Most Beefalo cattle have no detectable bison genetic ancestry

  1. Beth Shapiro
  2. Jonas Oppenheimer
  3. Michael P Heaton
  4. Kristen L Kuhn
  5. Richard E Green
  6. Harvey D Blackburn
  7. Timothy PL Smith

  • Curated by eLife

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    eLife Assessment

    This important study used whole-genome data to investigate Beefalo ancestry for the first time, providing insight into the genetics of Beefalo cattle and challenging the long-held claim of 37.5% bison ancestry reported by the American Beefalo Association. Despite some limitations regarding sequencing depth and sampling, the expert use of a comprehensive set of population-genomic methods allowed the authors to demonstrate convincingly that Beefalo and bison hybrid ancestry profiles are consistent with repeated backcrossing to either parental species. The work will be of significant interest to evolutionary biologists, population geneticists, animal breeders, and those involved in the conservation genetics of bovine species.

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Abstract

Hybridization is common among lineages in the genus Bos, often mediated through human management for the selection of adaptive or desirable traits. A recent example is the American Beefalo cattle breed, which was developed in the 1970s and defined as a hybrid between American bison (Bison bison) and cattle (Bos taurus). The American Beefalo Association (ABA) typically require ⅜ bison ancestry to qualify as Beefalo. Here, we sought to characterize admixed ancestry among Beefalo as a component of a larger project to understand the role of hybridization in shaping present-day diversity in bison and cattle. We generated genomic data from 50 historical and present-day Beefalo and bison hybrids, including several important founding animals, as well as from 10 bison originating from commercial herds that represent potential sources of bison ancestry in Beefalo. We found that most Beefalo did not contain detectable bison ancestry. No individual Beefalo within our data set satisfies the ancestry requirements specified by the ABA, although several Beefalo had smaller proportions of bison ancestry (2-18%). Some beefalo had detectable zebu cattle ancestry (2-38%), suggesting that hybridization of taurine and zebu cattle may contribute to morphological similarity between some Beefalo and bison. Overall, ancestry profiles of Beefalo and bison hybrid genomes are consistent with repeated backcrossing to either parental species rather than the breeding between hybrids themselves, implying significant barriers to gene flow between bison and cattle. Our results call into question the ⅜ bison ancestry targeted by the breed association and demonstrate the value of genomic information in examining claims of interspecies gene flow among Bos species.

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  1. Version published to 10.7554/elife.102750.2 on eLife
  2. Version published to 10.7554/elife.102750 on eLife
  3. eLife

    eLife Assessment

    This important study used whole-genome data to investigate Beefalo ancestry for the first time, providing insight into the genetics of Beefalo cattle and challenging the long-held claim of 37.5% bison ancestry reported by the American Beefalo Association. Despite some limitations regarding sequencing depth and sampling, the expert use of a comprehensive set of population-genomic methods allowed the authors to demonstrate convincingly that Beefalo and bison hybrid ancestry profiles are consistent with repeated backcrossing to either parental species. The work will be of significant interest to evolutionary biologists, population geneticists, animal breeders, and those involved in the conservation genetics of bovine species.

  4. eLife

    Reviewer #1 (Public review):

    Summary:

    This study used whole genome data to investigate Beefalo ancestry for the first time, filling the gap in the field of Beefalo ancestry. The authors used preserved semen samples to generate genomic data on 47 registered Beefalo and 3 bison hybrids, further questioning the ABA's stated goal of ⅜ bison ancestry. In addition, the authors also show that ancestry profiles of Beefalo and bison hybrid genomes are consistent with repeated backcrossing to either parental species, demonstrate the value of genomic information in examining gene flow between species in the genus Bison. Overall, these data thus demonstrate the utility of genomic information in validating specific breeding claims for a more complete understanding of gene flow and genetic variation among bovine species. This is an interesting study, but there are still some major weaknesses that exist.

    Strengths:

    Numerous genetic analysis methods such as PCA, ADMIXTURE, F4 ratios, and local ancestry inference techniques revealed that no single Beefalo set meets the ancestry requirements set by the American Beefalo Association (ABA) and some beefalo had detectable indicine cattle ancestry.

    Comments on revised version:

    The authors have made further revisions in the revised manuscript, and these revisions have undoubtedly helped improve the article. No further comments.

  5. eLife

    Reviewer #2 (Public review):

    Summary:

    Shapiro et al. set out to verify the American Beefalo Association's claim that Beefalo cattle possess 37.5% bison ancestry. They employ a comprehensive range of well-established population genomics methods to estimate ancestry in these hybrid populations, including PCA, ADMIXTURE, D and F statistics, and local ancestry inference. Their findings conclusively demonstrate that most Beefalo lack the claimed bison ancestry, with only 8 out of 47 samples showing any detectable bison ancestry, ranging from 2-18%.

