by Daniel R. Stahler, Bridgett M. vonHoldt, Rena M. Schweizer, & Robert K. Wayne
Nearly four decades following the eradication of the gray wolf in Yellowstone National Park, a new microbial species was discovered in the Lower Geyser Basin which revolutionized the world of molecular biology and wolf genetics (Guyer and Koshland 1989, Varley 1993). This microbial species was a heat-loving bacterium, Thermus aquaticus. It produces a heat-stable enzyme known as Taq polymerase that, for the first time, enabled scientists to replicate DNA on a massive scale using a series of simple steps involving the heating and cooling of DNA. Only bacteria adapted to the hot springs' environment produced this heat stable Taq polymerase that could survive these temperature fluctuations. Taq polymerase was featured as the molecule of the year (1989) on the cover of Science magazine (Guyer and Koshland 1989); and sales of Taq polymerase, related products, and equipment generated billions of dollars in revenue. The inventor of the DNA amplification process (the polymerase chain reaction or PCR) using Taq polymerase, Kary Mullis, was awarded the Noble Prize in 1993. Since the 1980s, PCR has revolutionized the fields of forensics, human medicine, infectious diseases, and molecular and population genetics. In a fascinating chain of events, the discovery of an obscure species adapted to Yellowstone's harsh geothermal waters opened the portals of scientific investigations for many organisms on the tree of life, and highlighted the importance of biological diversity for human society.
Beyond the obvious benefits to our own species, we need look no further than the Canidae family (wolves, dogs, coyotes, jackals, and foxes) to see where such molecular techniques have significantly advanced our understanding of ecology, evolution, and conservation. From unraveling the complex evolutionary histories of wolf-like canids and origins of dog domestication, to revealing the structure and function of genes, to addressing questions significant to canid conservation, few non-human species have been at the frontiers of genetic research as have wolves and their relatives. For Yellowstone wolves, the application of molecular DNA techniques puts this population in a particularly bright spotlight. Here, we provide an overview of the role genetics has played in the recovery of Yellowstone wolves and the variety of questions addressed by an ever-advancing field of molecular research.
The importance of genetics was first applied during the reintroduction years as founding individuals were selected from different packs in different parts of Canada and released into both Yellowstone National Park (YNP) and the central Idaho wilderness. As a result, a genetically diverse population comprised of many unrelated individuals became the foundation for what resulted in a rapidly expanding breeding population throughout Idaho and the Greater Yellowstone Ecosystem (GYE). This met an important conservation goal of any successful species recovery program by establishing a genetically diverse network of subpopulations (von-Holdt et al. 2010). When maintained at adequate sizes and connected through genetically effective dispersal, populations are found to be more resilient to the fitness costs associated with inbreeding, small population size, and isolation, and better equipped to adapt to changing environmental conditions.
With all founders genetically sampled, the next critical component to the YNP's genetic research was establishing a sampling protocol for their progeny. From the beginning, efforts have been made to collect genetic samples from all wolves handled during capture and radio-collaring events (figure 1), as well as from any deceased wolves discovered. Additionally, scats and hair left behind on the landscape have been collected and used, depending on sample quality or degree of degradation. From these sampling methods, DNA is extracted and amplified from whole blood, tissue, or scat, and banked for a variety of molecular applications. In addition to the genetic sampling, the Wolf Project team and collaborators have remained committed to collecting a variety of demographic, life history, morphological, behavioral, disease, and ecological datasets through time. The integration of such rich data over a significant period of years has rarely been achieved for any species in the wild, let alone for a reintroduced population of known founders and their descendants.
