Here, we investigate repeated evolution driven by habitat specialization in the common bottlenose dolphin. Parapatric pelagic and coastal ecotypes of common bottlenose dolphins have repeatedly formed across the oceans. Analyzing whole genomes of 57 individuals, we find that ecotype evolution involved a complex reticulated evolutionary history. We find parallel linked selection acted upon ancient alleles in geographically distant coastal populations, which were present as standing genetic variation in the pelagic populations. Candidate loci evolving under parallel linked selection were found in ancient tracts, suggesting recurrent bouts of selection through time. Therefore, despite the constraints of small effective population size and long generation time on the efficacy of selection, repeated adaptation in long-lived social species can be driven by a combination of ecological opportunities and selection acting on ancestral standing genetic variation.

Adaptation is typically studied by comparing modern populations with contrasting environments. Individuals persisting in the ancestral habitat are typically used to represent the ancestral founding population; however, it has been questioned whether these individuals are good proxies for the actual ancestors.(1) To address this, we applied a paleogenomics approach(2) to directly access the ancestral genepool: partially sequencing the genomes of two 11- to 13,000-year-old stickleback recovered from the transitionary layer between marine and freshwater sediments of two Norwegian isolation lakes(3) and comparing them with 30 modern stickleback genomes from the same lakes and adjacent marine fjord, in addition to a global dataset of 20 genomes.(4) The ancient stickleback shared genome-wide ancestry with the modern fjord population, whereas modern lake populations have lost substantial ancestral variation following founder effects, and subsequent drift and selection. Freshwater-adaptive alleles found in one ancient stickleback genome have not risen to high frequency in the present-day population from the same lake. Comparison to the global dataset suggested incomplete adaptation to freshwater in our modern lake populations. Our findings reveal the impact of population bottlenecks in constraining adaptation due to reduced efficacy of selection on standing variation present in founder populations.

What are the mechanisms that allow species to extend their ranges and adapt to the novel environmental conditions they find in the newly available habitat? The study of parallel adaptation of pairs of populations to similar environments can provide great insights into this question. Here, we test for parallel evolution driven by niche specialization in a highly social marine mammal, the common bottlenose dolphin,Tursiops truncatus, and investigate the origins of the genetic variation driving local adaptation. Coastal ecotypes of common bottlenose dolphins have recurrently emerged in multiple regions of the world from pelagic ecotype populations, when novel habitat became available. Analyzing the whole genomes of 57 individuals using comparative population genomics approaches, we found that coastal ecotype evolution was relatively independent between the Atlantic and Pacific, but related between different regions within the Atlantic. We show that parallel adaptation to coastal habitat was facilitated by repeated selection on ancient alleles present as standing genetic variation in the pelagic populations. Genes under parallel adaptation to coastal habitats have roles in cognitive abilities and feeding. Therefore, parallel adaptation in long-lived social species may be driven by a combination of ecological opportunities, selection acting on ancient variants, and stable behavioural transmission of ecological specialisations. Tried and tested genetic variation that has been subject to repeated bouts of selection, may promote linked adaptive variants with minimal pleiotropic effects, thereby facilitating their persistence at low frequency in source populations and enabling parallel evolution.

Parallel evolution is typically studied by comparing modern populations from contrasting environments, therefore the chronology of adaptive changes remains poorly understood. We applied a paleogenomics approach to investigate this temporal component of adaptation by sequencing the genomes of 11-13,000-year-old stickleback recovered from the transitionary layer between marine and freshwater sediments of two Norwegian isolation lakes, and comparing them with 30 modern stickleback genomes from the same lakes and adjacent marine fjord. The ancient stickleback shared genome-wide ancestry with the modern fjord population, whereas modern lake populations have lost substantial ancestral variation following founder effects. We found modern lake stickleback had lost freshwater-adaptive alleles found in the ancient stickleback genomes, and showed incomplete adaptation, revealing the hitherto underappreciated stochastic nature of selection on standing variation present in founder populations.One Sentence Summary‘Pleistocene threespine stickleback genomes reveal insights into the earliest stages of freshwater adaptation’

