Analyze Diet
Nature ecology & evolution2025; 9(12); 2248-2258; doi: 10.1038/s41559-025-02859-5

Mitochondrial genomes of Middle Pleistocene horses from the open-air site complex of Schöningen.

Abstract: Deep-time palaeogenomics offers rare insights into macroevolutionary events for both extant and extinct species. Aside from a Middle Pleistocene genome from North American permafrost (780-560 ka) and a number of Late Pleistocene specimens, most ancient horse DNA studies have focused on tracing the origins of domestication and subsequent periods. Here we present mitochondrial genomes from two Equus mosbachensis specimens from Schöningen, Germany, a Middle Pleistocene archaeological site complex with direct and repeated evidence of hominin-horse interactions on the shore of a palaeolake. Using petrous bone sampling, targeted enrichment and damage-aware and polarization-free mitochondrial DNA reconstruction methods, we extend the range of genome recovery in open-air sites to ~300,000 years ago. Phylogenetic analyses position these mitochondrial DNAs in two distinct, deeply divergent lineages, basal to both previously sequenced ancient Eurasian specimens and all modern-day horses. The Schöningen horse mitochondrial DNA data reveal a previously unrecognized diversification event within the clade, ultimately giving rise to modern-day horses, that is molecularly dated to ~570 ka and provides genetic support for the morphological species assignment. By extending the recoverable limits of ancient DNA from Middle Pleistocene open-air sites, our molecular findings bridge a temporal and geographic gap, providing insights on early evolutionary events within the genus Equus.
Publication Date: 2025-10-01 PubMed ID: 41034648PubMed Central: PMC12680542DOI: 10.1038/s41559-025-02859-5Google Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
  • Journal Article

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

Overview

  • This study reports the recovery and analysis of mitochondrial genomes from Middle Pleistocene horses (~300,000 years old) found at the open-air archaeological site complex of Schöningen, Germany.
  • The findings reveal new insights into the evolutionary history and diversification of horses, identifying ancient lineages that predate previously known ancient and modern horses.

Background and Importance of the Study

  • Ancient DNA studies typically focus on relatively recent specimens, especially those relating to domestication or Late Pleistocene eras.
  • Middle Pleistocene DNA data are very rare, especially from open-air archaeological sites, due to DNA degradation over time and environmental exposure.
  • Before this research, only a few ancient horse genomes were available, notably one from North American permafrost dated between 780,000 to 560,000 years ago.
  • The Schöningen site is significant for its repeated evidence of early humans (hominins) interacting with horses near an ancient lake shore, providing a valuable ecological and anthropological context.

Methodology

  • Two specimens identified as Equus mosbachensis (an extinct Middle Pleistocene horse species) were sampled.
  • Sampling focused on the petrous bone, known to preserve DNA better due to its dense structure.
  • Targeted enrichment techniques were used to isolate mitochondrial DNA (mtDNA) from the samples.
  • The researchers employed damage-aware and polarization-free methods for reconstructing the mtDNA, enhancing the accuracy of sequences from highly degraded ancient DNA.

Key Findings

  • Successfully recovered mitochondrial genomes date back around 300,000 years, extending the known range for DNA recovery from open-air sites.
  • Phylogenetic analysis revealed two distinct and deeply divergent mitochondrial lineages in the Schöningen horses.
  • These lineages are basal, meaning that they appear at the root of the evolutionary tree, prior to all previously sequenced ancient Eurasian horses and all modern horses.
  • The Schöningen mtDNA indicates a previously unrecognized diversification event within the horse lineage approximately 570,000 years ago.
  • This diversification is consistent with the morphology-based species identification of the specimens, confirming that the genetic data supports the species classification of Equus mosbachensis.

Implications and Significance

  • The study bridges a critical temporal and geographical gap in the horse evolutionary record by providing genomic data from a Middle Pleistocene, European open-air site.
  • The discovery of distinct, basal horse lineages increases understanding of early horse evolution and population dynamics before the Late Pleistocene and domestication phases.
  • Provides a molecular timeline for early diversification events within genus Equus, contributing to the broader field of macroevolutionary studies of mammals.
  • Demonstrates that ancient DNA can be successfully retrieved from challenging contexts like open-air sites, not only permafrost or caves, potentially expanding the scope of future palaeogenomic research.