Molecular Techniques & Markers Applied to Yellowstone Wolves
Advances in the molecular tools and techniques, and their application to ecological, evolutionary, and behavioral studies, have risen dramatically over the last two decades. Since wolves returned to Yellowstone, we have assessed the genetic composition, or genotypes, of individuals using a variety of molecular markers and approaches. Using PCR to amplify and sequence nuclear DNA, which individuals inherit from both parents, the nuclear genome has been surveyed for short repetitive DNA fragments known as microsatellite loci, single nucleotide polymorphisms (SNPs), or genome sequences. Microsatellites are often highly variable and are useful for resolving recent population dynamics, such as dispersal, paternity, and relationships. This information is used to estimate reproductive success through parent-assignments, although it requires a relatively large proportion of the breeding population be genetically represented. One drawback to using such repetitive DNA is that it is limited with regard to the proportion of the genome that can be surveyed. Often, a study consists of 8-40 loci, a small fraction of the genome, which limits the ability to make inferences about evolutionary patterns, complex population structure, or even resolve parentage in closely kin-structured animals like wolves. The next level at which YNP genetic data has been surveyed is with respect to variation in SNP loci. SNPs are single nucleotide polymorphisms, or variable sites, that segregate alleles (a variant form of a gene) in a population and can be genotyped across the entire genome. This data type is part of the next generation of genotyping techniques and can provide valuable information regarding regions of the genome that contain recent changes putatively linked to adaptations or are preserved stably over long evolutionary periods. The last level at which Yellowstone wolves have contributed towards conservation genetics is in genome sequencing. Here, we can scan the entire collection of >2 billion nucleotides and address similar questions but with much deeper resolution, as well as sequence genes that are expressed, or transcribed, in response to specific biotic or environmental conditions. Collectively, these genetic markers have allowed us to explore questions ranging from population structure and gene flow, to reconstructing pedigree relationships, understanding the dynamics of coat color, life-history strategies, and natural selection on the genome.
Major Projects & Findings
Pedigree construction and application
Scientists and the public alike have been eager to explore the genetic lineages of Yellowstone wolves. Using genotypes from 26 microsatellite loci, we assessed the relationships and genetic similarity of 200 Yellowstone wolves to infer patterns of parentage, breeding pair characteristics, and general pack formation assembly rules (vonHoldt et al. 2008). From the microsatellite data, we reconstructed the population genealogy, allowing us to detail pack formation, dissolution, and assess kinship ties among packs (figure 2). We also found Yellowstone wolves avoid inbreeding with close relatives. Knowing genealogical structure and how it influences behavior and demography allowed us to better evaluate the success of this wolf recovery—a valuable contribution to the field of conservation genetics (vonHoldt et al. 2008, 2010). We have also integrated the wolf pedigree with life-history data and demographics to demonstrate powerful new methods for exploring co-evolutionary dynamics (Coulson et al. 2011). Using Yellowstone's genealogy, we can calculate the level of heritability (the degree to which a trait measured in an individual is correlated with the same trait measured in its parents) for various life-history traits (e.g., reproductive success, fecundity, lifespan, behavior, coat color). As more wolves are sampled and newer genotyping methods applied, we will continue to build and improve the accuracy of the Yellowstone wolf pedigree for a variety of applications.
In order to evaluate the success of wolf recovery on a broader regional scale, we assessed genetic diversity and connectivity across the three recovery areas (Greater Yellowstone Ecosystem, Montana, and Idaho; vonHoldt et al. 2010). We analyzed DNA samples from 555 Northern Rocky Mountain wolves, including all 66 reintroduced founders in Idaho and YNP, for variation in 26 microsatellite loci over the initial 10-year recovery period (1995–2004). The population maintained high levels of variation with low levels of inbreeding and throughout this period expanded rapidly. We found significant genetic differences between the three recovery areas, and we verified effective dispersal (dispersal with subsequent reproduction) events between each of the recovery areas. Genetic connectivity was one of the primary stipulations for wolf delisting in the Rocky Mountains. This provided great insight into the landscape ecology of wolves through the scope of molecular genetics, and gives us a baseline and methodology by which to evaluate genetic structure and connectivity in the future.
