Radiocarbon and genomic evidence for the survival of Equus Sussemionus until the late Holocene
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Evaluation Summary:
This paper represents multiple milestones in our understanding of the evolution and extinction of Pleistocene equids, including revising the timing of extinction and clarifying the evolutionary history of Equus (Sussemionus) ovodovi. The discovery of the late persistence of non-caballine equid taxa in northern China until deep into the late Holocene is particularly important. This finding will be of broad interest to the paleontology, paleoecology, archaeology, paleogenomic communities and should stimulate important future research into equid extinction processes.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
The exceptionally rich fossil record available for the equid family has provided textbook examples of macroevolutionary changes. Horses, asses, and zebras represent three extant subgenera of Equus lineage, while the Sussemionus subgenus is another remarkable Equus lineage ranging from North America to Ethiopia in the Pleistocene. We sequenced 26 archaeological specimens from Northern China in the Holocene that could be assigned morphologically and genetically to Equus ovodovi , a species representative of Sussemionus . We present the first high-quality complete genome of the Sussemionus lineage, which was sequenced to 13.4× depth of coverage. Radiocarbon dating demonstrates that this lineage survived until ~3500 years ago, despite continued demographic collapse during the Last Glacial Maximum and the great human expansion in East Asia. We also confirmed the Equus phylogenetic tree and found that Sussemionus diverged from the ancestor of non-caballine equids ~2.3–2.7 million years ago and possibly remained affected by secondary gene flow post-divergence. We found that the small genetic diversity, rather than enhanced inbreeding, limited the species’ chances of survival. Our work adds to the growing literature illustrating how ancient DNA can inform on extinction dynamics and the long-term resilience of species surviving in cryptic population pockets.
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Author Response:
Reviewer #1 (Public Review):
In this manuscript, Cai and authors offer a new and important discovery demonstrating the persistence of a clade on non-caballine equids, Sussemionus, well into the later millennia of the Holocene in northern China. My expertise does not lie with the genomics analysis, so I will not offer detailed comment - but as an outsider, the arguments seemed well-supported and convincing.
We thank the reviewer for the positive assessment.
The primary weakness of the article lies in the omission of detailed archaeological context, and in the failure to consider implications for and from human societies. All specimens were taken directly from archaeological sites, but no information is given about the archaeological sites and cultures the specimens were derived from. In early China, ca. 3500 BP, the …
Author Response:
Reviewer #1 (Public Review):
In this manuscript, Cai and authors offer a new and important discovery demonstrating the persistence of a clade on non-caballine equids, Sussemionus, well into the later millennia of the Holocene in northern China. My expertise does not lie with the genomics analysis, so I will not offer detailed comment - but as an outsider, the arguments seemed well-supported and convincing.
We thank the reviewer for the positive assessment.
The primary weakness of the article lies in the omission of detailed archaeological context, and in the failure to consider implications for and from human societies. All specimens were taken directly from archaeological sites, but no information is given about the archaeological sites and cultures the specimens were derived from. In early China, ca. 3500 BP, the persistence of wild equid taxa is a very significant finding. This time period was a very dynamic period across northern East Asia, with the first introduction of domestic horses and the first spread of other livestock pastoralism (see Brunson et al, https://www.sciencedirect.com/science/article/abs/pii/S2352409X20300535). And, as summarized in Yuan and Flad (2006), many of the earliest sites speculatively linked with domestic horses that predate the final Shang Dynasty are isolated equid bones from archaeological sites, without definitive archaeological data to determine domestic or wild status. Therefore, the archaeological context of these finds is really important - how were each of the bones originally identified in archaeological reports? Is there associated evidence that the equids were hunted and eaten? The authors must add a section describing the archaeological context in greater detail, and considering the possible implications of the finds. For example, the persistence of sussemione equids through the 2nd millennium BCE implies that researchers must be exceedingly careful in zooarchaeological identifications prior to this period.