M.N. was funded by a Crawford‐Hayes studentship and National Parks and Wildlife Service (NPWS), Department of Culture, Heritage and Gaeltacht, Ireland. E.D. was supported by the Beaufort Marine Research Award in Fish Population Genetics funded by the Irish Government under the Sea Change Programme. Financial support for A.D.F. was provided by the Welsh Government and Higher Education Funding Council for Wales through the Sêr Cymru National Research Network for Low Carbon, Energy and Environment, and from the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska‐Curie grant agreement No. 663830. The functioning of marine protected areas (MPAs) designated for marine megafauna has been criticized due to the high mobility and dispersal potential of these taxa. However, dispersal within a network of small MPAs can be beneficial as connectivity can result in increased effective population size, maintain genetic diversity, and increase robustness to ecological and environmental changes making populations less susceptible to stochastic genetic and demographic effects (i.e., Allee effect). Here, we use both genetic and photo-identification methods to quantify gene flow and demographic dispersal between MPAs of a highly mobile marine mammal, the bottlenose dolphin Tursiops truncatus. We identify three populations in the waters of western Ireland, two of which have largely nonoverlapping core coastal home ranges and are each strongly spatially associated with specific MPAs. We find high site fidelity of individuals within each of these two coastal populations to their respective MPA. We also find low levels of demographic dispersal between the populations, but it remains unclear whether any new gametes are exchanged between populations through these migrants (genetic dispersal). The population sampled in the Shannon Estuary has a low estimated effective population size and appears to be genetically isolated. The second coastal population, sampled outside of the Shannon, may be demographically and genetically connected to other coastal subpopulations around the coastal waters of the UK. We therefore recommend that the methods applied here should be used on a broader geographically sampled dataset to better assess this connectivity. Publisher PDF

Sympatric speciation has been of key interest to biologists investigating how natural and sexual selection drive speciation without the confounding variable of geographic isolation. The advent of the genomic era has provided a more nuanced and quantitative understanding of the different and often complex modes of speciation by which sympatric sister taxa arose, and a reassessment of some of the most compelling empirical case studies of sympatric speciation. However, I argue that genomic studies based on contemporary populations may never be able to provide unequivocal evidence of true primary sympatric speciation, and there is a need to incorporate palaeogenomic studies into this field. This inability to robustly distinguish cases of primary and secondary 'divergence with gene flow' may be inconsequential, as both are useful for understanding the role of large effect barrier loci in the progression from localised genic isolation to genome-wide reproductive isolation. I argue that they can be of equivalent interest due to shared underlying mechanisms driving divergence and potentially leaving similar patterns of coalescence.

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.

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.

[Robeck et al. (2015)][1] claim that reproductive and actuarial senescence is common in mammalian species and therefore not an unexpected finding in killer whales. However, in most mammals, reproductive and somatic senescence are aligned, and reproduction gradually declines with age. In contrast, there is a substantial evidence that reproductive senescence is unusually accelerated relative to somatic senescence in resident killer whales, resulting in a prolonged postreproductive lifespan. We demonstrate that a postreproductive lifespan is a key component of resident killer whale life history, and is robust to reasonable error in age estimates. [1]: #ref-32

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 dataset of killer whale genomes in a rare attempt to elucidate global population structure in a non-human 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 post-glacial 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.

While environmental DNA (eDNA) is becoming increasingly established in biodiversity monitoring of freshwater ecosystems, the use of eDNA surveys in the marine environment is still in its infancy. Here, we use two approaches: targeted quantitative PCR (qPCR) and whole-genome enrichment capture followed by shotgun sequencing in an effort to amplify killer whale DNA from seawater samples. Samples were collected in close proximity to killer whales in inshore and offshore waters, in varying sea conditions and from the surface and subsurface but none returned strongly positive detections of killer whale eDNA. We validated our laboratory methodologies by successfully amplifying a dilution series of a positive control of killer whale DNA. Furthermore, DNA of Atlantic mackerel, which was present at all sites during sampling, was successfully amplified from the same seawater samples, with positive detections found in ten of the eighteen eDNA extracts. We discuss the various eDNA collection and amplification methodologies used and the abiotic and biotic factors that influence eDNA detection. We discuss possible explanations for the lack of positive killer whale detections, potential pitfalls, and the apparent limitations of eDNA for genetic research on cetaceans, particularly in offshore regions.

Oscillations in the Earth’s temperature and the subsequent retreating and advancing of ice-sheets around the polar regions are thought to have played an important role in shaping the distribution and genetic structuring of contemporary high-latitude populations. After the Last Glacial Maximum (LGM), retreating of the ice-sheets would have enabled early colonizers to rapidly occupy suitable niches to the exclusion of other conspecifics, thereby reducing genetic diversity at the leading-edge. Bottlenose dolphins (genus Tursiops) form distinct coastal and pelagic ecotypes, with finer-scale genetic structuring observed within each ecotype. We reconstruct the postglacial colonization of the Northeast Atlantic (NEA) by bottlenose dolphins using habitat modeling and phylogenetics. The AquaMaps model hindcasted suitable habitat for the LGM in the Atlantic lower latitude waters and parts of the Mediterranean Sea. The time-calibrated phylogeny, constructed with 86 complete mitochondrial genomes including 30 generated for this study and created using a multispecies coalescent model, suggests that the expansion to the available coastal habitat in the NEA happened via founder events starting ~15 000 years ago (95% highest posterior density interval: 4 900–26 400). The founders of the 2 distinct coastal NEA populations comprised as few as 2 maternal lineages that originated from the pelagic population. The low effective population size and genetic diversity estimated for the shared ancestral coastal population subsequent to divergence from the pelagic source population are consistent with leading-edge expansion. These findings highlight the legacy of the Late Pleistocene glacial cycles on the genetic structuring and diversity of contemporary populations.