Coat color (K-locus)
The discovery of genetic variants that affect phenotypes (i.e., the observable traits influenced by an organism's genes and its environment) is a rarity when studying natural populations. Research that has done so used model species that can be bred in a laboratory allowing for the genetic basis of traits to be uncovered through phenotypic assessments of offspring. Although not model species, gray wolves are unique in that their relative, the domestic dog, has been the focus of extensive genetic research and was the fifth species to have their genome sequenced. Thus, we have a great library of genetic information on traits such as coat color, and what genes are contained in the genome that can be utilized for an understanding of phenotypic variation in wild wolves. Specifically, black coat color has been investigated in Yellowstone wolves and found to be due to a single gene (a b-defensin gene termed CBD103 or the K-locus), with all black coated individuals carrying a 3-nucleotide deletion linked to this coat color phenotype, a mutation believed to have originated in domestic dogs of the Old World (Candille et al. 2007, Anderson et al. 2009). We demonstrated through use of our pedigree data this single mutation was responsible for all black wolves in YNP and that the black allele was dominant over the gray (figure 3). This result set the stage for studies that explored the link between genetics, viability, fitness, and selection (e.g., Coulson et al., 2011, Stahler et al. 2013, Hedrick et al. 2014, Schweizer et al. unpublished data). Remarkably, it was found that the K-locus gene is involved in immune function, suggesting an additional role in pathogen defense. In fact, ongoing research is showing black wolves have greater survivorship during distemper outbreaks (Cubaynes, unpublished data).
Another study (Schweizer, unpublished data) used a "capture" array (a method that allows one to subsample and sequence regions of the genome) to sequence the K-locus in over 200 Yellowstone wolves, as well as wolves from other populations. This allowed for levels of genetic variation to be measured among different populations, as well as the ability to estimate how strong positive selection has been in each one. We found Yellowstone wolves have undergone selection for the mutation that causes black coat color to a greater extent than other populations of North American wolves, and selection strength may have increased since the founding of the Yellowstone population. This may be related to patterns of canine diseases, immunity, and fitness advantages of the black heterozygotes (Coulson et al. 2011). Furthermore, the origin of the K-locus in wolves may have come from hybridization events between dogs and wolves in northwest North America (Schweizer, unpublished data).
Finally, we are applying emerging techniques in cell culture science to study the adaptive value of coat color variation. This involves techniques that allow cell lines of wolf fibroblasts (skin) and keratinocytes (keratin producing cells) to be initiated from wolf ear biopsy punches taken during capture. Transformation of these cell lines provides an infinite source of DNA and RNA, and allows a non-invasive method to test the effects of specific cell level challenges on genes. For example, we are testing skin cultures with various viral and bacterial antigens to determine how black and gray wolves respond differently to these stressors. We can then test the response to specific antigens and our hypothesis that the black coat color gene is involved in immune response. This basic design can be used to probe the function of any genetic variant and will be a critical tool for understanding adaptation in future studies. The Yellowstone Wolf project is one of the only studies using stem cell lines in wild animal research.
Behavior & health
Behavioral studies of aggression in Yellowstone wolves are ongoing, with aggression linked to specific phenotypes and with possible links to genetic and regulatory variants (e.g., Janowitz et al., unpublished data). We have used genealogy to estimate the heritability of the level of aggression an individual will display during interpack interactions. Our preliminary heritability analysis identified that a simple additive genic model of inheritance was not sufficient to explain this complex behavioral trait. Rather, we found this behavior is influenced by both an intricate foundation of genetic and regulatory features, as well as an environmental influence. We can also apply these research strategies to explore the molecular influences on disease susceptibility, such as mange infections. To investigate the impact of genetic and epigenetic variation on mange infection, we are currently assessing how molecular variation segregates with infection severity classification, time to recovery, and frequency of reinfection.
