Population genomics of the killer whale; SNP discovery towards the assessment of population structure at functional genes

Lead Research Organisation: Durham University
Department Name: Biological and Biomedical Sciences


Since a Canadian research project in the 1970s first showed how the regional killer whale population could be tracked over time using photographs to identify and follow the associations and movement of individual whales, it has become increasingly apparent that this species offers great potential for advancing our understanding of key evolutionary and ecological processes. Despite their tremendous potential for long-distance dispersion, with social groups travelling 1000s of km in a season, identified populations of associating individuals (divided into social units of 20-50 individuals) are genetically differentiated at neutral markers over small geographic scales. The apparent mechanism is dependence on local or seasonal habitat resources, and in particular, prey resources. In this way picivorous populations can be separated by the direction of migration in their anadromous salmonid prey (migrating north or south of an estuary), and picivorous populations can be isolated from marine-mammal-eating populations even in sympatry. There is a pattern of differentiation associated with geographic distance (as seen for many species), but only within an ecotype. Differentiation among ecotypes is based on biological factors (especially behaviour associated with foraging and social structure). Although the effective size of regional populations is likely small, there are sometimes physical differences that may be associated with resource exploitation (as seen in various other delphinid species, especially associated with buccal morphology), suggesting local adaptation. In this study we will identify genes that are apparently under selection, and associate them where possible with ecological characteristics, especially known aspect of the life history of the different ecotype populations. A key deliverable will be an extensive set of SNP markers, with some identified as likely under selection, to be applied in a follow-on study to an extensive program of population screening. A preliminary assessment based on these SNP markers will be undertaken in the current study as part of the SNP discovery process. This is made possible by the chosen methodology. The initial step will be the sequencing of the full genome for one sample to a quality level sufficient to use as a reference scaffold (to be done through the commercial provider, Eurofins MWG/ Operon). This will then be used to facilitate the RAD-Tag sequencing work and the associated bio-informatics (to be undertaken under the supervision of partner Neil Hall at CGR Liverpool). The RAD-Tag data will then be analysed by the PI in Durham in collaboration with Prof. Hall and with the assistance of the RA. The RAD-Tag sequences will permit the identification of SNP markers, and the reference genome would permit identification of their location and linkage within the killer whale genome. Analysis of an initial 150 whales from 5 populations will provide the preliminary data on signals for selection and a high-resolution assessment of patterns of differentiation. Bayesian and multiple regression methods will be applied to allow interpretation of genetic variation in the context of environmental variables, and to assess which variables explain the largest proportion of the genetic variance. Taken together these analyses will advance our understanding of the evolutionary processes that generate biodiversity within and among species, facilitate more effective programs of biodiversity conservation, and provide the raw material for future studies that will advance these objectives further.