Recent exploration into the interactions and relationship between hosts and their microbiota has revealed a connection between many aspects of the host’s biology, health and associated microorganisms. Whereas amplicon sequencing has traditionally been used to characterise the microbiome, the increasing number of published population genomics datasets offer an underexploited opportunity to study microbial profiles from the host shotgun sequencing data. Here, we use sequence data originally generated from killer whale Orcinus orca skin biopsies for population genomics, to characterise the skin microbiome and investigate how host social and geographic factors influence the microbial community composition. Having identified 845 microbial taxa from 2.4 million reads that did not map to the killer whale reference genome, we found that both ecotypic and geographic factors influence community composition of killer whale skin microbiomes. Furthermore, we uncovered key taxa that drive the microbiome community composition and showed that they are embedded in unique networks, one of which is tentatively linked to diatom presence and poor skin condition. Community composition differed between Antarctic killer whales with and without diatom coverage, suggesting that the previously reported episodic migrations of Antarctic killer whales to warmer waters associated with skin turnover may control the effects of potentially pathogenic bacteria such as Tenacibaculum dicentrarchi. Our work demonstrates the feasibility of microbiome studies from host shotgun sequencing data and highlights the importance of metagenomics in understanding the relationship between host and microbial ecology.

The primary prey of killer whales (Orcinus orca) in the Strait of Gibraltar is the bluefin tuna (Thunnus thynnus). All killer whales observed in this area hunt tuna by chasing individual fish until they become exhausted and can be overcome. However, a subset of pods also interact with a dropline tuna fishery which has developed since 1995. Here, we investigated the social structure within and among social units (pods). Our data suggested that social structure was shaped by maternal kinship, which appears to be a species-specific trait, but also by foraging behavior, which is less common at the intra-population level. At the start of the study, only one cohesive pod interacted with the fishery, which during the course of the study underwent fission into two socially differentiated pods. Social structure within these two fishery-interacting pods was more compact and homogenous with stronger associations between individuals than in the rest of the population. Three other pods were never seen interacting with the fishery, despite one of these pods being regularly sighted in the area of the fishery during the summer. Sociality can influence the spread of the novel foraging behaviors and may drive population fragmentation, which, in this example, is already a critically small community. Observations of social changes in relation to changes in foraging at the earliest stages of diversification in foraging behavior and social segregation may provide insights into the processes that ultimately result in the formation of socially isolated discrete ecotypes in killer whales., This work was funded by Loro Parque Foundation, CEPSA, Ministerio de Medio Ambiente, Fundación Biodiversidad, LIFE+ Indemares (LIFE07NAT/E/000732) and LIFE “Conservación de Cetáceos y tortugas de Murcia y Andalucía” (LIFE02NAT/E/8610), and “Plan Nacional I+D+I ECOCET” (CGL2011-25543) of the Spanish “Ministerio de Economía y Competitividad.” RdS and JG were supported by the Spanish Ministry of Economy and Competitiveness, through the Severo Ochoa Programme for Centres of Excellence in R+D+I (SEV-2012-0262),” and also RdS by the “Subprograma Juan de la Cierva.”

Mitochondrial DNA has been heavily utilized in phylogeography studies for several decades. However, underlying patterns of demography and phylogeography may be misrepresented due to coalescence stochasticity, selection, variation in mutation rates and cultural hitchhiking (linkage of genetic variation to culturally-transmitted traits affecting fitness). Cultural hitchhiking has been suggested as an explanation for low genetic diversity in species with strong social structures, counteracting even high mobility, abundance and limited barriers to dispersal. One such species is the sperm whale, which shows very limited phylogeographic structure and low mtDNA diversity despite a worldwide distribution and large population. Here, we use analyses of 175 globally distributed mitogenomes and three nuclear genomes to evaluate hypotheses of a population bottleneck/expansion vs. a selective sweep due to cultural hitchhiking or selection on mtDNA as the mechanism contributing to low worldwide mitochondrial diversity in sperm whales. In contrast to mtDNA control region (CR) data, mitogenome haplotypes are largely ocean-specific, with only one of 80 shared between the Atlantic and Pacific. Demographic analyses of nuclear genomes suggest low mtDNA diversity is consistent with a global reduction in population size that ended approximately 125,000 years ago, correlated with the Eemian interglacial. Phylogeographic analysis suggests that extant sperm whales descend from maternal lineages endemic to the Pacific during the period of reduced abundance and have subsequently colonized the Atlantic several times. Results highlight the apparent impact of past climate change, and suggest selection and hitchhiking are not the sole processes responsible for low mtDNA diversity in this highly social species.

