A mitochondrial genome sequence of a hominin from Sima de los Huesos

Journal name:
Nature
Volume:
505,
Pages:
403–406
Date published:
DOI:
doi:10.1038/nature12788
Received
Accepted
Published online

Excavations of a complex of caves in the Sierra de Atapuerca in northern Spain have unearthed hominin fossils that range in age from the early Pleistocene to the Holocene1. One of these sites, the ‘Sima de los Huesos’ (‘pit of bones’), has yielded the world’s largest assemblage of Middle Pleistocene hominin fossils2, 3, consisting of at least 28 individuals4 dated to over 300,000 years ago5. The skeletal remains share a number of morphological features with fossils classified as Homo heidelbergensis and also display distinct Neanderthal-derived traits6, 7, 8. Here we determine an almost complete mitochondrial genome sequence of a hominin from Sima de los Huesos and show that it is closely related to the lineage leading to mitochondrial genomes of Denisovans9, 10, an eastern Eurasian sister group to Neanderthals. Our results pave the way for DNA research on hominins from the Middle Pleistocene.

At a glance

Figures

  1. Location of the Middle Pleistocene site of Sima de los Huesos (yellow) as well as Late Pleistocene sites that have yielded Neanderthal DNA (red) and Denisovan DNA (blue).
    Figure 1: Location of the Middle Pleistocene site of Sima de los Huesos (yellow) as well as Late Pleistocene sites that have yielded Neanderthal DNA (red) and Denisovan DNA (blue).
  2. Femur XIII reassembled from three parts after sampling.
    Figure 2: Femur XIII reassembled from three parts after sampling.

    The natural fractures are visible in the proximal third of the femur.

  3. Patterns of cytosine deamination in the libraries constructed from the Sima de los Huesos hominin femur.
    Figure 3: Patterns of cytosine deamination in the libraries constructed from the Sima de los Huesos hominin femur.

    a, C to T substitution frequencies are shown for the terminal positions of the aligned sequences for all sequences (black), those sequences carrying a C to T substitutions at their 5′ ends (blue), at their 3′ ends (red), and for all Sima de los Huesos cave bear sequences from the U. deningeri sample9 (dotted line). b, C to T substitution frequencies at the first and last base of sequences in different fragment length bins.

  4. Bayesian phylogenetic tree of hominin mitochondrial relationships based on the Sima de los Huesos mtDNA sequence determined using the inclusive filtering criteria.
    Figure 4: Bayesian phylogenetic tree of hominin mitochondrial relationships based on the Sima de los Huesos mtDNA sequence determined using the inclusive filtering criteria.

    All nodes connecting the denoted hominin groups are supported with posterior probability of 1. The tree was rooted using chimpanzee and bonobo mtDNA genomes. The scale bar denotes substitutions per site.

  5. Size distribution of all overlap-merged sequences generated by shotgun sequencing (before mapping).
    Extended Data Fig. 1: Size distribution of all overlap-merged sequences generated by shotgun sequencing (before mapping).
  6. 5[prime] and 3[prime] C to T substitution frequencies plotted against the number of unique mitochondrial sequences retrieved from each sample library.
    Extended Data Fig. 2: 5′ and 3′ C to T substitution frequencies plotted against the number of unique mitochondrial sequences retrieved from each sample library.

    Libraries prepared from re-extracted pellets or surface material are highlighted in colour.

  7. Sequence length distribution of unique sequences.
    Extended Data Fig. 3: Sequence length distribution of unique sequences.

    The distribution obtained from the Sima de los Huesos cave bear is shown for comparison.

  8. Sequence coverage of the mitochondrial genome obtained from sequences with terminal C to T substitutions.
    Extended Data Fig. 4: Sequence coverage of the mitochondrial genome obtained from sequences with terminal C to T substitutions.
  9. Sequence coverage of the mitochondrial genome plotted separately for both capture probe sets used (based on sequences with a C to T substitution at the first or last alignment position).
    Extended Data Fig. 5: Sequence coverage of the mitochondrial genome plotted separately for both capture probe sets used (based on sequences with a C to T substitution at the first or last alignment position).
  10. Complete view of the mid-point rooted phylogenetic tree constructed with a Bayesian approach under a GTR[thinsp]+[thinsp]I[thinsp]+[thinsp][Gamma] model of sequence evolution using the Sima de los Huesos consensus sequence generated with inclusive filters as well as 54 present-day humans, 9 ancient humans, 7 Neanderthals, 2 Denosivans, 22 bonobos and 24 chimpanzees.
    Extended Data Fig. 6: Complete view of the mid-point rooted phylogenetic tree constructed with a Bayesian approach under a GTR+I+Γ model of sequence evolution using the Sima de los Huesos consensus sequence generated with inclusive filters as well as 54 present-day humans, 9 ancient humans, 7 Neanderthals, 2 Denosivans, 22 bonobos and 24 chimpanzees.