    Strengths:

    The primary strength of this analysis lies in the comprehensive dataset available to the authors, which includes important foundational Beefalo individuals and various reference populations. The rigorous and multi-faceted methodological approach employs several well-established techniques in population genomics for detecting and measuring admixture. Each method used has a firm basis in the field, providing consistent and robust results. The authors' approach of using PCA to initially assess the data within a global context, followed by more specific analyses using ADMIXTURE and D-statistics, provides a clear and logical progression of evidence. The presentation of these results in figures is particularly effective, clearly illustrating the key findings of the study. Additionally, the examination of both autosomal and sex chromosome ancestry offers a more complete understanding of Beefalo genetic composition and the mechanics of bison-cattle hybridisation.

    Weaknesses:

    One limitation of this analysis is the relatively low coverage (~2x) of many Beefalo samples. However, the authors have taken steps to mitigate biases that may arise from this, and their downsampling experiment demonstrates that this level of coverage is appropriate for summarising species-level ancestry across Bos. Another potential weakness is the limited sampling of contemporary Beefalo populations, as the study focuses primarily on historical samples. The authors have justified this choice on the grounds that contemporary Beefalo breeding involves no further bison input, so founder-era individuals are the most informative samples for addressing the study's central question.

    Appraisal:

    The authors have clearly achieved their primary aim using a rigorous and comprehensive methodology. Their extensive dataset and multi-faceted analytical approach provide strong support for their conclusions. The study not only addresses its main research question but also reveals unexpected insights into Beefalo genetics, particularly the presence of zebu ancestry, predominantly from Brahman cattle.

    Discussion:

    This study is valuable for several reasons beyond its primary findings. First, it definitively addresses and refutes the claim of 37.5% bison ancestry in Beefalo, providing crucial information for those studying these interspecies hybrids and the viability of their offspring. Second, it reveals the unexpected presence of zebu ancestry, predominantly from Brahman cattle, in many Beefalo, raising intriguing questions about the breed's development and the potential role of zebu cattle in achieving desired traits. This finding suggests that the distinctive appearance of Beefalo may be due in part to zebu admixture rather than bison ancestry. Third, the study highlights the significant barriers to admixture between bison and cattle, both in controlled breeding programs and potentially in wild populations. This has important implications for conservation genetics and our understanding of gene flow between these species. Lastly, the study demonstrates the power of genomic analysis in verifying breed claims and understanding the complex history of domestic animal breeds. These findings open new avenues for research in bovine genomics, breed development, and the dynamics of interspecies hybridisation.

    Comments on revised version:

    Thanks for the responses, which address my comments in full. I have no further concerns.

  6. eLife

    Reviewer #3 (Public review):

    Summary:

    The American beefalo cattle breed was developed as a mixture of 5/8 domestic cattle and 3/8 (or 37.5%) bison ancestry. The authors sequenced 50 genomes from bison and hybrids (historical and present-day). They found that most animals did not carry any detectable bison ancestry, with only a few between 2-18%, while other beefalo had taurine/zebu cattle ancestry, which may explain morphological traits. Breeding design was likely each time to a parental instead of to other admixtures.

    The authors utilize whole genome sequence data to explore the ancestry of beefalo with respect to expected and possible contributions from cattle lineages. Using molecular and analytical methods central to questions exploring genomic ancestry and identity, the authors very nicely show evidence that calls into question ability of ancestry to be deduced from breed club documentation without considering reproductive challenges that are known in hybridization between cattle lineages.

    Comments on revised version:

    The authors have addressed all my comments to help improve presentation of specific details, results, and readability. Thank you!

  7. eLife

    Author response:

    The following is the authors’ response to the original reviews.

    Public Reviews:

    Reviewer #1 (Public review):

    Summary:

    This study used whole genome data to investigate Beefalo ancestry for the first time, filling the gap in the field of Beefalo ancestry. The authors used preserved semen samples to generate genomic data on 47 registered Beefalo and 3 bison hybrids, further questioning the ABA's stated goal of ⅜ bison ancestry. In addition, the authors also show that ancestry profiles of Beefalo and bison hybrid genomes are consistent with repeated backcrossing to either parental species, demonstrating the value of genomic information in examining gene flow between species in the genus Bison. This is an interesting study that still has some major weaknesses that exist, but overall, the work demonstrates the utility of genomic information in validating specific breeding claims for a more complete understanding of gene flow and genetic variation among bovine species.

    We thank the reviewer for their thoughtful assessment of our work.

    Strengths:

    Numerous genetic analysis methods such as PCA, ADMIXTURE, F4 ratios, and local ancestry inference techniques revealed that no single Beefalo set meets the ancestry requirements set by the American Beefalo Association (ABA) and some beefalo had detectable indicine cattle ancestry.

    Weaknesses:

    While this study contributes to our knowledge of Beefalo ancestry, there are some key issues that need to be addressed in terms of analysing the specific results as well as writing the article.

    We have followed the reviewer’s suggestions for improving our study in detail (specified below), and appreciate their close reading of the manuscript.

