Development and evaluation of a novel single nucleotide polymorphism panel for North American bison.

Genome-wide single nucleotide polymorphism (SNP) genotyping platforms have become increasingly popular in characterizing livestock and wildlife populations, replacing traditional methods such as microsatellite fragment analysis. Herein, we report the development and evaluation of a novel bison SNP panel for population management and conservation. Initially, 2474 autosomal SNPs were selected from existing bison whole-genome sequences and variable sites among bison on the GGSP bovine 50K Chip, based on minor allele frequency, data completeness, and chromosome location. Additionally, 20 mitochondrial SNPs were chosen to identify known mitochondrial haplotypes in bison according to previous research. The SNPs were further evaluated using genotyping-by-sequencing with 190 bison, representing the historical lineages that survived the major population crash of the late 1800s. Variants with high potential for genotyping error were filtered out, and the remaining SNPs were placed on a custom Illumina™ array. The final panel consisting of 798 autosomal and 13 mitochondrial SNPs was used to establish baseline genetic parameters, compare populations, and assign mitochondrial haplotypes in 995 bison across ten populations. These SNPs were also found to be highly informative for individual animal identification and parentage assignment. This SNP panel provides a powerful new method to establish a baseline for estimating genetic health of bison populations and a new tool for bison managers to make informed management decisions based on genetic information specific to their populations.

Chromosome-level reference genome for North American bison (Bison bison) and variant database aids in identifying albino mutation.

We developed a highly contiguous chromosome-level reference genome for North American bison to provide a platform to evaluate the conservation, ecological, evolutionary, and population genomics of this species. Generated from a F1 hybrid between a North American bison dam and a domestic cattle bull, completeness and contiguity exceed that of other published bison genome assemblies. To demonstrate the utility for genome-wide variant frequency estimation, we compiled a genomic variant database consisting of 3 true albino bison and 44 wild-type pelage color bison. Through the examination of genomic variants fixed in the albino cohort and absent in the controls, we identified a nonsynonymous single nucleotide polymorphism (SNP) mutation on chromosome 29 in exon 3 of the tyrosinase gene (c.1114C>T). A TaqMan SNP Genotyping Assay was developed to genotype this SNP in a total of 283 animals across 29 herds. This assay confirmed the absence of homozygous variants in all animals except 7 true albino bison included in this study. In addition, the only heterozygous animals identified were 2 wild-type pelage color dams of albino offspring. Therefore, we propose that this new high-quality bison genome assembly and incipient variant database provides a highly robust and informative resource for genomics investigations for this iconic North American species.

Evolution of immunogenetic components encoding ultralong CDR H3.

The genomes of most vertebrates contain many V, D, and J gene segments within their Ig loci to construct highly variable CDR3 sequences through combinatorial diversity. This nucleotide variability translates into an antibody population containing extensive paratope diversity. Cattle have relatively few functional VDJ gene segments, requiring innovative approaches for generating diversity like the use of ultralong-encoding IGHV and IGHD gene segments that yield dramatically elongated CDR H3. Unique knob and stalk microdomains create protracted paratopes, where the antigen-binding knob sits atop a long stalk, allowing the antibody to bind both surface and recessed antigen epitopes. We examined genomes of twelve species of Bovidae to determine when ultralong-encoding IGHV and IGHD gene segments evolved. We located the 8-bp duplication encoding the unique TTVHQ motif in ultralong IGHV segments in six Bovid species (cattle, zebu, wild yak, domestic yak, American bison, and domestic gayal), but we did not find evidence of the duplication in species beyond the Bos and Bison genera. Additionally, we analyzed mRNA from bison spleen and identified a rich repertoire of expressed ultralong CDR H3 antibody mRNA, suggesting that bison use ultralong IGHV transcripts in their host defense. We found ultralong-encoding IGHD gene segments in all the same species except domestic yak, but again not beyond the Bos and Bison clade. Thus, the duplication event leading to this ultralong-encoding IGHV gene segment and the emergence of the ultralong-encoding IGHD gene segment appears to have evolved in a common ancestor of the Bos and Bison genera 5-10 million years ago.

Unsuccessful Chemical Immobilization of a Roan Antelope (Hippotragus equinus) Using a Premixed Medetomidine-Ketamine Combination.