Population genetic studies of non-model organisms often rely on initial ascertainment of genetic markers from a single individual or a small pool of individuals. This initial screening has been a significant barrier to beginning population studies on non-model organisms (Aitken et al., Mol Ecol 13:1423-1431, 2004; Morin et al., Trends Ecol Evol 19:208-216, 2004). As genomic data become increasingly available for non-model species, SNP ascertainment from across the genome can be performed directly from published genome contigs and short-read archive data. Alternatively, low to medium genome coverage from shotgun NGS library sequencing of single or pooled samples, or from reduced-representation libraries (e.g., capture enrichment; see Ref. "Hancock-Hanser et al., Mol Ecol Resour 13:254-268, 2013") can produce sufficient new data for SNP discovery with limited investment. We describe protocols for assembly of short read data to reference or related species genome contig sequences, followed by SNP discovery and filtering to obtain an optimal set of SNPs for population genotyping using a variety of downstream high-throughput genotyping methods.

Here we present a comparison of saddle and eye patch patterns of killer whales from Norwegian, Icelandic, British, Spanish and Greenlandic waters. We found only a small amount of variation in saddle patch shapes, which may reflect a recent phylogenetic divergence from the most recent common ancestor. Eye patch shapes were more variable than saddle patches in small details. Most individuals had eye patches with parallel orientation, with the exception of a small group of killer whales from the Hebrides, which, as previously reported, had sloping eye patches that sloped downward at the posterior end. This differentiation in pigmentation patterns of the Hebridean killer whales from neighbouring populations could reflect one or more of several evolutionary processes, including a deeper phylogenetic divergence, low gene flow with other local populations and drift.

Speciation is a fundamental evolutionary process, the knowledge of which is crucial for understanding the origins of biodiversity. Genomic approaches are an increasingly important aspect of this research field. We review current understanding of genome-wide effects of accumulating reproductive isolation and of genomic properties that influence the process of speciation. Building on this work, we identify emergent trends and gaps in our understanding, propose new approaches to more fully integrate genomics into speciation research, translate speciation theory into hypotheses that are testable using genomic tools and provide an integrative definition of the field of speciation genomics.

An assemblage of killer whales that has been sighted in waters off the west coast of the British Isles and Ireland has previously been shown to be isolated from other North Atlantic killer whale communities based on association patterns. By applying a Bayesian formulation of the Jolly–Seber mark-recapture model to the photo-identification data compiled from opportunistic photographic encounters with this population of killer whales, we show that such sparse and opportunistically-collected data can still be valuable in estimating population dynamics of small, wide-ranging groups. Good quality photo-identification data was collected from 32 encounters over 19 years. Despite a cumulative total of 77 identifications from these encounters, just ten individuals were identified and the remaining 67 identifications were re-sights of these ten animals. There was no detected recruitment through births during the study and, as a result, the population appears to be in a slight decline. The demography of the population was highly skewed towards older individuals and had an unusually high ratio of adult males, and we suggest that demographic stochasticity due to a small population size may be further impacting the population growth rate. We recommend that this population be managed as a separate conservation unit from neighbouring killer whale populations.

Identifying evolutionary divergent taxonomic units, e.g. species and subspecies, is important for conservation and evolutionary biology. The ‘type D’ killer whale, Orcinus orca, is a rarely observed morphotype with a pelagic, circumpolar subantarctic distribution, making dedicated research and therefore taxonomic study extremely difficult to date. In this study, we used DNA target enrichment hybridisation capture coupled to high throughput sequencing, to obtain the first DNA sequence from the only known museum specimen of this recently described morphotype. The high coverage, complete mitogenome sequence was compared to a previously published global dataset of 139 individuals, indicating that this type is highly divergent to all previously genetically sequenced killer whale forms. The estimated divergence time (390,000 years ago) from its most recent common ancestor with other extant killer whale lineages was the second oldest split within the killer whale phylogeny. This study provides the first genetic support of type D potentially being a distinct subspecies or species of killer whale, although further samples are needed to identify whether there is monophyly of mitogenome sequences and whether nuclear DNA also indicates reproductive isolation. These findings also highlight the value of natural history museum collections and new technologies to investigate the taxonomy of rare, cryptic or difficult to access species.

Methods to determine the sex from tissue samples of mammals include the amplification of Y chromosome specific regions, which should only amplify from males, or amplification of homologous regions of the X and Y chromosome containing XY specific SNPs. A disadvantage of the first approach is that PCR failure can be misinterpreted as the identification of a female. The latter approach is proposed to identify PCR failure through non-amplification of the X homologue, which should be present in both sexes. This method is therefore potentially more suitable for molecular sexing of degraded DNA with a high probability of PCR failure, such as for example, ancient DNA samples. Here, we investigate the validity of this assumption regarding the use of XY homologue PCR assays for molecular sexing of ancient DNA. We tested a primer set targeting the ZFX/ZFY alleles using ancient DNA extracts from 100 to 4500 years old bowhead whale samples, and for comparison on dilution series from modern bowhead whales of known sex. DNA sequencing of PCR products obtained from the ancient material confirmed a higher proportion of successful PCR amplifications of the X homologue over the Y homologue. This potentially biased sex determination was further assessed by testing highly diluted DNA extracts of modern samples, for which a consistently higher success rate of PCR amplification and lower PCR cycle threshold was found for the X homologue from females than either homologue from males. This is most likely due to the higher copy number of the X homologue in females, although other yet unknown attributes of the protocol may also cause the observed bias. The current case study provides a valuable example of a potential pitfall in molecular sex determination of ancient mammal DNA in zooarchaeology. High-throughput sequencing methods, in which sufficiently large numbers of reads can be unambiguously mapped to X and Y regions, should overcome such biases and be the most robust approach for molecular sex determination using degraded DNA.