    Reviewer #2 (Public review):

    Summary:

    Shapiro et al. set out to verify the American Beefalo Association's claim that Beefalo cattle possess 37.5% bison ancestry. They employ a comprehensive range of well-established population genomics methods to estimate ancestry in these hybrid populations, including PCA, ADMIXTURE, D and F statistics, and local ancestry inference. Their findings conclusively demonstrate that most Beefalo lack the claimed bison ancestry, with only 8 out of 47 samples showing any detectable bison ancestry, ranging from 2 - 18%.

    We thank the reviewer for their thoughtful assessment of our work.

    Strengths:

    The primary strength of this analysis lies in the comprehensive dataset available to the authors, which includes important foundational Beefalo individuals and various reference populations. The rigorous and multi-faceted methodological approach employs several well-established techniques in population genomics for detecting and measuring admixture. Each method used has a firm basis in the field, providing consistent and robust results. The authors' approach of using PCA to initially assess the data within a global context, followed by more specific analyses using ADMIXTURE and D-statistics, provides a clear and logical progression of evidence. The presentation of these results in figures is particularly effective, clearly illustrating the key findings of the study. Additionally, the examination of both autosomal and sex chromosome ancestry offers a more complete understanding of Beefalo genetic composition and the mechanics of bison-cattle hybridisation.

    Weaknesses:

    One limitation of this analysis is the relatively low coverage (~2x) of many Beefalo samples. However, the authors have taken steps to mitigate biases that may arise from this. Another weakness is the limited sampling of contemporary Beefalo populations, as the study focuses primarily on historical samples. This may limit our understanding of how Beefalo genetics may have changed over time.

    The reviewer is correct that the low coverage obtained for many Beefalo is one potential limitation, although we believe that the downsampling experiment we performed (Fig. S4) shows that this level of coverage is appropriate for summarizing species-level ancestry across Bos, as the reviewer notes.

    Sampling contemporary Beefalo individuals would be valuable, though as the focus of our study was to understand the origins of bison ancestry in Beefalo, we prioritized sampling individuals which played an important role in establishing the breed. We also note that contemporary Beefalo breeding involves crossing between Beefalo individuals or backcrossing to cattle, with no additional bison ancestry input since the formation of the Beefalo. As such, sampling individuals that existed close to the breed’s founding should provide the most insight into bison ancestry in Beefalo.

    Appraisal:

    The authors have clearly achieved their primary aim using a rigorous and comprehensive methodology. Their extensive dataset and multi-faceted analytical approach provide strong support for their conclusions. The study not only addresses its main research question but also reveals unexpected insights into Beefalo genetics, particularly the presence of zebu ancestry.

    Discussion:

    This study is valuable for several reasons beyond its primary findings. First, it definitively addresses and refutes the claim of 37.5% bison ancestry in Beefalo, providing crucial information for those studying these interspecies hybrids and the viability of their offspring. Second, it reveals the unexpected presence of zebu ancestry in many Beefalo, raising intriguing questions about the breed's development and the potential role of zebu cattle in achieving desired traits. This finding suggests that the distinctive appearance of Beefalo may be due in part to zebu admixture rather than bison ancestry. Third, the study highlights the significant barriers to admixture between bison and cattle, both in controlled breeding programs and potentially in wild populations. This has important implications for conservation genetics and our understanding of gene flow between these species. Lastly, the study demonstrates the power of genomic analysis in verifying breed claims and understanding the complex history of domestic animal breeds. These findings open new avenues for research in bovine genomics, breed development, and the dynamics of interspecies hybridisation.

    Reviewer #3 (Public review):

    Summary:

    I really like this topic and study. But I think much can be more focused and tightened up. All the components are here - just some more refining to really make the storyline clear, the journey of discovery, and the impact of such knowledge.

    We thank the reviewer for their thoughtful assessment of our work.

    Strengths:

    The authors dive directly into the question of genomic ancestry as compared to the breed club's reported ancestry with heavy, quantitative data and critical analytical methods. The questioning line is direct and does not meander. The reader learns about the challenges of breeding associations, and values of understood ancestry, and presents a clear need of re-evaluating the breed standards and expectations of beefalo (if ancestry is indeed the primary goal instead of a phenotype-driven breed mission).

    Weaknesses:

    Much of the quantitative results are only referred to in the main text with qualitative language. Please incorporate more written quantitative results to highlight evidence that underlines the study narrative because it is quite an interesting study!

    The reviewer highlights an important point, and we agree that the qualitative language used to describe the results was generally lacking. We have now described the results quantitatively throughout the manuscript where possible.

    Recommendations for the authors:

    Reviewer #1 (Recommendations for the authors):

    (1) This study is not the first to question claims surrounding bison ancestry in the breed and is the sample size too small to be representative of the entire genetic structure of Beefalo?