Summary

  • This research advances paleo-genomics by successfully extracting and analyzing mitochondrial genomes from Middle Pleistocene horses at Schöningen, an open-air site in Germany.
  • It uncovers ancient evolutionary lineages, deepening our understanding of horse ancestry and evolution that occurred hundreds of thousands of years ago.
  • The methodological innovations and findings highlight the potential for future studies to explore ancient DNA across wider temporal and environmental settings, offering new perspectives on extinct and extant species’ history.

Cite This Article

APA
Weingarten A, Häusler M, Serangeli J, Verheijen I, Reiter E, Radzevičiūtė R, Stoessel A, Krause J, Spyrou MA, Conard NJ, Nieselt K, Posth C. (2025). Mitochondrial genomes of Middle Pleistocene horses from the open-air site complex of Schöningen. Nat Ecol Evol, 9(12), 2248-2258. https://doi.org/10.1038/s41559-025-02859-5

Publication

ISSN: 2397-334X
NlmUniqueID: 101698577
Country: England
Language: English
Volume: 9
Issue: 12
Pages: 2248-2258

Researcher Affiliations

Weingarten, Arianna
  • Archaeo- and Palaeogenetics, Institute for Archaeological Sciences, Department of Geosciences, University of Tübingen, Tübingen, Germany. arianna.weingarten@uni-tuebingen.de.
  • Senckenberg Centre for Human Evolution and Palaeoenvironment, University of Tübingen, Tübingen, Germany. arianna.weingarten@uni-tuebingen.de.
  • Senckenberg Centre for Human Evolution and Palaeoenvironment, Schöningen, Germany. arianna.weingarten@uni-tuebingen.de.
Häusler, Meret
  • Archaeo- and Palaeogenetics, Institute for Archaeological Sciences, Department of Geosciences, University of Tübingen, Tübingen, Germany.
  • Integrative Transcriptomics, Institute for Bioinformatics and Medical Informatics, University of Tübingen, Tübingen, Germany.
Serangeli, Jordi
  • Senckenberg Centre for Human Evolution and Palaeoenvironment, Schöningen, Germany.
  • Early Prehistory and Quaternary Ecology, Department of Geosciences, University of Tübingen, Tübingen, Germany.
Verheijen, Ivo
  • Senckenberg Centre for Human Evolution and Palaeoenvironment, Schöningen, Germany.
  • Early Prehistory and Quaternary Ecology, Department of Geosciences, University of Tübingen, Tübingen, Germany.
  • Cultural Heritage Office of Lower Saxony, Hanover, Germany.
Reiter, Ella
  • Archaeo- and Palaeogenetics, Institute for Archaeological Sciences, Department of Geosciences, University of Tübingen, Tübingen, Germany.
Radzevičiūtė, Rita
  • Department of Archaeogenetics, Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany.
Stoessel, Alexander
  • Department of Archaeogenetics, Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany.
  • Institute of Zoology and Evolutionary Research, Friedrich Schiller University Jena, Jena, Germany.
Krause, Johannes
  • Department of Archaeogenetics, Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany.
Spyrou, Maria A
  • Archaeo- and Palaeogenetics, Institute for Archaeological Sciences, Department of Geosciences, University of Tübingen, Tübingen, Germany.
  • Senckenberg Centre for Human Evolution and Palaeoenvironment, University of Tübingen, Tübingen, Germany.
  • Department of Archaeogenetics, Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany.
Conard, Nicholas J
  • Senckenberg Centre for Human Evolution and Palaeoenvironment, University of Tübingen, Tübingen, Germany.
  • Early Prehistory and Quaternary Ecology, Department of Geosciences, University of Tübingen, Tübingen, Germany.
Nieselt, Kay
  • Integrative Transcriptomics, Institute for Bioinformatics and Medical Informatics, University of Tübingen, Tübingen, Germany.
Posth, Cosimo
  • Archaeo- and Palaeogenetics, Institute for Archaeological Sciences, Department of Geosciences, University of Tübingen, Tübingen, Germany. cosimo.posth@uni-tuebingen.de.
  • Senckenberg Centre for Human Evolution and Palaeoenvironment, University of Tübingen, Tübingen, Germany. cosimo.posth@uni-tuebingen.de.