The ocean is hypothesized to be where life on earth originated, and subsequent evolutionary transitions between marine and terrestrial environments have been key events in the origin of contemporary biodiversity. Here, we review how comparative genomic approaches are an increasingly important aspect of understanding evolutionary processes, such as physiological and morphological adaptation to the diverse habitats within the marine environment. In addition, we highlight how population genomics has provided unprecedented resolution for population structuring, speciation and adaptation in marine environments, which can have a low cost of dispersal and few physical barriers to gene flow, and can thus support large populations. Building upon this work, we outline the applications of genomics tools to conservation and their relevance to assessing the wide-ranging impact of fisheries and climate change on marine species.

A key goal for wildlife managers is identifying discrete, demographically independent conservation units. Previous genetic work assigned killer whales that occur seasonally in the Strait of Gibraltar (SoG) and killer whales sampled off the Canary Islands (CI) to the same population. Here we present new analyses of photo-identification and individual genotypes to assess the level of contemporary gene flow and migration between study areas, and analyses of biomarkers to assess ecological differences. We identified 47 different individuals from 5 pods in the SoG and 16 individuals in the CI, with no matches found between the areas. Mitochondrial DNA control region haplotype was shared by all individuals sampled within each pod, suggesting that pods have a matrifocal social structure typical of this species, whilst the lack of shared mitogenome haplotypes between the CI and SoG individuals suggests that there was little or no female migration between groups. Kinship analysis detected no close kin between CI and SoG individuals, and low to zero contemporary gene flow. Isotopic values and organochlorine pollutant loads also suggest ecological differences between study areas. We further found that one individual from a pod within the SoG not seen in association with the other four pods and identified as belonging to a potential migrant lineage by genetic analyses, had intermediate isotopic values and contaminant between the two study areas. Overall our results suggest a complex pattern of social and genetic structuring correlated with ecological variation. Consequently at least CI and SoG should be considered as two different management units. Understanding this complexity appears to be an important consideration when monitoring and understanding the viability of these management units. Understand the viability will help the conservation of these threatened management units., This work was funded by Loro Parque Foundation, CEPSA, Ministerio de Medio Ambiente, Fundación Biodiversidad, LIFE+ Indemares (LIFE07NAT/E/000732) and LIFE “Conservación de Cetáceos y tortugas de Murcia y Andalucía” (LIFE02NAT/E/8610), “Plan Nacional I+D+I ECOCET” (CGL2011-25543) of the Spanish “Ministerio de Economía y Competitividad”. R. d. S. and J. G. were supported by the Spanish Ministry of Economy and Competitiveness, through the Severo Ochoa Programme for Centres of Excellence in R+D+I (SEV-2012-0262)>, and also R. d. S. by the “Subprograma Juan de la Cierva”. Genetic analysis at the University of British Columbia was supported by the Vancouver Aquarium and assisted by Allyson Miscambell.

Organochlorine (OC) pesticides and the more persistent polychlorinated biphenyls (PCBs) have well-established dose-dependent toxicities to birds, fish and mammals in experimental studies, but the actual impact of OC pollutants on European marine top predators remains unknown. Here we show that several cetacean species have very high mean blubber PCB concentrations likely to cause population declines and suppress population recovery. In a large pan-European meta-analysis of stranded (n = 929) or biopsied (n = 152) cetaceans, three out of four species:- striped dolphins (SDs), bottlenose dolphins (BNDs) and killer whales (KWs) had mean PCB levels that markedly exceeded all known marine mammal PCB toxicity thresholds. Some locations (e.g. western Mediterranean Sea, south-west Iberian Peninsula) are global PCB “hotspots” for marine mammals. Blubber PCB concentrations initially declined following a mid-1980s EU ban, but have since stabilised in UK harbour porpoises and SDs in the western Mediterranean Sea. Some small or declining populations of BNDs and KWs in the NE Atlantic were associated with low recruitment, consistent with PCB-induced reproductive toxicity. Despite regulations and mitigation measures to reduce PCB pollution, their biomagnification in marine food webs continues to cause severe impacts among cetacean top predators in European seas., The research was conducted by the UK Cetacean Strandings Investigation Program (CSIP) and the Centre for Environment, Fisheries and Aquaculture Science (Cefas) under contract to the UK Department for Environment, Food and Rural Affairs (Defra) and the Devolved Governments of Scotland and Wales. Additional funding for toxicological analyses was provided by ASCOBANS under contract “SSFA2010-3” and by the Fundació Barcelona Zoo. The Gulf of Cádiz and Strait of Gibraltar samples collection were sponsored by Loro Parque Foundation, Fundación Biodiversidad, CEPSA and ECOCET Project (CGL2011-25543) and logistical support for sample sexing was provided by Laboratorio de Ecología Molecular, Estación Biológica de Doñana, CSIC (LEM-EBD). Samples from Galicia were obtained with the support of Direccion Xeral de Conservacion da Natureza-Xunta de Galicia, co-financed with European Regional Development Funds (ERDF/FEDER). S. Murphy was supported by a Marie Curie International Outgoing Fellowship within the Seventh European Community Framework Programme. R. de Stephanis and J. Giménez were supported by the Severo Ochoa Programme for Centres of Excellence in R + D + I (SEV-2012-0262). A. D. Foote was supported by a Lawski Foundation stipend.