    The reviewer correctly points out that this study is not the first to address uncertainty in the amount of bison ancestry present across beefalo. All earlier studies, to our knowledge, have been highlighted in the introduction and discussion (Lenoir and Lichtenberger, 1978 and Stormont et al, 1986). However, these studies examined a narrow range of Beefalo sources and used older methods (karyotyping and blood typing), such that comprehensive statements about the proportion of bison ancestry in Beefalo could not be made.

    We also agree that an appropriate sampling scheme is crucial for making definitive statements about Beefalo ancestry across the breed. As Beefalo breeding typically involves breeding select “full-blood” individuals with cattle, the ancestry across contemporary Beefalo is likely complex, with the cattle component coming from a wide range of breeds. Therefore, our sampling emphasized “full-blood” representatives, especially those that were involved in the founding of the breed and from which later Beefalo descend. This involved an exhaustive survey of the Beefalo individuals contained within the USDA’s National Animal Germplasm Program. Although we did not extensively evaluate current Beefalo diversity, we believe this approach is most suited for characterizing bison ancestry within Beefalo, as bison ancestry is maintained primarily through the continued use of genetic material from these “full-blood” individuals rather than repeated hybridization between bison and cattle.

    (2) Although genomic information is important for breeding research, this requires quality of data. The coverage of the data used in this study was mainly ~2X, and although multiple methods of analysis gave similar results, the ability to identify rare variants (e.g. insertions or deletions of long segments of the genome) may be limited at low coverage, affecting the confidence of the results.

    This is an important consideration, and we agree with the reviewer that the sequencing depth obtained for most individuals in our study precludes accurate genotype calling. Therefore, we did not attempt to perform traditional genotype calling. Rather, we used a pseudohaploid calling approach in which a random base was selected to represent the genotype at each position for each individual, using a pre-ascertained set of variants discovered in gaur, a closely related outgroup to bison and cattle. This pseudohaploid approach is common in other situations where coverage is low, for example in analyzing ancient DNA.

    Furthermore, our ancestry analyses focused on biallelic SNPs which were discovered in gaur and we did not attempt to call structural variants, given the limitations in coverage. As this outgroup ascertainment approach seeks to target SNPs which were polymorphic in the ancestor of both bison and cattle, which should yield unbiased results in population genetic analyses, we were less interested in discovering rare variation within the species and populations we examined here.

    Finally, we performed downsampling experiments comparing low coverage read data to genotypes called from high coverage data, and obtained consistent results between low and high coverage analyses using read-level data and called genotypes (Fig. S7).

    (3) Missing from the conclusions is the very important presentation of the results of genomic calling, the basics of what these data look like, coverage histograms, number of SNPs, categorization, annotations, and so on. These are necessary prerequisites for subsequent population analysis.

    The reference to “5.29M” on page 14 has been replaced with the exact number of SNPs used in analyses (5,291,534). The average sequencing depth for each sample is also included in Table S1.

    (4) The manuscript mentions "most" in a number of places, but can the authors give an accurate number based on the current data? "Most" is not a rigorous description. Based on the simulations of genomic data, how many Beefalo cattle were not detected as hybridized? This may be related to both sample size and where the authors sampled.

    We thank the reviewer for this important suggestion. We have now replaced vague summaries of results with precise numbers. However, we are unsure what “simulations” means in this context, as all results were obtained by analyzing empirical data from Beefalo, bison, cattle, and other bovines, rather than simulations.

    (5) The information in the third and fourth paragraphs of the Introduction is not sufficiently coherent and could be further consolidated into a more logical presentation.

    We have now condensed these paragraphs and edited them for clarity.

    (6) "For some analyses we also incorporated published genomes from outgroups". The description here is unclear as to what criteria were used to select these data, and it is possible that the choice of outgroups could lead to different conclusions from the analyses. In addition, ancient DNA data from cattle may be useful for this study and the authors are encouraged to consider it.

    Outgroup choice can certainly have a large impact on population genetic analyses. For the species examined in our study, we considered other Bos species, including yak, gaur, and banteng, as suitable outgroups, along with water buffalo, which is the closest outgroup outside of Bos. We have added comparisons of D-statistics using yak as an outgroup as a supplementary figure (Fig. S4), in addition to those using water buffalo as the outgroup which were presented in Figure 2.

    As we were examining species-level ancestry, and given the high level of divergence between bison and cattle, relative to that between published ancient and modern cattle genomes, we believed that it was most appropriate to use high quality modern cattle data, rather than poorer quality ancient cattle genomes, for analyses. Additionally, as any hybridization which took place between bison and cattle in the formation of Beefalo would have occurred within the past ~50 years, modern cattle are likely to be the most appropriate proxy for the cattle ancestry in Beefalo, especially given the lack of published historical North American cattle genomes.

    (7) The coordinates of the PCA plot need to be further supported by providing values.

    We have now updated axis labels for the PCA in Fig. 1A to include the proportion of variance explained for the first two components.