We thank the reviewer for pointing this out. We have provided more details about the archaeological context in the revised manuscript: “Nearly 20,000 square meters of Honghe (47.20°N, 123.62°E) have been excavated, revealing a late Neolithic settlement site dated to approximately ~3,400-4,400 years ago and belonging to a unique, rich fishing and hunting culture characteristic of northeastern China (Figure 1—figure supplement 1). The scale of the moated settlement indicates that there was already social management and relatively high productivity and building technology. The Muzhuzhuliang site (38.83°N, 110.50°E) belongs to the “Longshan Culture” dated to approximately ~3,800-4,300 years ago. It is the most complete moated settlement hitherto excavated in the late Neolithic Age of Northern China, and showed a subsistence economy based on agriculture, animal husbandry and hunting. The Shatangbeiyuan site (35.63°N, 105.11°E) belongs to the early cultural relics of “Qijia culture” in the Neolithic Age, which is dated to approximately ~3,900-4,200 years ago. Millet represented the main crop produced at that time, stone and bone arrowheads have also indicated that hunting was also performed. The rise and decline of these cultures were substantially influenced by the regional environmental conditions. And no traces of domestication but consumption were found in the equine specimens of three sites, indicating that they were hunted for food”.
And we have added “And given that the persistence of Sussemiones through the second millennium BCE, researchers must be exceedingly careful in zooarchaeological identifications prior to this period.” at the end of the article.
Moreover, the result might also warrant a discussion about the role of pastoral cultures, or the introduction of domestic horses, in the final extinction of the sussemiones. Without such a summary, it is incomplete to suggest that their final extinction is a result of inbreeding and reduced genetic diversity.
We agree that this is an interesting point to consider. We have added the sentence “Considering the knowledge of environmental and human archaeology, our results imply that the extinction of this lineage may be affected by the combination of climatic change and human mediation.”
Reviewer #2 (Public Review):
Dawei Cai and colleagues present a series of firsts and new discoveries including (1) the first high coverage genome from an equid that is unequivocally an extinct species and (2) demonstrating that Equus (Sussemionus) ovodovi survived into the late Holocene, belonged to a lineage sister to all extant non-caballine equids, and underwent extensive admixture soon after its divergence from non-caballine equids.
The manuscript is clearly laid out and well written. The analyses are conducted logically and to a high standard, which includes testing the impacts of reference genome choice and DNA misincorporations in nearly all analyses. The conclusions are mostly supported by the data but some methodological clarifications and discussion of conflicting results are required.
Thanks for your comments.
Strengths/weaknesses of the five main findings:
(1) Sussemiones survived into the late Holocene. Strengths: It is remarkable that Sussemiones survived so late into the Holocene, but the authors present radiocarbon evidence from multiple skeletal elements and sites supporting the late survival hypothesis. Combined with the genomic evidence, there is very strong support for this assertion. Weaknesses: The manuscript does not describe the radiocarbon methods, such as which laboratory these analyses were conducted in and whether samples were ultrafiltered or not. A description of the calibration methods and curve version used is also lacking.
Thank you for this suggestion. We have provided more details about the radiocarbon methods in the revision and Supplementary Table S2. “Radiocarbon dating of the samples was performed at the Beta Analytic Radiocarbon Dating Laboratory, Miami, Florida. Bone or tooth pieces about 2g were sampled in the bone and sent for subsequent dating of collagen (not ultrafiltered). Calibration was carried out using OxCalOnline (https://c14.arch.ox.ac.uk/oxcal.html) and the IntCal20 calibration curve.”
(2) Equus (Sussemionus) ovodovi is a sister lineage to all extant non-caballine equids. Strengths: The authors construct both exome and candidate neutral loci phylogenies from across the nuclear genome, including testing the impact of two different reference genomes. All analyses support the same placement of E. ovodovi with 100% bootstrap support. The assertion is therefore strongly supported. Weaknesses: No weaknesses identified.
We thank the reviewer for the positive assessment.
(3) The early evolution of the lineages leading to the E. ovodovi and the three main extant equid groups was characterised by extensive admixture. Strengths: The authors use three different methods to infer the presence, extent, and/or direction of admixture. Weaknesses: A major weakness here is the incongruence between the TreeMix models and the D-statistics and G-PhoCS analyses (the latter two give a coherent story). Given the large admixture events determined by G-PhoCS, it seems concerning that these events are not recovered as migration edges in the TreeMix analyses.