Analysing population genomic data from killer whale ecotypes, which we estimate have globally radiated within less than 250,000 years, we show that genetic structuring including the segregation of potentially functional alleles is associated with socially inherited ecological niche. Reconstruction of ancestral demographic history revealed bottlenecks during founder events, likely promoting ecological divergence and genetic drift resulting in a wide range of genome-wide differentiation between pairs of allopatric and sympatric ecotypes. Functional enrichment analyses provided evidence for regional genomic divergence associated with habitat, dietary preferences and post-zygotic reproductive isolation. Our findings are consistent with expansion of small founder groups into novel niches by an initial plastic behavioural response, perpetuated by social learning imposing an altered natural selection regime. The study constitutes an important step towards an understanding of the complex interaction between demographic history, culture, ecological adaptation and evolution at the genomic level., Killer whales have evolved into specialized ecotypes based on hunting strategies and ecological niches. Here, Andrew Foote and colleagues sequenced the whole genome of individual killer whales representing 5 different ecotypes from North Pacific and Antarctic, and show expansion of small founder groups to adapt to specific ecological niches.

Epidermal skin samples from eastern North Atlantic killer whales, Orcinus orca, were analyzed for carbon and nitrogen stable isotope ratios. From those, comparisons within a data set of 17 samples collected from Tysfjord, Norway, in November suggested that diet is relatively specialized during this time period at this location. There were significant differences between a small set of samples from Iceland and those collected from Norway, which had all been assigned to the same population by a previous population genetics study. The results would be consistent with matrilines feeding on either the Norwegian or Icelandic stocks of Atlantic herring (Clupea harengus). There was no significant difference within Icelandic samples between those assigned to the population known to feed upon herring and those assigned to a population hypothesized to follow Atlantic mackerel (Scomber scombrus). The greatest differences were between the epidermal samples analyzed in this study and tooth and bone collagen samples from the North Sea that were analyzed previously, which also showed significantly more variation in isotopic ratios than found for skin samples. These differences could reflect differences in turnover rate, differences in diet-tissue fractionation and discrimination due to the amino acid composition of the different tissues, and/or greater competition promoting dietary variation between groups in the North Sea.

The persistence and size of social groups can be plastic and governed by ecological selection or be under greater genetic control and constrained by phylogenetic inertia. Comparing sociality of phylogenetically divergent populations under the same ecological conditions or between groups within a population under different ecological conditions can identify the relative influence of ecological selection on group formation. Here, we compare the size and persistence of social groups within a community of Atlantic killer whales, comparing between data collected from an area around Scotland where the whales have mainly been seen to hunt seals and data collected from an area around Iceland where the whales have mainly been seen to hunt herring. Additionally, we compare the observed social structure with that of previously studied Pacific ecotypes. Atlantic killer whale groups in both locations had a stable long-term primary social tier (association index level . 0.8) similar to that of Pacific killer whales. However, associations between these groups were much lower when hunting for seals than for fish in both the Atlantic and the Pacific. The occurrence of these differences in sociality between Atlantic groups, which are linked in a single social network, suggests that ecological selection partially determines sociality in this species. Furthermore, if sociality was constrained by phylogenetic inertia, then the Atlantic killer whales would all be expected to be more similar to the Pacific fish-eating ecotype than the more phylogenetically distant Pacific mammal-eating ecotype. Our study suggests that sociality in killer whales is to some extent plastic and can be adapted to the local ecological conditions. Key words: ecology, killer whale, orca, orcinus, sociality. [Behav Ecol]