Planned Impact

The application of molecular genetic data to problems in evolutionary biology and conservation began in the 1960s with the advent of protein electrophoresis. Now, not quite 50 years later, we can investigate whole genomes, even at the population level. Each advance is important because it allows us to test previous hypotheses more rigorously, and explore open questions that earlier technologies were unable to address. A particular enabling feature of population genomics is the ability to much more inclusively address the role of natural selection in shaping population structure, and to assess what genomic resources are essential to conserve. These studies are only just beginning, and so one major impact of our proposed study is to contribute to the advance of this field of study. The positive impacts for the scientific community have been discussed in the academic beneficiaries summary. To policy makers and managers interested in conservation strategy, these studies will provide key data on management stock structure, cryptic diversity, and a better understanding of the tools needed to maximise the impact of their conservation efforts. With limited resources, it will become increasingly important to invest in projects that will have maximum positive influence on the welfare of target populations. This means that a better understanding will be required about the relationship between environment, phenotype and genotype. Genomic approaches are the way to achieve these objectives. For the general public, it is important to remain informed about how science progresses in ways that can address problems important to them. In this case that potential is especially in the contributions the study will make to biodiversity conservation in general (with respect to the development of methods with greater potential), and to the conservation of the killer whale in particular. This is one of a few key species that has particular power to capture the public imagination. Nearly everyone knows what a killer whale is, and many will have seen them on TV, in aquaria, or even in the wild. Data from this species therefore provides a very useful hook to facilitate the public presentation of science. At the same time, the killer whale is very much in need of the attentions of conservation biologists, managers and policy makers, and some local populations have already be recognised in the US as endangered. The contribution to impact from this project will be divided into three aspects. 1) Dissemination to the scientific community will be through the usual mechanisms associated with publication in peer review literature and presentation at national and international conferences. All sequence data will be provided to public databases, and we will present our results broadly in the popular media. 2) To help facilitate dissemination both to the public and to relevant conservation agencies, such as those involved in marine ecosystem management, we propose the development of a web site (linked to the ConGRESS web site, and established as pages associated with the PI's lab website). 3) Finally, in addition to presentations at Evolution and Conservation meetings to scientific audiences, the PI will also provide a public lecture towards the end of the study that will take advantage of the high profile status of the study species to help promote the message of biodiversity conservation, and convey the need for new methods to address these problems, including conservation genomics.
Description A key objective of this study was to apply genomic methods to better understand the evolution of diversity and population structure in the killer whale, a species known to show consistent differences among 'ecotypes', sometimes among ecotype populations living in the same geographic area (in 'sympatry'). Using whole genome analyses we found evidence for a severe population bottleneck during the last ice age, helping to explain the very low genetic diversity found for this species in most parts of its global range (Moura et al. 2014a). We also confirmed that our genome (from an individual from the North Pacific) showed very low levels of diversity compared to other species of large mammals. Investigating a local population off South Africa we found uniquely high levels of diversity, suggesting that this may represent a refugial population that was less affected by the bottleneck event (Moura et al. 2014a). Constructing phylogenetic evolutionary histories using 1.7 million base pairs we found evidence for the Southern Ocean populations being ancestral, and for the evolution of ecotype differentiation (between populations that pursue fish prey and those that pursue marine mammals) in sympatry (Moura et al. 2014b). This revised earlier interpretations based only on mitochondrial DNA that these ecotypes may have evolved in allopatry. Single-locus interpretations, as provided by mitochondrial DNA, can provide misleading inference due to stochastic events. Our interpretations based on whole nuclear genomic data are therefore more robust.

Further investigation of population structure used 3500 loci distributed across the genome. Some of these loci could be shown to be under directional selection, providing evidence for differentiation in sympatry based on ecological processes isolating ecotypes (Moura et al. 2014c). This included the identification of loci with fixed differences among populations linked to functional genes with relevant functions (e.g. associated with digestion and reproduction). This especially included genes that control the expression of other genes, such as 'GATA4' for which there was a change in the amino acid sequence of the protein produced by the gene. This fixed difference distinguished the two main ecotypes, whales preying on marine mammals compared to those preying on fish. These very high resolution analyses also provided new insight into the pattern and extent of differentiation among populations, and into the demographic history of regional populations. The signal for a historical population bottleneck was again recovered by this independent method, followed by a period of expansion, and then population contraction as local founder populations were established (Moura et al. 2014c). Taken together these data show that both random processes associated with small population size (genetic drift) and strong selection have been important in the evolution of phenotypic diversity and the differentiation of populations with distinct habitat and resource requirements. Importantly, our data imply that adaptation to ecological factors can promote isolation and differentiation even in sympatry for this species. There is the potential for incipient speciation, however our data suggest that if killer whale populations are speciating, this is an ongoing process that began recently (within the current geologic era).

An important aspect of our broader contribution is a draft genome for the killer whale, and marker data for 3500 useful loci (single nucleotide polymorphisms, SNPs), all now freely available on public databases. In the case for support key deliverables were identified as 'an initial understanding of the evolution of functional diversity in this species, and the development of SNP markers useful for a more extensive (standard NERC proposal) study.' We now have a good foundation for understanding the types of loci that are being selected for in distinct ecotype populations, and the key role that population size and population dynamics have played in determining the pattern and level of diversity. We also made the unexpected discovery that a population off South Africa has retained diversity lost elsewhere, and that the loss of diversity elsewhere was associated with the coldest period of the last glaciation (Moura et al. 2014a). The long-term stability of the Benguela upwelling system off southern Africa may have been important, especially in contract to the less stable systems elsewhere in the world. A natural extension, therefore, will be to investigate the relatively diverse population off South Africa in more detail to better understand the adaptive potential of the species and the factors and mechanisms that promote the evolution of diversity in large marine predators. Since the conclusion of the project we have extended the work off South Africa in collaboration with South African colleagues, and are currently working on further publications. In 2019 and 2020 we have been undertaking research in South Africa funded by the International Whaling Commission that will provide materials and resources for the further development of this work, including further genomic analyses.