    The posterior probabilities are provided for the major nodes.

Tables

  1. Characteristics of all libraries prepared for this study
    Extended Data Table 1: Characteristics of all libraries prepared for this study
  2. Results from shallow shotgun sequencing of a subset of libraries
    Extended Data Table 2: Results from shallow shotgun sequencing of a subset of libraries
  3. Number of sequences retained in the sample libraries after each step of processing and filtering
    Extended Data Table 3: Number of sequences retained in the sample libraries after each step of processing and filtering
  4. Inferred time to the most recent common ancestor (TMRCA) of the modern human, Neanderthal, chimpanzee and bonobo mtDNAs, as well as divergence estimates for human/chimpanzee and bonobo/chimpanzee mtDNA (continuation of Table 1)
    Extended Data Table 4: Inferred time to the most recent common ancestor (TMRCA) of the modern human, Neanderthal, chimpanzee and bonobo mtDNAs, as well as divergence estimates for human/chimpanzee and bonobo/chimpanzee mtDNA (continuation of Table 1)

Accession codes

Referenced accessions

GenBank/EMBL/DDBJ

References

  1. Carbonell, E. et al. The first hominin of Europe. Nature 452, 465469 (2008)
  2. Arsuaga, J. L., Martinez, I., Gracia, A. & Lorenzo, C. The Sima de los Huesos crania (Sierra de Atapuerca, Spain). A comparative study. J. Hum. Evol. 33, 219281 (1997)
  3. Arsuaga, J. L. et al. Size variation in Middle Pleistocene humans. Science 277, 10861088 (1997)
  4. Bermúdez de Castro, J. M. & Nicolas, M. E. Palaeodemography of the Atapuerca-SH Middle Pleistocene hominid sample. J. Hum. Evol. 33, 333355 (1997)
  5. Bischoff, J. L. et al. Geology and preliminary dating of the hominid-bearing sedimentary fill of the Sima de los Huesos Chamber, Cueva Mayor of the Sierra de Atapuerca, Burgos, Spain. J. Hum. Evol. 33, 129154 (1997)
  6. Martínez, I. & Arsuaga, J. L. The temporal bones from Sima de los Huesos Middle Pleistocene site (Sierra de Atapuerca, Spain). A phylogenetic approach. J. Hum. Evol. 33, 283318 (1997)
  7. Martinón-Torres, M., Bermudez de Castro, J. M., Gomez-Robles, A., Prado-Simon, L. & Arsuaga, J. L. Morphological description and comparison of the dental remains from Atapuerca-Sima de los Huesos site (Spain). J. Hum. Evol. 62, 758 (2012)
  8. Stringer, C. The status of Homo heidelbergensis (Schoetensack 1908). Evol. Anthropol. 21, 101107 (2012)
  9. Reich, D. et al. Genetic history of an archaic hominin group from Denisova Cave in Siberia. Nature 468, 10531060 (2010)
  10. Meyer, M. et al. A high-coverage genome sequence from an archaic Denisovan individual. Science 338, 222226 (2012)
  11. Ortega, A. I. et al. Evolution of multilevel caves in the Sierra de Atapuerca (Burgos, Spain) and its relation to human occupation. Geomorphology 196, 122137 (2013)
  12. Arsuaga, J. L. et al. Sima de los Huesos (Sierra de Atapuerca, Spain). The site. J. Hum. Evol. 33, 109127 (1997)
  13. Valdiosera, C. et al. Typing single polymorphic nucleotides in mitochondrial DNA as a way to access Middle Pleistocene DNA. Biol. Lett. 2, 601603 (2006)
  14. Dabney, J. et al. Complete mitochondrial genome sequence of a Middle Pleistocene cave bear reconstructed from ultrashort DNA fragments. Proc. Natl Acad. Sci. USA 110, 1575815763 (2013)
  15. Willerslev, E. et al. Ancient biomolecules from deep ice cores reveal a forested southern Greenland. Science 317, 111114 (2007)
  16. Orlando, L. et al. Recalibrating Equus evolution using the genome sequence of an early Middle Pleistocene horse. Nature 499, 7478 (2013)
  17. Carretero, J. M. et al. Stature estimation from complete long bones in the Middle Pleistocene humans from the Sima de los Huesos, Sierra de Atapuerca (Spain). J. Hum. Evol. 62, 242255 (2012)
  18. Gansauge, M. T. & Meyer, M. Single-stranded DNA library preparation for the sequencing of ancient or damaged DNA. Nature Protocols 8, 737748 (2013)
  19. Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 17541760 (2009)
  20. Briggs, A. W. et al. Patterns of damage in genomic DNA sequences from a Neandertal. Proc. Natl Acad. Sci. USA 104, 1461614621 (2007)
  21. Krause, J. et al. A complete mtDNA genome of an early modern human from Kostenki, Russia. Curr. Biol. 20, 231236 (2010)