    (8) In Figure 1, Beefalo has one individual, NAGP9109, which belongs exclusively to the indicine group. For this individual, wouldn't it be nicer to label it separately in the PCA and ADMIXTURE plots, like Joe's Pride (JP), to make the presentation of the results clearer?

    This individual was one which was determined to be mislabeled as Beefalo within the NAGP and is actually a Brahman cattle. Therefore, we have relabeled it as zebu, rather than Beefalo, throughout the figures.

    (9) As the sex chromosome data do not fully support the authors' claims, some caution may be needed in describing the results.

    We interpret the sex chromosomal results as being fully consistent with patterns seen in the autosomes. However, they do shed some light on the dynamics of bison-cattle hybridization, and suggest male-mediated gene flow in which bison ancestry in Beefalo was introduced primarily through bison bulls.

    (10) Would it be appropriate to analyse the results at K = 3 only? The admixture analysis of all bison, cattle, bison hybrids, and buffalo individuals at different K values should further refine the results.

    We now also show ADMIXTURE results at K=2 and K=4 (Fig. S2) and present the cross-validation results from ADMIXTURE (Fig. S3).

    (11) The conclusions of this article about bison ancestry in Beefalo individuals are completely inconsistent with the American Beefalo Association, and should a description of possible reasons for this discrepancy be added to the discussion?

    Our analyses make it clear that there was much less hybridization between bison and cattle leading to the formation of the Beefalo that was previously believed. As the genetic data does not provide insight into exactly why this might be the case, we can only speculate on the precise reasons bison-cattle hybridization did not take place, which we have avoided here.

    Reviewer #2 (Recommendations for the authors):

    The manuscript is well written, the figures are easily understandable, and the claims made are justified by the results obtained.

    It is need to clarify cattle breeding terminology, particularly concerning breeds like the Brahman. While often described as zebu-taurine hybrids, Brahman cattle typically show over 90% zebu ancestry when analysed using ADMIXTURE against panels including European Bos taurus, African Bos taurus, and Bos indicus animals. This context would help explain why "NAGP9109" clusters with the Zebu group.

    We thank the reviewer for this useful context, and agree that most Brahman cattle have a high proportion of zebu ancestry. In fact, the zebu group we included primarily consists of Brahman individuals, which we have now clarified in the text, which now reads:

    “The reported pedigree in the NAGP for this animal lists its composition as 1/2 Brahman, 1/4 Charolais, 1/8 bison, 1/16th Hereford, and 1/16th Shorthorn, but the American Brahman Breeders Association records this animal (#309519) as purebred Brahman, which is a zebu breed (5 of the other 6 zebu individuals analyzed here are Brahman cattle).”

    I suggest three other improvements:

    (1) Standardise terminology: The manuscript alternates between "zebu" and "indicine" when referring to these cattle. While both terms are correctly defined in the introduction as "indicine (zebu; Bos indicus)" using one term consistently throughout would improve readability. I prefer "zebu" but leave this choice to the authors.

    We agree that this mixed terminology was confusing and have replaced all instances of “indicine” with “zebu.”

    (2) Add PCA metrics, including the percentage of variance explained by each principal component would demonstrate the genetic distinctiveness between bison and cattle, and between Taurus and zebu cattle. This would also support the selection of K=3 for the ADMIXTURE analysis.

    The axis labels for the PCA have been updated to include the proportion of variance explained for each component. We now also show ADMIXTURE results at K=2 and K=4 (Fig. S2) and present the cross-validation results from ADMIXTURE (Fig. S3).

    (3) Improve quantitative precision: The authors could improve precision by replacing qualitative statements with exact counts. For example "39 of 47 Beefalo showed no detectable bison ancestry." The same suggestion applies when describing how many Beefalo had zebu ancestry.

    We thank the reviewer for this useful suggestion, and agree that the manuscript used imprecise language in describing the results of certain analyses. We have now added quantitative detail throughout the Results section.

    Reviewer #3 (Recommendations for the authors):

    (1) Introduction

    The introduction sets a tone that is heavily focused on the genetic revelation that the economics of beefalo are somewhat of a facade. Beefalo are indeed not part-buffalo (bison). It is unclear to me if the introduction also could benefit from motivating this with more of a theoretical framework based on evolution, inheritance, or trait transmission. If this is really meant to be an economics-focused article, then lean more heavily into that. As it stands, it straddles a bit of economics, a bit of legacies that appear false (beefalo are not part bison at all!), and a bit of admixture genetics theory.

    We intended the focus of this study to be on documenting the species-level ancestry of Beefalo, and concentrated the information presented in the Introduction on this topic. Given that less hybridization between bison and cattle appears to have taken place to form the Beefalo breed than was previously described, we believe that broader theoretical statements about admixture are less relevant here, beyond highlighting examples of successful and failed interspecies hybridization in Bos. We also avoided speculating on the history of the establishment of the breed beyond what could be understood from the genetic data.

    Can the authors give a bit more details about beefalo breeding? Did the breeders select for any quantitative traits and is there a targeted phenotype for beefalo they used as a standard?