MeSH Terms

  • Animals
  • Horses / genetics
  • Genome, Mitochondrial
  • Phylogeny
  • Fossils
  • DNA, Ancient / analysis
  • Germany
  • DNA, Mitochondrial / genetics
  • Biological Evolution

Conflict of Interest Statement

Competing interests: The authors declare no competing interests.

References

This article includes 68 references
  1. Green RE. A draft sequence of the Neandertal genome.. 710–722 (2010).
    doi: 10.1126/science.1188021pmc: PMC5100745pubmed: 20448178google scholar: lookup
  2. Krause J. The complete mitochondrial DNA genome of an unknown hominin from southern Siberia.. 894–897 (2010).
    doi: 10.1038/nature08976pmc: PMC10152974pubmed: 20336068google scholar: lookup
  3. Wang M-S. A polar bear paleogenome reveals extensive ancient gene flow from polar bears into brown bears.. 936–944 (2022).
    doi: 10.1038/s41559-022-01753-8pubmed: 35711062google scholar: lookup
  4. Barlow A. Middle Pleistocene genome calibrates a revised evolutionary history of extinct cave bears.. 1771–1779.e7 (2021).
    doi: 10.1016/j.cub.2021.01.073pubmed: 33592193google scholar: lookup
  5. Orlando L. Ancient DNA analysis.. 14 (2021).
  6. Liu Y, Mao X, Krause J, Fu Q. Insights into human history from the first decade of ancient human genomics.. 1479–1484 (2021).
    doi: 10.1126/science.abi8202pubmed: 34554811google scholar: lookup
  7. Dalén L, Heintzman PD, Kapp JD, Shapiro B. Deep-time paleogenomics and the limits of DNA survival.. 48–53 (2023).
    doi: 10.1126/science.adh7943pmc: PMC10586222pubmed: 37797036google scholar: lookup
  8. Orlando L, Gilbert MTP, Willerslev E. Reconstructing ancient genomes and epigenomes.. 395–408 (2015).
    doi: 10.1038/nrg3935pubmed: 26055157google scholar: lookup
  9. Kjær KH. A 2-million-year-old ecosystem in Greenland uncovered by environmental DNA.. 283–291 (2022).
    doi: 10.1038/s41586-022-05453-ypmc: PMC9729109pubmed: 36477129google scholar: lookup
  10. Lindahl T. Instability and decay of the primary structure of DNA.. 709–715 (1993).
    doi: 10.1038/362709a0pubmed: 8469282google scholar: lookup
  11. Kistler L, Ware R, Smith O, Collins M, Allaby RG. A new model for ancient DNA decay based on paleogenomic meta-analysis.. 6310–6320 (2017).
    doi: 10.1093/nar/gkx361pmc: PMC5499742pubmed: 28486705google scholar: lookup
  12. Van Der Valk T. Million-year-old DNA sheds light on the genomic history of mammoths.. 265–269 (2021).
    doi: 10.1038/s41586-021-03224-9pmc: PMC7116897pubmed: 33597750google scholar: lookup
  13. Orlando L. Recalibrating evolution using the genome sequence of an early Middle Pleistocene horse.. 74–78 (2013).
    doi: 10.1038/nature12323pubmed: 23803765google scholar: lookup
  14. Dabney J. Complete mitochondrial genome sequence of a Middle Pleistocene cave bear reconstructed from ultrashort DNA fragments.. 15758–15763 (2013).
    doi: 10.1073/pnas.1314445110pmc: PMC3785785pubmed: 24019490google scholar: lookup
  15. Meyer M. Nuclear DNA sequences from the Middle Pleistocene Sima de los Huesos hominins.. 504–507 (2016).
    doi: 10.1038/nature17405pubmed: 26976447google scholar: lookup
  16. Meyer M. A mitochondrial genome sequence of a hominin from Sima de los Huesos.. 403–406 (2014).
    doi: 10.1038/nature12788pubmed: 24305051google scholar: lookup