We unsuccessfully attempted to safely chemically immobilize a roan antelope (Hippotragus equinus) with a premixed combination of medetomidine (5 mg/mL) and ketamine (150 mg/mL) for injury treatment. This dose (0.066 mg/kg medetomidine and 1.96 mg/kg ketamine) produced poor quality of immobilization, probably exacerbated by stimulation before completing induction.

Genomic evaluation of hybridization in historic and modern North American Bison (Bison bison).

During the late nineteenth century North American bison underwent a significant population bottleneck resulting in a reduction in population size of over 99% and a species-level near-extinction event. Factors responsible for this destruction included indiscriminate killing, loss of access to suitable habitat, and diseases. At the nadir of this population crash, very few wild plains bison survived and were restricted to Yellowstone National Park, USA and a small number of wild wood bison remained in Wood Buffalo National Park, Canada. However, most surviving bison in the late 1800's were maintained by cattle ranchers in private herds where hybridization between bison with various breeds of domestic cattle was often encouraged. Over the last 20 years, the legacy of this introgression has been identified using mitochondrial DNA and limited nuclear microsatellite analyses. However, no genome-wide assessment has been performed, and some herds were believed to be free of introgression based on current genetic testing strategies. Herein, we report detailed analyses using whole genome sequencing from nineteen modern and six historical bison, chosen to represent the major lineages of bison, to identify and quantitate signatures of nuclear introgression in their recent (within 200 years) history. Both low and high coverage genomes provided evidence for recent introgression, including animals from Yellowstone, Wind Cave, and Elk Island National Parks which were previously thought to be free from hybridization with domestic cattle. We employed multiple approaches, including one developed for this work, to identify putative cattle haplotypes in each bison genome. These regions vary greatly in size and frequency by sample and herd, though we detected domestic cattle introgression in all bison genomes tested. Since our sampling strategy spanned across the diversity of modern bison populations, these finding are best explained by multiple historical hybridization events between these two species with significant genetic recombination over the last 200 years. Our results demonstrate that whole genome sequencing approaches are required to accurately quantitate cattle introgression in bison.

Spatiotemporal Genetic Diversity of Lions Reveals the Influence of Habitat Fragmentation across Africa.

Direct comparisons between historical and contemporary populations allow for detecting changes in genetic diversity through time and assessment of the impact of habitat fragmentation. Here, we determined the genetic architecture of both historical and modern lions to document changes in genetic diversity over the last century. We surveyed microsatellite and mitochondrial genome variation from 143 high-quality museum specimens of known provenance, allowing us to directly compare this information with data from several recently published nuclear and mitochondrial studies. Our results provide evidence for male-mediated gene flow and recent isolation of local subpopulations, likely due to habitat fragmentation. Nuclear markers showed a significant decrease in genetic diversity from the historical (HE = 0.833) to the modern (HE = 0.796) populations, whereas mitochondrial genetic diversity was maintained (Hd = 0.98 for both). Although the historical population appears to have been panmictic based on nDNA data, hierarchical structure analysis identified four tiers of genetic structure in modern populations and was able to detect most sampling locations. Mitogenome analyses identified four clusters: Southern, Mixed, Eastern, and Western and were consistent between modern and historically sampled haplotypes. Within the last century, habitat fragmentation caused lion subpopulations to become more geographically isolated as human expansion changed the African landscape. This resulted in an increase in fine-scale nuclear genetic structure and loss of genetic diversity as lion subpopulations became more differentiated, whereas mitochondrial structure and diversity were maintained over time.

Genetic analysis of African lions (Panthera leo) in Zambia support movement across anthropogenic and geographical barriers.

The Luangwa Valley in eastern Zambia is a transverse offshoot of the Great Rift Valley system. This region appears to have an isolating effect as evidenced by suspected endemic subspecies, such as the Cookson's wildebeest and Thornicroft's giraffe. Recent mitochondrial DNA studies demonstrated that African lions in Zambia consist of two highly diverse eastern and western sub-populations. Herein, we report nuclear and mitochondrial DNA results from 409 lions that support this population substructure across Zambia but proposes only partial isolation of the Luangwa Valley with more movement between the populations than previously thought. Population assignment analysis identifies two populations with little evidence of admixture assigning lions to either the eastern or western sub-populations. A high occurrence of private alleles and clear evidence for a Wahlund effect further justify the presence of a highly structured population. But, while mitochondrial DNA analysis still shows little to no matrilineal gene flow (FST = 0.53) between sub-populations, microsatellite analysis suggests there is gene flow (FST = 0.04) with low but significant isolation-by-distance and an average of 6 migrants per generation. Evidence of isolation-by-distance is also found in factorial correspondence analysis with the Lower Zambezi National Park and eastern corridor clusters overlapping isolated clusters of the Luangwa Valley and western sub-population. From this evidence, the Luangwa Valley appears separated from the western sub-population with some dispersal through the southern regions of the eastern sub-population. Both the eastern and western sub-populations have high heterozygosity (0.68 and 0.69, respectively) and genetic diversity (0.47 and 0.50, respectively) values, indicative of genetically healthy populations.