    Limited information exists about the precise origins of Beefalo, which were never publicly shared—possibly in part for reasons this manuscript addresses. The only criteria defining Beefalo is the proportion of bison ancestry, and so no quantitative traits or specific phenotypes are related to breed standard.

    Can the authors provide a few examples of what is known about the incompatibilities and reproductive challenges? What is known from past research or from the Beefalo Association documenting the breeding history?

    We provided a general summary of hybridization and incompatibility across Bos, but unfortunately cannot provide details about incompatibilities in Beefalo specifically. Though there is a long history of challenges interbreeding bison and cattle (referenced in the third paragraph of the Introduction), to our knowledge no examination has been carried out of Beefalo specifically and little is known about Beefalo pedigrees (again, perhaps for reasons related to information presented in this study).

    (2) Results Section Sequencing Beefalo genomes

    Please report the number of polymorphic sites to accompany the genomic read depth averages. It seems the authors could include a larger summary of the genomic data that was used for downstream analyses (like the PCA in the next section). Also, does this dataset include the sex chromosomes? How many variants that are retained for analyses are autosomal, sex-linked, or haploid? Please provide more characteristics of the data that was generated after QC and filtering.

    We have now replaced “5.29M” on page 14 with the exact number of SNPs (5,291,534) and added a description of genotype calling to the Results section. We have also included the number of SNPs used for sex chromosomal analyses.

    (3) Results section Estimating bison ancestry in beefalo

    What is a "foundational" individual? Is this a beefalo pedigree founder, a common sire, or an individual with remarkably high bison content? I see in the introduction Joe's Pride was the "most expensive cattle" but there are surely other aspects of "foundational" that the reader should understand as the results are presented.

    We agree that this terminology was imprecise, and have now clarified that we use foundational to mean an early individual that was important in the founding of the Beefalo breed, such as those that were first bred by Bud Basolo.

    For the sentence "The reported pedigree in the NAGP for this animal [NAGP9109] lists its composition as 1⁄2 Brahman, 1⁄4 Charolais, 1⁄8 bison, 1/16th Hereford, and 1/16th Shorthorn, but the American Brahman Breeders Association records this animal (#309519) as purebred Brahman.", this is difficult for a reader with limited cattle breed knowledge to infer significance of this. What is the origination of Brahman breed cattle? Does Brahman ancestry come from another mixed origin that could explain this discrepancy? Does the PCA have references to resolve the origin of Brahman? I realize this may sound extraneous but if membership to a breed that is recently formed from several other lineages or breeds, could you be seeing the deeper parts that compose Brahman cattle? How could one validate that the contributors erroneously labeled this individual as a beefalo?

    We have now noted that the Brahman breed has primarily zebu ancestry. The placement of this individual in the PCA supports the American Brahman Breeders Association metadata, and suggests that the NAGP labeling is incorrect:

    “The reported pedigree in the NAGP for this animal lists its composition as 1/2 Brahman, 1/4 Charolais, 1/8 bison, 1/16th Hereford, and 1/16th Shorthorn, but the American Brahman Breeders Association records this animal (#309519) as purebred Brahman, which is a zebu breed (5 of the other 6 zebu individuals analyzed here are Brahman cattle). We believe NAGP9109 was erroneously labeled as Beefalo by the contributors.”

    Figure 1A: Please add % explained by each PC.

    We have now updated axis labels for the PCA to include the proportion of variance explained for each component.

    Figures 1B and 1C are identical except for the Y axis. Please combine them into a graph with 2 Y-axes (one for PC1 and one for ADMIXTURE). Also, please include the bison in this panel as well.

    We have now updated these panels to include bison, although have kept the labeling so that they may be referenced separately in the text.

    I see that the authors did both unsupervised and supervised. Can the main text have the supervised graphical result instead of the unsurprised? That is more relevant for ancestry proportions via an assignment probability to ancestry groups. Or, if possible, could the authors consider STRUCTURE to also obtain the probability of assignment to a prior defied parental up to 2-generations back? This is by far the best way to leverage the ancestry information of the cattle and bison parental references in addition to the known F1/bison hybrids. Swap the Supplementary Figure 1 with Figure 1D!

    The supervised and unsupervised ADMIXTURE results are highly consistent, as could be expected given the high levels of divergence between species. We prefer to show the unsupervised results in the main text, as this makes the fewest assumptions about the ancestry of the examined individuals, and so also shows that the panels used to represent each species (taurine cattle, zebu cattle, and bison) do not contain individuals which were themselves highly admixed, which could have influenced the supervised ADMIXTURE analyses.

    For the unsupervised ADMIXTURE analyses, what were the cross-validation values per K value tested? How did the authors decide that K=3 was the best one to show?

    We now also show ADMIXTURE results at K=2 and K=4 (Fig. S2) and present the cross-validation results from ADMIXTURE (Fig. S3).