Moura, A.E., Janse van Rensburg, C., Pilot, M., Tehrani, A., Best, P.B., Thornton, M., Plon, S., de Bruyn P.J.N., Worley, K.C., Gibbs, R.A., Dahlheim, M.E. & Hoelzel, A.R. 2014a. Killer Whale Nuclear Genome and mtDNA Reveal Widespread Population Bottleneck During the Last Glacial Maximum. Mol. Biol. Evol. 31, 1121-1131.

Moura, A.E., Kenny, J.G., Chaudhuri, R., Hughes, M.A., Reisinger, R.R., de Bruyn, P.J.N., Dahlheim, M.E., Hall, N., Hoelzel, A.R. 2014b. Phylogenomics of the killer whale indicates ecotype divergence in sympatry. Heredity doi:10.1038/hdy.2014.67

Moura, A.E., Kenny, J.G., Chaudhuri, R., Hughes, M.A., Welch, A., Reisinger, R.R., de Bruyn, P.J.N., Dahlheim, M.E., Hall, N., Hoelzel, A.R. 2014c. Population genomics of the killer whale indicates ecotype evolution in sympatry involving both selection and drift. Mol. Ecol. 31, 1121-1131.
Exploitation Route These genomic data will provide the raw material for further studies to advance our understanding of the processes associated with the evolution and loss of diversity in natural populations. They will also promote a better understanding of the mechanisms involved in the evolution of new species. Our data raise questions about the interaction between environment, social structure and dispersal behaviour that will help identify key evolutionary processes affecting biodiversity. New studies can build on our findings to explore male-mediated connectivity, female philopatry and the process of incipient speciation. We also provide data that can be further exploited to learn about the genetics that determine functional adaptation to local environments. We provide insight into the long-term behaviour and resource requirements of the ocean's top predator, and thereby provide new hypotheses to test about the impact of climate change on marine ecosystems over the course of the Pleistocene climate cycles. These data also provide transferable inference about the conservation and management needs of large marine species, and about the critical role of environmental change, but this is described in more detail in the narrative impact section.
Sectors Environment,Leisure Activities, including Sports, Recreation and Tourism,Government, Democracy and Justice,Other