Killer whales Orcinus orca are sighted regularly off Shetland, UK, but little is known about their numbers, diet and population identity. We aimed to relate vocal behaviour to diet of killer whales around Shetland in order to investigate population structure and differences in feeding strategies. Fieldwork was conducted in the summers of 2008 and 2009. We located killer whales through a sightings network and shore-based scans and collected photo-ID data, behavioural information, feeding data and acoustic recordings from a small boat. The majority of encounters (n = 14) were of small groups (1 to 15 individuals) travelling close to shore and feeding on marine mammals. Two encounters were with large groups (20+ individuals) feeding on herring Clupea harengus farther offshore. Seal-hunting groups vocalised rarely, producing pulsed calls, echolocation clicks and whistles almost exclusively when surface-active or milling after a kill. Herring-eating groups were largely silent during one encounter, but very vocal during the other. Analysis of pulsed calls identified 6 stereotyped call types for seal-hunting groups and 7 for herring-eating groups. No call types were shared between both kinds of groups. The vocal behaviour of seal-hunting groups showed striking parallels to that of Pacific marine mammal specialists and presumably evolved to decrease detection by acoustically sensitive prey. One call type produced by Shetland herring-eating killer whales matched a vocalisation that a previous study had described from Iceland and identified as a possible herding call that may function to concentrate herring during feeding. These findings point to behavioural and dietary specialisation among Shetland killer whales, which should be taken into account when making management decisions affecting these animals.

Population genetic structure of North Atlantic killer whale samples was resolved from differences in allele frequencies of 17 microsatellite loci, mtDNA control region haplotype frequencies and for a subset of samples, using complete mitogenome sequences. Three significantly differentiated populations were identified. Differentiation based on microsatellite allele frequencies was greater between the two allopatric populations than between the two pairs of partially sympatric populations. Spatial clustering of individuals within each of these populations overlaps with the distribution of particular prey resources: herring, mackerel and tuna, which each population has been seen predating. Phylogenetic analyses using complete mitogenomes suggested two populations could have resulted from single founding events and subsequent matrilineal expansion. The third population, which was sampled at lower latitudes and lower density, consisted of maternal lineages from three highly divergent clades. Pairwise population differentiation was greater for estimates based on mtDNA control region haplotype frequencies than for estimates based on microsatellite allele frequencies, and there were no mitogenome haplotypes shared among populations. This suggests low or no female migration and that gene flow was primarily male mediated when populations spatially and temporally overlap. These results demonstrate that genetic differentiation can arise through resource specialization in the absence of physical barriers to gene flow.

Killer whales (Orcinus orca) currently comprise a single, cosmopolitan species with a diverse diet. However, studies over the last 30 yr have revealed populations of sympatric ‘‘ecotypes’’ with discrete prey preferences, morphology, and behaviors. Although these ecotypes avoid social interactions and are not known to interbreed, genetic studies to date have found extremely low levels of diversity in the mitochondrial control region, and few clear phylogeographic patterns worldwide. This low level of diversity is likely due to low mitochondrial mutation rates that are common to cetaceans. Using killer whales as a case study, we have developed a method to readily sequence, assemble, and analyze complete mitochondrial genomes from large numbers of samples to more accurately assess phylogeography and estimate divergence times. This represents an important tool for wildlife management, not only for killer whales but for many marine taxa. We used highthroughput sequencing to survey whole mitochondrial genome variation of 139 samples from the North Pacific, North Atlantic, and southern oceans. Phylogenetic analysis indicated that each of the known ecotypes represents a strongly supported clade with divergence times ranging from ;150,000 to 700,000 yr ago. We recommend that three named ecotypes be elevated to full species, and that the remaining types be recognized as subspecies pending additional data. Establishing appropriate taxonomic designations will greatly aid in understanding the ecological impacts and conservation needs of these important marine predators. Wepredict that phylogeographic mitogenomics will become an important tool for improved statistical phylogeography and more precise estimates of divergence times. [Supplemental material is available online at http://www.genome.org. The sequence data from this study have been submitted to GenBank (http://www.ncbi.nlm.nih.gov/genbank) under accession nos. GU187153–GU187164, GU187166– GU187219, and HM060332–HM060334.]

Ecological divergence has a central role in speciation and is therefore an important source of biodiversity. Studying the micro-evolutionary processes of ecological diversification at its early stages provides an opportunity for investigating the causative mechanisms and ecological conditions promoting divergence. Here we use morphological traits, nitrogen stable isotope ratios and tooth wear to characterize two disparate types of North Atlantic killer whale. We find a highly specialist type, which reaches up to 8.5 m in length and a generalist type which reaches up to 6.6 m in length. There is a single fixed genetic difference in the mtDNA control region between these types, indicating integrity of groupings and a shallow divergence. Phylogenetic analysis indicates this divergence is independent of similar ecological divergences in the Pacific and Antarctic. Niche-width in the generalist type is more strongly influenced by between-individual variation rather than within-individual variation in the composition of the diet. This first step to divergent specialization on different ecological resources provides a rare example of the ecological conditions at the early stages of adaptive radiation.