    Regarding "D-statistics ..... are consistent with 0 for most individual Beefalos....", I have two comments. First, by "consistent with", do you mean "are not significantly different from 0", indicating that (explain what this means in your words). Next, "most individual beefalos" means how many? Please provide numbers and values to highlight points or specific findings.

    The interpretation of the D-statistics has been clarified and Z-scores and numbers of individuals to quantitatively describe these results have been added. The text now reads:

    “D-statistics of the form D (taurus, Beefalo; bison, water buffalo), which test whether Beefalo share more alleles with bison than taurine cattle, again show 39 Beefalo have no excess affinity with bison compared to taurine cattle (-13.04 < Z < 3.14), although the same eight Beefalo identified in PCA and ADMIXTURE as having bison ancestry also have an excess of bison alleles (6.16 < Z < 34.86), confirming their bison ancestry (Fig. 2A).”

    "In Beefalo with bison ancestry, that ancestry tends to be present in large contiguous blocks, often tens of megabases in size, indicative of recent admixture (Figure 3A, B)". Please display the quantitative results (mean, max, range, standard deviation, etc.) in the main text and point the reader to the table that contains the values for each individual. The rest of this paragraph also uses the words "most' or "always" - please provide numbers. Is most 30/46 beefalo? Is it always exactly all 47 beefalo? Readers want to see numbers!

    The reviewer is correct that this section lacked specificity. We have now provided the exact number of individuals identified with bison and zebu ancestry.

    The section starting "Several lines of evidence attest to the efficacy of using these source panels..." could realistically come first in the Results section and before beefalo results are presented. This would build confidence for the reader that this panel of samples passes a QC and will indeed be able to resolve ancestry-based questions.

    This section specifically refers to the local ancestry analyses, which we have now clarified in the text.

    Figure 3A-C: Please include on each of these figure panels the documented (breeder association) ancestry percentage and the percentage of bison ancestry you obtained from your genomic analyses. Moving it from the legend to the figure is more immediately powerful for the reader. If the authors dated the admixture events as well, please include the meta-data of the association pedigree reporting when bison entered the target individual's genome versus the genome-estimated number of generations since admixture.

    Figure 3 has now been updated to include the reported bison ancestry. No attempt was made to date the admixture event or compare with reported pedigrees, as documented Beefalo pedigrees are typically very sparse (and may be unreliable, as our results suggest).

    Figure 3 legend: Move the following text from the figure legend to the Results section: "Three bison hybrids are inferred to have ~75% bison ancestry, while eight Beefalo have detectable bison ancestry, ranging from 2-18%. Indicine ancestry is detected in most Beefalo at variable levels, ranging from 2-38%, with most Beefalo having between 2-18%.".

    This sentence has been removed from the legend and is now worked into the main text. The corresponding paragraph in the results now reads:

    “Local ancestry inference across individual Beefalo and bison-cattle hybrid genomes provides similar estimates of overall Beefalo ancestry, inferring an absence of bison ancestry across the 37 Beefalo that lacked evidence for such ancestry in previous analyses (Fig. 3). Three bison hybrids are inferred to have ~75% bison ancestry, while eight Beefalo have detectable bison ancestry, ranging from 2-18%. Zebu ancestry is detected in 38 Beefalo at variable levels, ranging from 2-38%, with all but two of Beefalo having between 2-18%.”

    (4) Results section Beefalo sex chromosome ancestry

    Check that the authors do not reference Figure 4B before Figure 4A.

    Thank you to the reviewer for noticing this, it has now been corrected.

    Figure 4A: Could this panel be considered to merge with the autosomal admixture plot? It helps with comparison. Not a firm request - but it is nice to see what is consistent versus what is discordant.

    To avoid cluttering the figure with two highly similar plots, we preferred to separate the autosomal and sex chromosomal results.

    Figure 4C: Could this panel be merged with the autosomal ancestry bar graph to help the reader with visual comparisons?

    We thank the reviewer for this suggestion, but do not understand exactly which figures they are suggesting to be merged.

    (5) Materials and Methods: Modeling Beefalo ancestry:

    The language used in this sentence "This approach allows for directly understanding the ancestry of Beefalo individuals relative to these three groups while mitigating the effects of the low sequencing depth obtained for many Beefalo." conflicts with a sentence later in this paragraph which called PCA a model-free analysis. Please correct.

    Unfortunately, we are unsure what the reviewer refers to here and believe that this sentence does not conflict with the characterization of PCA as a model-free analytical approach.

  8. eLife

    eLife Assessment

    This important study used whole genome data to investigate Beefalo ancestry for the first time. It provides insight into the genetics of Beefalo cattle, definitively challenging the long-held claim of 37.5% buffalo ancestry reported by the American Beefalo Association. This results are convincing, with a comprehensive range of well-established population genomics methods being used to estimate ancestry in these animals. This work will be of significant interest to evolutionary biologists, population geneticists, animal breeders, and those involved in the conservation genetics of bovine species.