Description Our lab has had a long-term involvement in the promotion of effective conservation and management for cetacean species, and for the killer whale in particular. We were instrumental in supporting the work that led to the listing of the 'Southern Resident' killer whale population as a Distinct Population Segment (DPS) under the US Endangered Species Act (ESA, which requires genetic data to confirm reproductive isolation; based on multiple publications and commissioned reports between 1991 and 2004). As part of that process the office responsible for the listing (National Marine Fisheries Service office in Seattle) is required to generate a 'determination of critical habitat' and a 'species recovery plan' for listed populations. They are also required to respond to any related petitions, including petitions for de-listing (as came up in 2012). We have continued to contribute to the development of the action plan and to the resolution of issues associated with petitions. The data generated during our NERC study will be highly valuable in support of that process and towards providing accurate assessments. For example, our data greatly improves understanding of the historical population dynamics of regional populations, reinforcing the idea that founder groups established local populations quite recently (within the Holocene; Moura et al. 2014a,b,c), helping to explain the pattern of diversity within and among populations. We also provide new understanding of the functional diversity that defines ecotype populations, and we identify fixed differences linked to functional loci between regional populations. The Southern Residents were most differentiated in this respect for all comparisons, reinforcing the value of their independent protection under the ESA legislation (Moura et al. 2014c). This status as a recognised DPS has led to further policy, such as the introduction of new regulations governing vessel traffic in the vicinity of this population of killer whales. Our data also have further transferable relevance to the conservation of killer whale populations world-wide, for example through the recognition of the unique diversity found in the population off South Africa, making this a high priority region for conservation efforts, and the suggestion that most other populations have been through a recent population bottleneck (Moura et al. 2014a). Transferable inference for other species is gained through the demonstration of the value of these methods applied towards addressing key questions with important conservation implications. We also provide inference about the potential impact of past climatic fluctuations on the ocean ecosystem, by demonstrating a likely impact on a key alpha predator with a global distribution. Such inference can help predict and mitigate the impact of future climate change. Data from this study has been presented in high-profile scientific publications and in the popular media, which expanded on the conservation issues. Presentations to both scientific and popular audiences have been made in the US, South Africa and in the UK. I will continue to promote the biodiversity conservation impact resulting from this study through the media, through my web pages online, in expected further publications, and through my involvement with intergovernmental conservation organisations such as the International Whaling Commission. Research assistant Andre Moura has gained extensive training and experience in genomic methodologies, and has now obtained a University Lectureship at Lincoln University. A student intern, Annalora Irvine, also received training and work experience associated with next generation sequencing methodologies and wrote an excellent report on her project and findings associated with the study. Our research published in Heredity generated some discussion leading to the publication of two letters (references added below). Most recently funding from the International Whaling Commission has allowed us to obtain data that will further support the more effective conservation of this species in South African waters. Moura, A.E., Janse van Rensburg, C., Pilot, M., Tehrani, A., Best, P.B., Thornton, M., Plon, S., de Bruyn P.J.N., Worley, K.C., Gibbs, R.A., Dahlheim, M.E. & Hoelzel, A.R. 2014a. Killer Whale Nuclear Genome and mtDNA Reveal Widespread Population Bottleneck During the Last Glacial Maximum. Mol. Biol. Evol. 31, 1121-1131. Moura, A.E., Kenny, J.G., Chaudhuri, R., Hughes, M.A., Reisinger, R.R., de Bruyn, P.J.N., Dahlheim, M.E., Hall, N., Hoelzel, A.R. 2014b. Phylogenomics of the killer whale indicates ecotype divergence in sympatry. Heredity doi:10.1038/hdy.2014.67 Moura, A.E., Kenny, J.G., Chaudhuri, R., Hughes, M.A., Welch, A., Reisinger, R.R., de Bruyn, P.J.N., Dahlheim, M.E., Hall, N., Hoelzel, A.R. 2014c. Population genomics of the killer whale indicates ecotype evolution in sympatry involving both selection and drift. Mol. Ecol. 23, 5179-5192 Hoelzel, A.R. & Moura, A.E. 2015. Resource specialisation and the divergence of killer whale populations. Heredity 115, 93-95. Hoelzel, A.R. & Moura, A.E. 2016. Killer whales differentiating in geographic sympatry facilitated by divergent behavioural traditions. Heredity 117, 481-482.
First Year Of Impact 2014
Sector Environment,Leisure Activities, including Sports, Recreation and Tourism,Government, Democracy and Justice,Other
Impact Types Societal

Description Genomics 
Organisation University of Liverpool
Department Liverpool Centre for Genomic Research
Country United Kingdom 
Sector Academic/University 
PI Contribution Genomic work on this project was done in collaboration with Prof. Neal Hall and the Genomics Centre at Liverpool University.
Collaborator Contribution RAD sequencing was undertaken at the Genomics Centre in Liverpool for project NE/J014443/1. For project NE/K005359/1 the collaboration is ongoing, and genome sequencing, re-sequencing, genome annotation and associated bioinformatics work is being done in Liverpool.
Impact The data generated for NE/J014443/1 on population genomics and phylogenomics (see publications) were an outcome of this collaboration. Outputs for the current project are currently in preparation.
Start Year 2012
Description South African killer whale research 
Organisation University of Pretoria
Country South Africa 
Sector Academic/University 
PI Contribution We generated genetic data based on materials provided.
Collaborator Contribution Materials for research and ongoing collaborations enhanced the scope of the study and our ability to interpret findings.
Impact This association contributed to each of the published studies generated so far.
Start Year 2012