  22. Sawyer, S., Krause, J., Guschanski, K., Savolainen, V. & Paabo, S. Temporal patterns of nucleotide misincorporations and DNA fragmentation in ancient DNA. PLoS ONE 7, e34131 (2012)
  23. Krause, J. et al. The complete mitochondrial DNA genome of an unknown hominin from southern Siberia. Nature 464, 894897 (2010)
  24. Ronquist, F. & Huelsenbeck, J. P. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19, 15721574 (2003)
  25. Shapiro, B. et al. A Bayesian phylogenetic method to estimate unknown sequence ages. Mol. Biol. Evol. 28, 879887 (2011)
  26. Arsuaga, J. L., Martinez, I., Gracia, A., Carretero, J. M. & Carbonell, E. Three new human skulls from the Sima de los Huesos Middle Pleistocene site in Sierra de Atapuerca, Spain. Nature 362, 534537 (1993)
  27. Arsuaga, J. L. Colloquium paper: terrestrial apes and phylogenetic trees. Proc. Natl Acad. Sci. USA 107 (Suppl. 2). 89108917 (2010)
  28. Hublin, J. J. Out of Africa: Modern human origins special feature: The origin of Neandertals. Proc. Natl Acad. Sci. USA 106, 1602216027 (2009)
  29. Mounier, A., Marchal, F. & Condemi, S. Is Homo heidelbergensis a distinct species? New insight on the Mauer mandible. J. Hum. Evol. 56, 219246 (2009)
  30. Carbonell, E. et al. An Early Pleistocene hominin mandible from Atapuerca-TD6, Spain. Proc. Natl Acad. Sci. USA 102, 56745678 (2005)
  31. Dabney, J. & Meyer, M. Length and GC-biases during sequencing library amplification: a comparison of various polymerase-buffer systems with ancient and modern DNA sequencing libraries. Biotechniques 52, 8794 (2012)
  32. Kircher, M., Sawyer, S. & Meyer, M. Double indexing overcomes inaccuracies in multiplex sequencing on the Illumina platform. Nucleic Acids Res. 40, e3 (2012)
  33. Fu, Q. et al. DNA analysis of an early modern human from Tianyuan Cave, China. Proc. Natl Acad. Sci. USA 110, 22232227 (2013)
  34. Rohland, N. & Reich, D. Cost-effective, high-throughput DNA sequencing libraries for multiplexed target capture. Genome Res. 22, 939946 (2012)
  35. Maricic, T., Whitten, M. & Paabo, S. Multiplexed DNA sequence capture of mitochondrial genomes using PCR products. PLoS ONE 5, e14004 (2010)
  36. Renaud, G., Kircher, M., Stenzel, U. & Kelso, J. freeIbis: an efficient basecaller with calibrated quality scores for Illumina sequencers. Bioinformatics 29, 12081209 (2013)
  37. Kircher, M. Analysis of high-throughput ancient DNA sequencing data. Methods Mol. Biol. 840, 197228 (2012)
  38. Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 20782079 (2009)
  39. Ingman, M., Kaessmann, H., Paabo, S. & Gyllensten, U. Mitochondrial genome variation and the origin of modern humans. Nature 408, 708713 (2000)
  40. Fu, Q. et al. A revised timescale for human evolution based on ancient mitochondrial genomes. Curr. Biol. 23, 553559 (2013)
  41. Briggs, A. W. et al. Targeted retrieval and analysis of five Neandertal mtDNA genomes. Science 325, 318321 (2009)
  42. Zsurka, G. et al. Distinct patterns of mitochondrial genome diversity in bonobos (Pan paniscus) and humans. BMC Evol. Biol. 10, 270 (2010)
  43. Bjork, A., Liu, W., Wertheim, J. O., Hahn, B. H. & Worobey, M. Evolutionary history of chimpanzees inferred from complete mitochondrial genomes. Mol. Biol. Evol. 28, 615623 (2011)
  44. Katoh, K. & Standley, D. M. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772780 (2013)
  45. Posada, D. & Crandall, K. A. MODELTEST: testing the model of DNA substitution. Bioinformatics 14, 817818 (1998)
  46. Drummond, A. J. & Rambaut, A. BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evol. Biol. 7, 214 (2007)
  47. Kass, R. E. & Raftery, A. E. Bayes Factors. J. Am. Stat. Assoc. 90, 773795 (1995)
  48. Horai, S. et al. Man’s place in Hominoidea revealed by mitochondrial DNA genealogy. J. Mol. Evol. 35, 3243 (1992)
  49. Green, R. E. et al. A complete Neandertal mitochondrial genome sequence determined by high-throughput sequencing. Cell 134, 416426 (2008)
  50. Stone, A. C. et al. More reliable estimates of divergence times in Pan using complete mtDNA sequences and accounting for population structure. Phil. Trans. R. Soc. Lond. B 365, 32773288 (2010)
  51. Soares, P. et al. Correcting for purifying selection: an improved human mitochondrial molecular clock. Am. J. Hum. Genet. 84, 740759 (2009)