  9. eLife

    Reviewer #1 (Public review):

    Summary:

    This study used whole genome data to investigate Beefalo ancestry for the first time, filling the gap in the field of Beefalo ancestry. The authors used preserved semen samples to generate genomic data on 47 registered Beefalo and 3 bison hybrids, further questioning the ABA's stated goal of ⅜ bison ancestry. In addition, the authors also show that ancestry profiles of Beefalo and bison hybrid genomes are consistent with repeated backcrossing to either parental species, demonstrating the value of genomic information in examining gene flow between species in the genus Bison. This is an interesting study that still has some major weaknesses that exist, but overall, the work demonstrates the utility of genomic information in validating specific breeding claims for a more complete understanding of gene flow and genetic variation among bovine species.

    Strengths:

    Numerous genetic analysis methods such as PCA, ADMIXTURE, F4 ratios, and local ancestry inference techniques revealed that no single Beefalo set meets the ancestry requirements set by the American Beefalo Association (ABA) and some beefalo had detectable indicine cattle ancestry.

    Weaknesses:

    While this study contributes to our knowledge of Beefalo ancestry, there are some key issues that need to be addressed in terms of analysing the specific results as well as writing the article.

  10. eLife

    Reviewer #2 (Public review):

    Summary:

    Shapiro et al. set out to verify the American Beefalo Association's claim that Beefalo cattle possess 37.5% bison ancestry. They employ a comprehensive range of well-established population genomics methods to estimate ancestry in these hybrid populations, including PCA, ADMIXTURE, D and F statistics, and local ancestry inference. Their findings conclusively demonstrate that most Beefalo lack the claimed bison ancestry, with only 8 out of 47 samples showing any detectable bison ancestry, ranging from 2 - 18%.

    Strengths:

    The primary strength of this analysis lies in the comprehensive dataset available to the authors, which includes important foundational Beefalo individuals and various reference populations. The rigorous and multi-faceted methodological approach employs several well-established techniques in population genomics for detecting and measuring admixture. Each method used has a firm basis in the field, providing consistent and robust results. The authors' approach of using PCA to initially assess the data within a global context, followed by more specific analyses using ADMIXTURE and D-statistics, provides a clear and logical progression of evidence. The presentation of these results in figures is particularly effective, clearly illustrating the key findings of the study. Additionally, the examination of both autosomal and sex chromosome ancestry offers a more complete understanding of Beefalo genetic composition and the mechanics of bison-cattle hybridisation.

    Weaknesses:

    One limitation of this analysis is the relatively low coverage (~2x) of many Beefalo samples. However, the authors have taken steps to mitigate biases that may arise from this. Another weakness is the limited sampling of contemporary Beefalo populations, as the study focuses primarily on historical samples. This may limit our understanding of how Beefalo genetics may have changed over time.

    Appraisal:

    The authors have clearly achieved their primary aim using a rigorous and comprehensive methodology. Their extensive dataset and multi-faceted analytical approach provide strong support for their conclusions. The study not only addresses its main research question but also reveals unexpected insights into Beefalo genetics, particularly the presence of zebu ancestry.

    Discussion:

    This study is valuable for several reasons beyond its primary findings. First, it definitively addresses and refutes the claim of 37.5% bison ancestry in Beefalo, providing crucial information for those studying these interspecies hybrids and the viability of their offspring. Second, it reveals the unexpected presence of zebu ancestry in many Beefalo, raising intriguing questions about the breed's development and the potential role of zebu cattle in achieving desired traits. This finding suggests that the distinctive appearance of Beefalo may be due in part to zebu admixture rather than bison ancestry. Third, the study highlights the significant barriers to admixture between bison and cattle, both in controlled breeding programs and potentially in wild populations. This has important implications for conservation genetics and our understanding of gene flow between these species. Lastly, the study demonstrates the power of genomic analysis in verifying breed claims and understanding the complex history of domestic animal breeds. These findings open new avenues for research in bovine genomics, breed development, and the dynamics of interspecies hybridisation.

  11. eLife

    Reviewer #3 (Public review):

    Summary:

    I really like this topic and study. But I think much can be more focused and tightened up. All the components are here - just some more refining to really make the storyline clear, the journey of discovery, and the impact of such knowledge.

    Strengths:

    The authors dive directly into the question of genomic ancestry as compared to the breed club's reported ancestry with heavy, quantitative data and critical analytical methods. The questioning line is direct and does not meander. The reader learns about the challenges of breeding associations, and values of understood ancestry, and presents a clear need of re-evaluating the breed standards and expectations of beefalo (if ancestry is indeed the primary goal instead of a phenotype-driven breed mission).

    Weaknesses:

    Much of the quantitative results are only referred to in the main text with qualitative language. Please incorporate more written quantitative results to highlight evidence that underlines the study narrative because it is quite an interesting study!

  12. Version published to 10.7554/elife.102750.1 on eLife
  13. Version published to 10.1101/2024.09.16.613218 on bioRxiv