Evaluation of fecal samples as a valid source of DNA by comparing paired blood and fecal samples from American bison (Bison bison).

BACKGROUND: The collection and analysis of fecal DNA is a common practice, especially when dealing with wildlife species that are difficult to track or capture. While fecal DNA is known to be lower quality than traditional sources of DNA, such as blood or other tissues, few investigations have verified fecal samples as a valid source of DNA by directly comparing the results to high quality DNA samples from the same individuals. Our goal was to compare DNA from fecal and blood samples from the same 50 American plains bison (Bison bison) from Yellowstone National Park, analyze 35 short tandem repeat (STR) loci for genotyping efficiency, and compare heterozygosity estimates. RESULTS: We discovered that some of the fecal-derived genotypes obtained were significantly different from the blood-derived genotypes from the same bison. We also found that fecal-derived DNA samples often underestimated heterozygosity values, in some cases by over 20%. CONCLUSIONS: These findings highlight a potential shortcoming inherent in previous wildlife studies that relied solely on a multi-tube approach, using exclusively low quality fecal DNA samples with no quality control to account for false alleles and allelic dropout. Herein, we present a rigorous marker selection protocol that is applicable for a wide range of species and report a set of 15 STR markers for use in future bison studies that yielded consistent results from both fecal and blood-derived DNA.

Mitochondrial Genome Analysis Reveals Historical Lineages in Yellowstone Bison.

Yellowstone National Park is home to one of the only plains bison populations that have continuously existed on their present landscape since prehistoric times without evidence of domestic cattle introgression. Previous studies characterized the relatively high levels of nuclear genetic diversity in these bison, but little is known about their mitochondrial haplotype diversity. This study assessed mitochondrial genomes from 25 randomly selected Yellowstone bison and found 10 different mitochondrial haplotypes with a haplotype diversity of 0.78 (± 0.06). Spatial analysis of these mitochondrial DNA (mtDNA) haplotypes did not detect geographic population subdivision (FST = -0.06, p = 0.76). However, we identified two independent and historically important lineages in Yellowstone bison by combining data from 65 bison (defined by 120 polymorphic sites) from across North America representing a total of 30 different mitochondrial DNA haplotypes. Mitochondrial DNA haplotypes from one of the Yellowstone lineages represent descendants of the 22 indigenous bison remaining in central Yellowstone in 1902. The other mitochondrial DNA lineage represents descendants of the 18 females introduced from northern Montana in 1902 to supplement the indigenous bison population and develop a new breeding herd in the northern region of the park. Comparing modern and historical mitochondrial DNA diversity in Yellowstone bison helps uncover a historical context of park restoration efforts during the early 1900s, provides evidence against a hypothesized mitochondrial disease in bison, and reveals the signature of recent hybridization between American plains bison (Bison bison bison) and Canadian wood bison (B. b. athabascae). Our study demonstrates how mitochondrial DNA can be applied to delineate the history of wildlife species and inform future conservation actions.

Mitochondrial Haplotype Diversity in Zambian Lions: Bridging a Gap in the Biogeography of an Iconic Species.

Analysis of DNA sequence diversity at the 12S to 16S mitochondrial genes of 165 African lions (Panthera leo) from five main areas in Zambia has uncovered haplotypes which link Southern Africa with East Africa. Phylogenetic analysis suggests Zambia may serve as a bridge connecting the lion populations in southern Africa to eastern Africa, supporting earlier hypotheses that eastern-southern Africa may represent the evolutionary cradle for the species. Overall gene diversity throughout the Zambian lion population was 0.7319 +/- 0.0174 with eight haplotypes found; three haplotypes previously described and the remaining five novel. The addition of these five novel haplotypes, so far only found within Zambia, nearly doubles the number of haplotypes previously reported for any given geographic location of wild lions. However, based on an AMOVA analysis of these haplotypes, there is little to no matrilineal gene flow (Fst = 0.47) when the eastern and western regions of Zambia are considered as two regional sub-populations. Crossover haplotypes (H9, H11, and Z1) appear in both populations as rare in one but common in the other. This pattern is a possible result of the lion mating system in which predominately males disperse, as all individuals with crossover haplotypes were male. The determination and characterization of lion sub-populations, such as done in this study for Zambia, represent a higher-resolution of knowledge regarding both the genetic health and connectivity of lion populations, which can serve to inform conservation and management of this iconic species.