We thank the reviewer for the suggestion. As the reviewer notes, two reasons may cause the incongruence between the TreeMix models and the G-PhoCS analyses. First, the TreeMix models will work best when gene flow between populations is restricted to a relatively short time period, situations of continuous migration violate this assumption and lead to unclear results (see Pickrell, Joseph K., & Pritchard, Jonathan K. (2012), https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1002967). Second, two different datasets were used in the analyses. The tree topologies and gene flow were recovered using whole-genome SNPs by TreeMix, while the G-PhoCS analyses of selected samples based on 15,324 candidate ‘neutral’ loci.
(4) Population size of E. ovodovi over the past 2 Myr. Strengths: The authors correct for differences in genome coverage to allow for the PSMC profiles between four equid taxa to be comparable, allowing for comparison of population size trajectories. Weaknesses: In Figure 4, the presented PSMC profiles are a mix of those with or without transitions (comparing profiles to Figure - 4 figure supplement 1). Given that the exclusion of transitions impacts the PSMC profiles, these should be standardized in Figure 4 to give a fair comparison.
We thank the reviewer for this suggestion as well. As for the possible mis-incorporation pattern and high error rate of four equids, we compared the PSMC analyses performed with and without transitions. A consistent pattern was observed regarding two datasets expect for the PSMC bootstrap pseudo-replicates for HH06D, and we therefore only presented PSMC profiles without transitions when considering the ancient HH06D specimen. Meanwhile, we applied a correction based on an empirical uniform false-negative rate for low coverage genomes (<20×). All three Eurasian equine species genomes were rescaled following the same procedure (see L. Orlando et al. (2013), https://www.nature.com/articles/nature12323).
(5) Inbreeding was a contributing factor to the extinction of E. ovodovi. Strengths: The authors determine heterozygosity and runs-of-homozygosity in E. ovodovi and compare these to all living equids, and find that E. ovodovi had low heterozygosity although not excessive runs-of-homozygosity. Weaknesses: The authors should be more cautious with their interpretation/phrasing on L383-384, given that inbreeding and/or reduced genetic diversity has not been demonstrated as the extinction driver.
Thanks for the suggestion, and we have now re-written this sentence: “So combined with a degree of inbreeding, the reduced genetic diversity available may have contributed to the subsequent extinction of the lineage”).
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Evaluation Summary:
This paper represents multiple milestones in our understanding of the evolution and extinction of Pleistocene equids, including revising the timing of extinction and clarifying the evolutionary history of Equus (Sussemionus) ovodovi. The discovery of the late persistence of non-caballine equid taxa in northern China until deep into the late Holocene is particularly important. This finding will be of broad interest to the paleontology, paleoecology, archaeology, paleogenomic communities and should stimulate important future research into equid extinction processes.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
-
Reviewer #1 (Public Review):
In this manuscript, Cai and authors offer a new and important discovery demonstrating the persistence of a clade on non-caballine equids, Sussemionus, well into the later millennia of the Holocene in northern China. My expertise does not lie with the genomics analysis, so I will not offer detailed comment - but as an outsider, the arguments seemed well-supported and convincing.
The primary weakness of the article lies in the omission of detailed archaeological context, and in the failure to consider implications for and from human societies. All specimens were taken directly from archaeological sites, but no information is given about the archaeological sites and cultures the specimens were derived from. In early China, ca. 3500 BP, the persistence of wild equid taxa is a very significant finding. This time …
Reviewer #1 (Public Review):
In this manuscript, Cai and authors offer a new and important discovery demonstrating the persistence of a clade on non-caballine equids, Sussemionus, well into the later millennia of the Holocene in northern China. My expertise does not lie with the genomics analysis, so I will not offer detailed comment - but as an outsider, the arguments seemed well-supported and convincing.
The primary weakness of the article lies in the omission of detailed archaeological context, and in the failure to consider implications for and from human societies. All specimens were taken directly from archaeological sites, but no information is given about the archaeological sites and cultures the specimens were derived from. In early China, ca. 3500 BP, the persistence of wild equid taxa is a very significant finding. This time period was a very dynamic period across northern East Asia, with the first introduction of domestic horses and the first spread of other livestock pastoralism (see Brunson et al, https://www.sciencedirect.com/science/article/abs/pii/S2352409X20300535). And, as summarized in Yuan and Flad (2006), many of the earliest sites speculatively linked with domestic horses that predate the final Shang Dynasty are isolated equid bones from archaeological sites, without definitive archaeological data to determine domestic or wild status. Therefore, the archaeological context of these finds is really important - how were each of the bones originally identified in archaeological reports? Is there associated evidence that the equids were hunted and eaten? The authors must add a section describing the archaeological context in greater detail, and considering the possible implications of the finds. For example, the persistence of sussemione equids through the 2nd millennium BCE implies that researchers must be exceedingly careful in zooarchaeological identifications prior to this period. Moreover, the result might also warrant a discussion about the role of pastoral cultures, or the introduction of domestic horses, in the final extinction of the sussemiones. Without such a summary, it is incomplete to suggest that their final extinction is a result of inbreeding and reduced genetic diversity.