Movement, site fidelity and connectivity have important consequences for the evolution of population structure and therefore the conservation and management of a species. In this study photographs of naturally marked killer whales collected from sites across the Northeast Atlantic are used to estimate fidelity to sampling locations and movement between locations, expressed as transition probabilities, pt, using maximum likelihood methods. High transition probabilities suggest there is high inter-annual site fidelity to all locations, and large-scale movement between the spawning and wintering grounds of both Norwegian and Iceland stocks of Atlantic herring. There was no evidence of movement between the Norwegian herring grounds and Icelandic herring grounds, or between the mackerel fishing grounds and the herring fishing grounds. Thus the movement of predictable and abundant prey resources can lead to intrinsic isolation in this species We also find movement between the Northern Isles, Scotland and East Iceland. An association network indicates that killer whales predating seals around the Northern Isles, Scotland are linked to the community of killer whales that follow the Icelandic summer-spawning herring. This adds support to existing evidence of a broad niche width in some populations.

Sightings of killer whales around Shetland were recorded between 1991 and 2006 and for the whole of Scotland for 2007. The data were used to investigate temporal patterns in killer whale occurrence around Shetland and spatial patterns in occurrence around Scotland. 2. There was a strong seasonal peak in sightings around Shetland during June-July, coinciding with the harbour seal pupping season. 3. There was no clear trend in annual sightings around Shetland between 1991 and 2006. 4. Killer whales were sighted most frequently around Shetland and the Pentland Firth as well as around Mull and the Treshnish Isles. 5. These findings are discussed in terms of potential impacts upon local declining harbour seal populations and future research requirements. Copyrightr2009 John Wiley & Sons, Ltd.

Fish-eating killer whales Orcinus orca in the northeastern Pacific live in highly stable matrifocal social groups called pods. Each pod produces a repertoire of seven or more stereotyped call types. We compared the relative production of call types of free-ranging killer whale pods over time and between social contexts. The relative production of call types by each pod during directional travel was distinct over a 27-yr period; however, both temporal stability and pod distinctiveness were strongly influenced by a subset of dominant call types within the repertoire of each pod. Some call types within the repertoires contain biphonation (two overlapping independently modulated tones) and have a higher estimated active space than call types containing just one tone. In multi-pod aggregations the relative production of the dominant call types of each pod decreased and the relative production of a subset of call types that are rarely recorded from single-pod groupings increased. The majority of these contained biphonation. The data suggest a distinction between a subset of dominant call types that may function to identify the pod and a subset of less common call types including several call types containing biphonation that are more commonly produced during inter-pod affiliations.

The dramatic increase in the application of genomic techniques to non-model organisms (NMOs) over the past decade has yielded numerous valuable contributions to evolutionary biology and ecology, many of which would not have been possible with traditional genetic markers. We review this recent progression with a particular focus on genomic studies of marine mammals, a group of taxa that represent key macroevolutionary transitions from terrestrial to marine environments and for which available genomic resources have recently undergone notable rapid growth. Genomic studies of NMOs utilize an expanding range of approaches, including whole genome sequencing, restriction site-associated DNA sequencing, array-based sequencing of single nucleotide polymorphisms and target sequence probes (e.g., exomes), and transcriptome sequencing. These approaches generate different types and quantities of data, and many can be applied with limited or no prior genomic resources, thus overcoming one traditional limitation of research on NMOs. Within marine mammals, such studies have thus far yielded significant contributions to the fields of phylogenomics and comparative genomics, as well as enabled investigations of fitness, demography, and population structure. Here we review the primary options for generating genomic data, introduce several emerging techniques, and discuss the suitability of each approach for different applications in the study of NMOs.

Killer whales (Orcinus orca) in the Mediterranean Sea are currently restricted to the Strait of Gibraltar and surrounding waters. Thirty-nine individuals were present in 2011, with a well-differentiated social structure, organized into five pods. Killer whale occurrence in the Strait is apparently related to the migration of their main prey, Atlantic bluefin tuna (Thunnus thynnus). In spring, whale distribution was restricted to shallow waters off the western coast of the Strait where all pods were observed actively hunting tuna. In summer, the whales were observed in the shallow central waters of the Strait. A relatively new feeding strategy has been observed among two of the five pods. These two pods interact with an artisanal drop-line fishery. Pods depredating the fishery had access to larger tuna in comparison with pods that were actively hunting. The Strait of Gibraltar killer whales are socially and ecologically different from individuals in the Canary Islands. Molecular genetic research has indicated that there is little or no female-mediated gene migration between these areas. Conservation threats include small population size, prey depletion, vessel traffic, and contaminants. We propose the declaration of the Strait of Gibraltar killer whales as an endangered subpopulation. A conservation plan to protect the Strait of Gibraltar killer whales is urgently needed, and we recommend implementation of a seasonal management area where activities producing underwater noise are restricted, and the promotion of bluefin tuna conservation.

Marine mammals from different mammalian orders share several phenotypic traits adapted to the aquatic environment and therefore represent a classic example of convergent evolution. To investigate convergent evolution at the genomic level, we sequenced and performed de novo assembly of 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 of these substitutions 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, whereas convergent molecular evolution is relatively common, adaptive molecular convergence linked to phenotypic convergence is comparatively rare.