Genetic variation and differentiation of bison (Bison bison) subspecies and cattle (Bos taurus) breeds and subspecies.

The genetic relationship of American plains bison (Bison bison bison) and wood bison (Bison bison athabascae) was quantified and compared with that among breeds and subspecies of cattle. Plains bison from 9 herds (N = 136), wood bison from 3 herds (N = 65), taurine cattle (Bos taurus taurus) from 14 breeds (N = 244), and indicine cattle (Bos taurus indicus) from 2 breeds (N = 53) were genotyped for 29 polymorphic microsatellite loci. Bayesian cluster analyses indicate 3 groups, 2 of which are plains bison and 1 of which is wood bison with some admixture, and genetic distances do not show plains bison and wood bison as distinct groups. Differentiation of wood bison and plains bison is also significantly less than that of cattle breeds and subspecies. These and other genetic data and historical interbreeding of bison do not support recognition of extant plains bison and wood bison as phylogenetically distinct subspecies.

Phenotypic effects of cattle mitochondrial DNA in American bison.

Hybridization between endangered species and more common species is a significant problem in conservation biology because it may result in extinction or loss of adaptation. The historical reduction in abundance and geographic distribution of the American plains bison (Bison bison bison) and their recovery over the last 125 years is well documented. However, introgression from domestic cattle (Bos taurus) into the few remaining bison populations that existed in the late 1800s has now been identified in many modern bison herds. We examined the phenotypic effect of this ancestry by comparing weight and height of bison with cattle or bison mitochondrial DNA (mtDNA) from Santa Catalina Island, California (U.S.A.), a nutritionally stressful environment for bison, and of a group of age-matched feedlot bison males in Montana, a nutritionally rich environment. The environmental and nutritional differences between these 2 bison populations were very different and demonstrated the phenotypic effect of domestic cattle mtDNA in bison over a broad range of conditions. For example, the average weight of feedlot males that were 2 years of age was 2.54 times greater than that of males from Santa Catalina Island. In both environments, bison with cattle mtDNA had lower weight compared with bison with bison mtDNA, and on Santa Catalina Island, the height of bison with cattle mtDNA was lower than the height of bison with bison mtDNA. These data support the hypothesis that body size is smaller and height is lower in bison with domestic cattle mtDNA and that genomic integrity is important for the conservation of the American plains bison.

Genetic population substructure in bison at Yellowstone National Park.

The Yellowstone National Park bison herd is 1 of only 2 populations known to have continually persisted on their current landscape since pre-Columbian times. Over the last century, the census size of this herd has fluctuated from around 100 individuals to over 3000 animals. Previous studies involving radiotelemetry, tooth wear, and parturition timing provide evidence of at least 2 distinct groups of bison within Yellowstone National Park. To better understand the biology of Yellowstone bison, we investigated the potential for limited gene flow across this population using multilocus Bayesian clustering analysis. Two genetically distinct and clearly defined subpopulations were identified based on both genotypic diversity and allelic distributions. Genetic cluster assignments were highly correlated with sampling locations for a subgroup of live capture individuals. Furthermore, a comparison of the cluster assignments to the 2 principle winter cull sites revealed critical differences in migration patterns across years. The 2 Yellowstone subpopulations display levels of differentiation that are only slightly less than that between populations which have been geographically and reproductively isolated for over 40 years. The identification of cryptic population subdivision and genetic differentiation of this magnitude highlights the importance of this biological phenomenon in the management of wildlife species.

Genome-wide association study implicates testis-sperm specific FKBP6 as a susceptibility locus for impaired acrosome reaction in stallions.