-
Reviewer #2 (Public Review):
Dawei Cai and colleagues present a series of firsts and new discoveries including (1) the first high coverage genome from an equid that is unequivocally an extinct species and (2) demonstrating that Equus (Sussemionus) ovodovi survived into the late Holocene, belonged to a lineage sister to all extant non-caballine equids, and underwent extensive admixture soon after its divergence from non-caballine equids.
The manuscript is clearly laid out and well written. The analyses are conducted logically and to a high standard, which includes testing the impacts of reference genome choice and DNA misincorporations in nearly all analyses. The conclusions are mostly supported by the data but some methodological clarifications and discussion of conflicting results are required.
Strengths/weaknesses of the five main …
Reviewer #2 (Public Review):
Dawei Cai and colleagues present a series of firsts and new discoveries including (1) the first high coverage genome from an equid that is unequivocally an extinct species and (2) demonstrating that Equus (Sussemionus) ovodovi survived into the late Holocene, belonged to a lineage sister to all extant non-caballine equids, and underwent extensive admixture soon after its divergence from non-caballine equids.
The manuscript is clearly laid out and well written. The analyses are conducted logically and to a high standard, which includes testing the impacts of reference genome choice and DNA misincorporations in nearly all analyses. The conclusions are mostly supported by the data but some methodological clarifications and discussion of conflicting results are required.
Strengths/weaknesses of the five main findings:
(1) Sussemiones survived into the late Holocene.
Strengths: It is remarkable that Sussemiones survived so late into the Holocene, but the authors present radiocarbon evidence from multiple skeletal elements and sites supporting the late survival hypothesis. Combined with the genomic evidence, there is very strong support for this assertion.
Weaknesses: The manuscript does not describe the radiocarbon methods, such as which laboratory these analyses were conducted in and whether samples were ultrafiltered or not. A description of the calibration methods and curve version used is also lacking.(2) Equus (Sussemionus) ovodovi is a sister lineage to all extant non-caballine equids.
Strengths: The authors construct both exome and candidate neutral loci phylogenies from across the nuclear genome, including testing the impact of two different reference genomes. All analyses support the same placement of E. ovodovi with 100% bootstrap support. The assertion is therefore strongly supported.
Weaknesses: No weaknesses identified.(3) The early evolution of the lineages leading to the E. ovodovi and the three main extant equid groups was characterised by extensive admixture.
Strengths: The authors use three different methods to infer the presence, extent, and/or direction of admixture.
Weaknesses: A major weakness here is the incongruence between the TreeMix models and the D-statistics and G-PhoCS analyses (the latter two give a coherent story). Given the large admixture events determined by G-PhoCS, it seems concerning that these events are not recovered as migration edges in the TreeMix analyses.(4) Population size of E. ovodovi over the past 2 Myr.
Strengths: The authors correct for differences in genome coverage to allow for the PSMC profiles between four equid taxa to be comparable, allowing for comparison of population size trajectories.
Weaknesses: In Figure 4, the presented PSMC profiles are a mix of those with or without transitions (comparing profiles to Figure - 4 figure supplement 1). Given that the exclusion of transitions impacts the PSMC profiles, these should be standardized in Figure 4 to give a fair comparison.(5) Inbreeding was a contributing factor to the extinction of E. ovodovi.
Strengths: The authors determine heterozygosity and runs-of-homozygosity in E. ovodovi and compare these to all living equids, and find that E. ovodovi had low heterozygosity although not excessive runs-of-homozygosity.
Weaknesses: The authors should be more cautious with their interpretation/phrasing on L383-384, given that inbreeding and/or reduced genetic diversity has not been demonstrated as the extinction driver. -