  17. Meyer M. Palaeogenomes of Eurasian straight-tusked elephants challenge the current view of elephant evolution.. e25413 (2017).
    doi: 10.7554/eLife.25413pmc: PMC5461109pubmed: 28585920google scholar: lookup
  18. MacFadden BJ, Hulbert RC. Explosive speciation at the base of the adaptive radiation of Miocene grazing horses.. 466–468 (1988).
    doi: 10.1038/336466a0google scholar: lookup
  19. Forsten A. Middle Pleistocene replacement of stenonid horses by caballoid horses—ecological implications.. 23–33 (1988).
  20. Azzaroli A. Quaternary mammals and the “end-Villafranchian” dispersal event—a turning point in the history of Eurasia.. 117–139 (1983).
  21. Rook L. Mammal biochronology (land mammal ages) around the world from Late Miocene to Middle Pleistocene and major events in horse evolutionary history.. 278 (2019).
    doi: 10.3389/fevo.2019.00278google scholar: lookup
  22. Vershinina AO. Ancient horse genomes reveal the timing and extent of dispersals across the Bering Land Bridge.. 6144–6161 (2021).
    doi: 10.1111/mec.15977pubmed: 33971056google scholar: lookup
  23. Fages A. Tracking five millennia of horse management with extensive ancient genome time series.. 1419–1435.e31 (2019).
    doi: 10.1016/j.cell.2019.03.049pmc: PMC6547883pubmed: 31056281google scholar: lookup
  24. Orlando L. The evolutionary and historical foundation of the modern horse: lessons from ancient genomics.. 563–581 (2020).
  25. Librado P. Widespread horse-based mobility arose around 2200 BCE in Eurasia.. 819–825 (2024).
    doi: 10.1038/s41586-024-07597-5pmc: PMC11269178pubmed: 38843826google scholar: lookup
  26. Hutson JM, Villaluenga A, García-Moreno A, Turner E, Gaudzinski-Windheuser S. Persistent predators: zooarchaeological evidence for specialized horse hunting at Schöningen 13II-4.. 103590 (2024).
    doi: 10.1016/j.jhevol.2024.103590pubmed: 39357283google scholar: lookup
  27. Thieme H. Lower Palaeolithic hunting spears from Germany.. 807–810 (1997).
    doi: 10.1038/385807a0pubmed: 9039910google scholar: lookup
  28. Conard NJ. Excavations at Schöningen and paradigm shifts in human evolution.. 1–17 (2015).
    doi: 10.1016/j.jhevol.2015.10.003pubmed: 26653207google scholar: lookup
  29. Van Kolfschoten T. The Palaeolithic locality Schöningen (Germany): a review of the mammalian record.. 469–480 (2014).
  30. Urban B, Sierralta M. New palynological evidence and correlation of Early Palaeolithic sites Schöningen 12 B and 13 II, Schöningen open lignite mine.. 77–96 (Propylaeum, 2019).
  31. Rivals F. Investigation of equid paleodiet from Schöningen 13 II-4 through dental wear and isotopic analyses: archaeological implications.. 129–137 (2015).
    doi: 10.1016/j.jhevol.2014.04.002pubmed: 25242064google scholar: lookup
  32. Rohland N, Glocke I, Aximu-Petri A, Meyer M. Extraction of highly degraded DNA from ancient bones, teeth and sediments for high-throughput sequencing.. 2447–2461 (2018).
    doi: 10.1038/s41596-018-0050-5pubmed: 30323185google scholar: lookup
  33. Gansauge M-T, Aximu-Petri A, Nagel S, Meyer M. Manual and automated preparation of single-stranded DNA libraries for the sequencing of DNA from ancient biological remains and other sources of highly degraded DNA.. 2279–2300 (2020).
    doi: 10.1038/s41596-020-0338-0pubmed: 32612278google scholar: lookup