Impaired acrosomal reaction (IAR) of sperm causes male subfertility in humans and animals. Despite compelling evidence about the genetic control over acrosome biogenesis and function, the genomics of IAR is as yet poorly understood, providing no molecular tools for diagnostics. Here we conducted Equine SNP50 Beadchip genotyping and GWAS using 7 IAR-affected and 37 control Thoroughbred stallions. A significant (P<6.75E-08) genotype-phenotype association was found in horse chromosome 13 in FK506 binding protein 6 (FKBP6). The gene belongs to the immunophilins FKBP family known to be involved in meiosis, calcium homeostasis, clathrin-coated vesicles, and membrane fusions. Direct sequencing of FKBP6 exons in cases and controls identified SNPs g.11040315G>A and g.11040379C>A (p.166H>N) in exon 4 that were significantly associated with the IAR phenotype both in the GWAS cohort (n = 44) and in a large multi-breed cohort of 265 horses. All IAR stallions were homozygous for the A-alleles, while this genotype was found only in 2% of controls. The equine FKBP6 was exclusively expressed in testis and sperm and had 5 different transcripts, of which 4 were novel. The expression of this gene in AC/AG heterozygous controls was monoallelic, and we observed a tendency for FKBP6 up-regulation in IAR stallions compared to controls. Because exon 4 SNPs had no effect on the protein structure, it is likely that FKBP6 relates to the IAR phenotype via regulatory or modifying functions. In conclusion, FKBP6 was considered a susceptibility gene of incomplete penetrance for IAR in stallions and a candidate gene for male subfertility in mammals. FKBP6 genotyping is recommended for the detection of IAR-susceptible individuals among potential breeding stallions. Successful use of sperm as a source of DNA and RNA propagates non-invasive sample procurement for fertility genomics in animals and humans.

Complete mitochondrial DNA sequence analysis of Bison bison and bison-cattle hybrids: function and phylogeny.

Complete mitochondrial DNA (mtDNA) genomes from 43 bison and bison-cattle hybrids were sequenced and compared with other bovids. Selected animals reflect the historical range and current taxonomic structure of bison. This study identified regions of potential nuclear-mitochondrial incompatibilities in hybrids, provided a complete mtDNA phylogenetic tree for this species, and uncovered evidence of bison population substructure. Seventeen bison haplotypes defined by 66 polymorphic sites were discovered, whereas 728 fixed differences and 86 non-synonymous mutations were identified between bison and bison-cattle hybrid sequences. The potential roles of the mtDNA genome in the function of hybrid animals and bison taxonomy are discussed.

Patterns of genetic variation in US federal bison herds.

Like many wide-ranging mammals, American bison (Bison bison) have experienced significant range contraction over the past two centuries and are maintained in artificially isolated populations. A basic understanding of the distribution of genetic variation among populations is necessary to facilitate long-term germplasm preservation and species conservation. The 11 herds maintained within the US federal system are a critically important source of germplasm for bison conservation, as they include many of the oldest herds in the USA and have served as a primary resource for the establishment of private and public herds worldwide. In this study, we used a panel of 51 nuclear markers to investigate patterns of neutral genetic variation among these herds. Most of these herds have maintained remarkably high levels of variation despite the severe bottleneck suffered in the late 1800s. However, differences were noted in the patterns of variation and levels of differentiation among herds, which were compared with historical records of establishment, supplementation, herd size, and culling practices. Although some lineages have been replicated across multiple herds within the US federal system, other lineages with high levels of genetic variation exist in isolated herds and should be considered targets for the establishment of satellite herds. From this and other studies, it is clear that the genetic variation represented in the US federal system is unevenly distributed among National Park Service and Fish and Wildlife Service herds, and that these resources must be carefully managed to ensure long-term species conservation.

Assessing familial aggregation of paratuberculosis in beef cattle of unknown pedigree.

The objective of this study was to assess genetic similarity of beef cattle using microsatellite markers and to use this information to describe familial aggregation of paratuberculosis test results in Texas beef cattle. Paratuberculosis testing was performed on 2622 adult beef cattle using two commercially available serum ELISAs and radiometric fecal culture. Pedigree records were collected for registered purebred herds and herds with sufficiently detailed production records to identify parent-offspring pairs. Cases were defined as cattle with at least one positive paratuberculosis test result. Three controls were matched by herd of residence for each case. All parent-offspring pairs, cases, and controls were genotyped for 12 microsatellites. Bayesian analysis of allele frequency data was used to describe population substructure and assign individual cattle into groups of genetically similar cattle. The proportion of known parent-offspring pairs assigned to the same cluster was used to assess the validity of the approach to identify familial structure. Conditional logistic regression was used to describe the association between cluster assignment and paratuberculosis test-status matched by herd. Nine clusters of genetically similar individuals were identified and were supported by the proportion of parent-offspring pairs assigned to the same clusters. Increased odds of having at least one positive paratuberculosis test result were identified for two clusters compared to the cluster with the lowest proportion of positive paratuberculosis test results after conditioning on herd. The results of this study demonstrate that population substructure can be used to describe familial aggregation of paratuberculosis test results in beef cattle of unknown pedigree.