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Author information

Affiliations

  1. Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, Deutscher Platz 6, 04103 Leipzig, Germany

    • Matthias Meyer,
    • Qiaomei Fu,
    • Ayinuer Aximu-Petri,
    • Isabelle Glocke,
    • Birgit Nickel &
    • Svante Pääbo
  2. Key Laboratory of Vertebrate Evolution and Human Origins of Chinese Academy of Sciences, Institute of Vertebrate Paleontology and Paleoanthropology, Chinese Academy of Sciences, Beijing 100044, China

    • Qiaomei Fu
  3. Centro de Investigación Sobre la Evolución y Comportamiento Humanos, Universidad Complutense de Madrid–Instituto de Salud Carlos III, 28029 Madrid, Spain

    • Juan-Luis Arsuaga,
    • Ignacio Martínez &
    • Ana Gracia
  4. Departamento de Paleontología, Facultad de Ciencias Geológicas, Universidad Complutense de Madrid, 28040 Madrid, Spain

    • Juan-Luis Arsuaga
  5. Área de Paleontología, Depto. de Geografía y Geología, Universidad de Alcalá, Alcalá de Henares, 28871 Madrid, Spain

    • Ignacio Martínez &
    • Ana Gracia
  6. Centro Nacional de Investigación sobre la Evolución Humana, Paseo Sierra de Atapuerca, 09002 Burgos, Spain

    • José María Bermúdez de Castro
  7. Institut Català de Paleoecologia Humana i Evolució Social, C/Marcel·lí Domingo s/n (Edifici W3), Campus Sescelades, 43007 Tarragona, Spain

    • Eudald Carbonell
  8. Àrea de Prehistòria, Dept. d’Història i Història de l’Art, Univ. Rovira i Virgili, Fac. de Lletres, Av. Catalunya, 35, 43002 Tarragona, Spain

    • Eudald Carbonell

Contributions

M.M. designed the experiments and analysed the data; Q.F. performed phylogenetic analyses; A.A., I.G. and B.N. performed the experiments; J.-L.A., I.M., A.G., J.M.B. and E.C. excavated the fossil and provided expert archaeological and anthropological information; J.-L.A. and S.P. were involved in study design; and M.M., J.-L.A. and S.P. wrote the manuscript.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

The Sima de los Huesos mtDNA consensus sequence (based on the inclusive filtering criteria) is deposited in GenBank under accession number KF683087.

Author details

Extended data figures and tables

Extended Data Figures

  1. Extended Data Figure 1: Size distribution of all overlap-merged sequences generated by shotgun sequencing (before mapping). (67 KB)
  2. Extended Data Figure 2: 5′ and 3′ C to T substitution frequencies plotted against the number of unique mitochondrial sequences retrieved from each sample library. (127 KB)

    Libraries prepared from re-extracted pellets or surface material are highlighted in colour.

  3. Extended Data Figure 3: Sequence length distribution of unique sequences. (77 KB)

    The distribution obtained from the Sima de los Huesos cave bear is shown for comparison.

  4. Extended Data Figure 4: Sequence coverage of the mitochondrial genome obtained from sequences with terminal C to T substitutions. (351 KB)
  5. Extended Data Figure 5: Sequence coverage of the mitochondrial genome plotted separately for both capture probe sets used (based on sequences with a C to T substitution at the first or last alignment position). (323 KB)
  6. Extended Data Figure 6: Complete view of the mid-point rooted phylogenetic tree constructed with a Bayesian approach under a GTR+I+Γ model of sequence evolution using the Sima de los Huesos consensus sequence generated with inclusive filters as well as 54 present-day humans, 9 ancient humans, 7 Neanderthals, 2 Denosivans, 22 bonobos and 24 chimpanzees. (60 KB)

    The posterior probabilities are provided for the major nodes.

Extended Data Tables

  1. Extended Data Table 1: Characteristics of all libraries prepared for this study (510 KB)
  2. Extended Data Table 2: Results from shallow shotgun sequencing of a subset of libraries (485 KB)
  3. Extended Data Table 3: Number of sequences retained in the sample libraries after each step of processing and filtering (118 KB)
  4. Extended Data Table 4: Inferred time to the most recent common ancestor (TMRCA) of the modern human, Neanderthal, chimpanzee and bonobo mtDNAs, as well as divergence estimates for human/chimpanzee and bonobo/chimpanzee mtDNA (continuation of Table 1) (116 KB)

Additional data