  34. Maricic T, Whitten M, Pääbo S. Multiplexed DNA sequence capture of mitochondrial genomes using PCR products.. e14004 (2010).
  35. Furtwängler A. Ratio of mitochondrial to nuclear DNA affects contamination estimates in ancient DNA analysis.. 14075 (2018).
    doi: 10.1038/s41598-018-32083-0pmc: PMC6145933pubmed: 30232341google scholar: lookup
  36. Fu Q. A revised timescale for human evolution based on ancient mitochondrial genomes.. 553–559 (2013).
    doi: 10.1016/j.cub.2013.02.044pmc: PMC5036973pubmed: 23523248google scholar: lookup
  37. Peltzer A. EAGER: efficient ancient genome reconstruction.. 60 (2016).
    doi: 10.1186/s13059-016-0918-zpmc: PMC4815194pubmed: 27036623google scholar: lookup
  38. Posth C. Reconstructing the deep population history of Central and South America.. 1185–1197 (2018).
    doi: 10.1016/j.cell.2018.10.027pmc: PMC6327247pubmed: 30415837google scholar: lookup
  39. Lynch VJ. Elephantid genomes reveal the molecular bases of woolly mammoth adaptations to the Arctic.. 217–228 (2015).
    doi: 10.1016/j.celrep.2015.06.027pubmed: 26146078google scholar: lookup
  40. Jónsson H, Ginolhac A, Schubert M, Johnson PLF, Orlando L. mapDamage2.0: fast approximate Bayesian estimates of ancient DNA damage parameters.. 1682–1684 (2013).
  41. Neukamm J, Peltzer A, Nieselt K. DamageProfiler: fast damage pattern calculation for ancient DNA.. 3652–3653 (2021).
  42. Yuan J. Mitochondrial genomes of Late Pleistocene caballine horses from China belong to a separate clade.. 106691 (2020).
  43. Librado P. The origins and spread of domestic horses from the Western Eurasian steppes.. 634–640 (2021).
    doi: 10.1038/s41586-021-04018-9pmc: PMC8550961pubmed: 34671162google scholar: lookup
  44. Bouckaert R. BEAST 2.5: An advanced software platform for Bayesian evolutionary analysis.. e1006650 (2019).
  45. Schubert M, Lindgreen S, Orlando L. Prehistoric genomes reveal the genetic foundation and cost of horse domestication.. .
    pmc: PMC4284583pubmed: 25512547doi: 10.1073/pnas.1416991111google scholar: lookup
  46. Heintzman PD. A new genus of horse from Pleistocene North America.. e29944 (2017).
    doi: 10.7554/eLife.29944pmc: PMC5705217pubmed: 29182148google scholar: lookup
  47. To T-H, Jung M, Lycett S, Gascuel O. Fast dating using least-squares criteria and algorithms.. 82–97 (2016).
    doi: 10.1093/sysbio/syv068pmc: PMC4678253pubmed: 26424727google scholar: lookup
  48. Tucci M. Evidence for the age and timing of environmental change associated with a Lower Palaeolithic site within the Middle Pleistocene Reinsdorf sequence of the Schöningen coal mine, Germany.. 110309 (2021).
  49. Hutson JM. Revised age for Schöningen hunting spears indicates intensification of Neanderthal cooperative behavior around 200,000 years ago.. eadv0752 (2025).
    doi: 10.1126/sciadv.adv0752pmc: PMC12063642pubmed: 40344053google scholar: lookup
  50. Zavala EI. Pleistocene sediment DNA reveals hominin and faunal turnovers at Denisova Cave.. 399–403 (2021).
    doi: 10.1038/s41586-021-03675-0pmc: PMC8277575pubmed: 34163072google scholar: lookup
  51. Gelabert P. A sedimentary ancient DNA perspective on human and carnivore persistence through the Late Pleistocene in El Mirón Cave, Spain.. 107 (2025).
    doi: 10.1038/s41467-024-55740-7pmc: PMC11696082pubmed: 39747910google scholar: lookup