Familial associations with paratuberculosis ELISA results in Texas Longhorn cattle.

The objective of this cross-sectional study was to estimate familial associations with paratuberculosis ELISA status in beef cattle. Texas Longhorn cattle (n=715) greater than 2years of age were sampled for paratuberculosis testing using ELISA and fecal culture. Diagnostic test results were indicative of substantial numbers of false-positive serological reactions consistent with environmental exposure to non-MAP Mycobacterium spp. Associations between ancestors and paratuberculosis ELISA status of offspring were assessed using conditional logistic regression. The association between ELISA status of the dam and her offspring was assessed using linear mixed-effect models. Significant associations were identified between some ancestors and offspring ELISA status. The odds of being classified as "suspect" or greater based on ELISA results were 4.6 times greater for offspring of dams with similarly increased S:P ratios. A significant positive linear association was also observed between dam and offspring log-transformed S:P ratios. Results indicate that there is familial aggregation of paratuberculosis ELISA results in beef cattle and suggest that genetic selection based on paratuberculosis ELISA status may decrease seroprevalence. However, genetic selection may have minimal effect on paratuberculosis control in herds with exposure to non-MAP Mycobacterium spp.

Molecular characterization of the Rocky Mountain elk (Cervus elaphus nelsoni) PRNP putative promoter.

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSE) affecting deer (Odocoileus spp.), moose (Alces alces), and Rocky Mountain elk (Cervus elaphus nelsoni). Leucine homozygosity at elk PRNP codon 132 has been associated with reduced CWD susceptibility. However, naturally acquired CWD has been detected in elk possessing the 132 Leu/Leu genotype. Recent human and bovine studies indicate that PRNP regulatory polymorphisms may also influence TSE occurrence. Therefore, we generated sequences for the elk PRNP putative promoter (2.2 kb), exon 1 (predicted; 54 bp), intron 1 (predicted; 193 bp), and exon 3 (771 bp). Promoter prediction analysis using CpGProD yielded a single elk PRNP promoter that was homologous to regions of known promoter activity in cow and sheep. Molecular interrogation of the elk PRNP putative promoter revealed 32 diallelic single-nucleotide polymorphisms (SNPs). No variation was detected within the predicted exon 1 or intron 1 sequences. Evaluation of elk PRNP exon 3 revealed 3 SNPs (63Y, 312R, 394W-->Met/Leu). Bayesian haplotype reconstruction resulted in 3 elk PRNP haplotypes, with complete linkage disequilibrium observed between all PRNP putative promoter SNPs and codon 132. The results of this study provide the initial genomic foundation for future comparative and haplotype-based elk PRNP studies.

A comprehensive evaluation of cattle introgression into US federal bison herds.

Genetic introgression, especially from interspecies hybridization, is a significant threat to species conservation worldwide. In this study, 11 US federal bison populations were comprehensively examined for evidence of both mitochondrial and nuclear domestic cattle (Bos taurus) introgression. Mitochondrial introgression was examined using established polymerase chain reaction methods and confirmed through analysis of D-loop sequences. Nuclear introgression was assessed in 14 chromosomal regions through examination of microsatellite electromorph and sequence differences between bison and domestic cattle. Only one population was identified with domestic cattle mitochondrial DNA introgression. In contrast, evidence of nuclear introgression was found in 7 (63.6%) of the examined populations. Historic accounts of bison transfers among populations were corroborated with evidence of introgressed DNA transmission. While neither nuclear nor mitochondrial domestic cattle introgression was detected in bison from Grand Teton National Park, Sully's Hill National Game Preserve, Wind Cave National Park, or Yellowstone National Park, adequate sample sizes were available only from the last 2 populations to allow for statistical confidence (>90%) in nuclear introgression detection limits. The identification of genetically unique and undisturbed populations is critical to species conservation efforts, and this study serves as a model for the genetic evaluation of interspecies introgression.