It is commonly thought genetic variation is the key to adaptation and surviving environmental and biotic challenges, such as global warming and a change in predator or prey diversity. This is likely true for change occurring across generations, but the mechanisms for adaption across an individual's lifetime concerns gene expression. Whenever we eat, sleep, exercise, or relax, a select set of genes is turned on and off, like a molecular switch, which is critical for daily survival as a response to life challenges, such as strife, starvation, or disease. However, we are largely ignorant of the scale of gene expression response and its limits in natural systems. For the first time ever in a wild vertebrate population, we have characterized gene expression using next generation sequencing techniques (RNA-seq.). This effort involved a whole new collection scheme, as previous blood preservation techniques only preserved DNA. Our new focus is to also collect RNA, the set of molecules that reflect which genes are expressed. Charruau (Charruau et al. 2016) explains the factors that govern gene expression in model species and humans, such as sex and dominance rank, do not strongly influence gene expression in blood from wolves. This may reflect the highly cooperative and integrative nature of wolf society. Overwhelmingly, gene expression patterns in wolves are instead related to age and disease. Wolves age rapidly, as most individuals die by age five or six, and exhibit evidence of extreme injury and disease throughout this period. This rate of aging is matched by an equally rapid rate of gene expression senescence; a six-year-old wolf has experienced as much change in gene expression, over a similar subset of genes, as an elderly human. Our results also highlight how disease, such as mange, may cause secondary effects on gene expression, in addition to a primary pathogen response. We discovered wolves have a component of gene expression response related to mange that likely reflects tissue damage and healing due to scratching associated with the infection. These results establish a critical baseline for future studies of wolves across changing environments and in human dominated landscapes with distinct stressors. Further, we provide a new precedence and protocol for similar studies of other wild vertebrates.
Wolves & the age of genomics
We have entered the age of genomics, and wolves are at forefront being among >10 genomes having been described (Freedman et al. 2014, Zhang et al. 2014). However, these are genomes from Old World wolves used to study dog domestication and high altitude adaptation. North American wolves have a unique and divergent ancestry that needs a separate assessment. We finished the genome sequencing of 302M, a famous Yellowstone wolf that fathered many offspring. For this wolf, we have the highest sequencing resolution (an average of 50-fold coverage of each nucleotide) compared to any other wolf to-date. We also sequenced his mate (569F) and an offspring (570M), and are in the process of sequencing four more offspring. These sequences will provide a definitive estimate of mutation rate in wild wolves, a value that is an essential parameter in determining the evolutionary rate of genes and how selection alters the genome. It will enable us to build population models of how favorable and deleterious variation accumulates in populations (Marsden et al. 2015) and understand selection at the K-locus (black coat color gene). The complete genome also defines diagnostic North American wolf markers that can be used to probe admixture with dogs and coyotes (e.g., vonHoldt and Wayne 2012). The sequencing of 302M, a legacy wolf of the YNP population, will place genetic research in YNP in the limelight and exemplify its cutting edge nature to the public.
Genetic Research: Present & Future
The impact of humans has radically altered natural spaces worldwide and will be more severe in the future. This impact will disproportionately affect large predators, so we need to establish a genetic baseline and develop predictive tools in order to calibrate and predict genetic responses in the future. The genetic research done in YNP is in large part aimed at this need. We have established a genetic baseline using a wide variety of genetic markers. We have shown the population is genetically healthy and unlikely to suffer genetically in the future, if genetic exchange between populations continues. We have established a complete population pedigree of more than 350 wolves, which is the basis for understanding the genetic underpinning of wolf behavior and physiological traits. We have advanced the understanding of the genetic basis of coat color in YNP wolves and probed its role in survivorship and disease resistance. For the first time, we have a detailed map of the North American wolf genome and can measure mutation rates on individual genes that influence phenotype and adaptation. We now understand the basic factors influencing gene expression and regulation that can limit response to future environmental stressors. We plan to expand this research and integrate data on disease and behavior to explain genetic and epigenetic patterns. These efforts will continue to keep Yellowstone wolves on the frontier of genomic research.