  52. Evans S, Llamas B, Wood JR. Sedimentary ancient DNA from caves: challenges and opportunities.. 565–578 (2025).
    doi: 10.1002/jqs.3712google scholar: lookup
  53. Boulbes N, Van Asperen EN. Biostratigraphy and palaeoecology of European .. 301 (2019).
    doi: 10.3389/fevo.2019.00301google scholar: lookup
  54. Eisenmann V. Les métapodes d’Equus sensu lato (Mammalia, Périssodactyla).. 863–886 (1979).
  55. Orlando L. Revising the recent evolutionary history of equids using ancient DNA.. 21754–21759 (2009).
    doi: 10.1073/pnas.0903672106pmc: PMC2799835pubmed: 20007379google scholar: lookup
  56. Weinstock J. Evolution, systematics, and phylogeography of pleistocene horses in the new world: a molecular perspective.. e241 (2005).
  57. Urban B. Spatial interpretation of high‐resolution environmental proxy data of the Middle Pleistocene Palaeolithic faunal kill site Schöningen 13 II‐4, Germany.. 440–458 (2023).
    doi: 10.1111/bor.12619google scholar: lookup
  58. Krahn KJ. Temperature and palaeolake evolution during a Middle Pleistocene interglacial–glacial transition at the Palaeolithic locality of Schöningen, Germany.. 504–524 (2024).
    doi: 10.1111/bor.12670google scholar: lookup
  59. Kircher, M. in (eds Shapiro, B. & Hofreiter, M.) vol. 840 197–228 (Humana Press, 2012).
  60. Meyer M, Kircher M. Illumina sequencing library preparation for highly multiplexed target capture and sequencing.. pdb.prot5448 (2010).
    doi: 10.1101/pdb.prot5448pubmed: 20516186google scholar: lookup
  61. Schubert M, Lindgreen S, Orlando L. AdapterRemoval v2: rapid adapter trimming, identification, and read merging.. 88 (2016).
    doi: 10.1186/s13104-016-1900-2pmc: PMC4751634pubmed: 26868221google scholar: lookup
  62. Li H, Durbin R. Fast and accurate short read alignment with Burrows–Wheeler transform.. 1754–1760 (2009).
  63. Pečnerová P. Genome-based sexing provides clues about behavior and social structure in the woolly mammoth.. 3505–3510 (2017).
    doi: 10.1016/j.cub.2017.09.064pubmed: 29103934google scholar: lookup
  64. Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput.. 1792–1797 (2004).
    doi: 10.1093/nar/gkh340pmc: PMC390337pubmed: 15034147google scholar: lookup
  65. Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: molecular evolutionary genetics analysis across computing platforms.. 1547–1549 (2018).
    doi: 10.1093/molbev/msy096pmc: PMC5967553pubmed: 29722887google scholar: lookup
  66. Minh BQ. IQ-TREE 2: new models and efficient methods for phylogenetic inference in the genomic era.. 1530–1534 (2020).
    doi: 10.1093/molbev/msaa015pmc: PMC7182206pubmed: 32011700google scholar: lookup
  67. Diez-del-Molino D, Heintzman PD, Dalén L. A list of representative paleogenomic datasets derived from human and faunal remains.. (2023).
    doi: 10.5281/zenodo.8270285google scholar: lookup
  68. Lang J. The Pleistocene of Schöningen, Germany: a complex tunnel valley fill revealed from 3D subsurface modelling and shear wave seismics.. 86–105 (2012).

Citations

This article has been cited 1 times.
  1. Saldaña CL, Justo S, Murga L, Vásquez HV, Maicelo JL, Arbizu CI, Bardales W. Mitochondrial genome assembly of the Peruvian Paso horse through PacBio long-read sequencing.. Sci Rep 2025 Dec 21;16(1):85.
    doi: 10.1038/s41598-025-29107-xpubmed: 41423464google scholar: lookup