Understanding how extinct species are related to each other or to their living relatives is often a difficult task. Many extinct species have been identified only from incomplete fragments of some of their bones. However, even if complete skeletons have been found, determining the relationships between species can be tricky because researchers often have to rely solely on the shapes of the bones.

It is sometimes possible to retrieve DNA sequences from fossil bones. This is easier with younger fossils and those that have been recovered from cold environments. Ancient DNA sequences have been retrieved from only a few fossils older than 100,000 years, but such DNA sequences can be tremendously useful in determining how different species are related to each other.

Today there are three living elephant species: the African forest elephant, the African savanna elephant and the Asian elephant. However, there are many extinct elephant species. For example, the European straight-tusked elephant went extinct at least 30,000 years ago, although most of the fossils that have been discovered are at least 100,000 years old. Straight-tusked elephants are generally assumed to be closely related to the Asian elephant, but this conclusion had been based solely on reconstructing skeletons.

Meyer et al. have now obtained DNA sequences from fossils of four straight-tusked elephants ranging from around 120,000 to 240,000 years in age. These sequences were analysed to determine how straight-tusked elephants are related to the three living elephant species and the extinct mammoth, the DNA sequences for which can be found in public databases. The analyses revealed that straight-tusked elephants are in fact most closely related to the African forest elephant, not the Asian elephant as previously thought.

This result completely changes our picture of elephant evolution and suggests that it is extremely difficult to determine elephant relationships based on the shape of their skeleton alone. It also shows that the African elephant lineage was not restricted to the African continent (the place where all elephant lineages originated), but that it also left Africa.

Overall, the results presented by Meyer et al. confirm that DNA sequences are of critical importance for understanding the evolution of animals. Future research should include obtaining DNA sequences from additional extinct elephant species as well as careful re-evaluation of skeletal measurements for reconstructing elephant evolution.

In the late Miocene in Africa, the last of several major radiations within Proboscidea gave rise to the family Elephantidae, which comprises living elephants and their extinct relatives including mammoths (genus Mammuthus) and various dwarf elephant species from Mediterranean islands. The three living elephant species (the African savanna elephant, Loxodonta africana, the African forest elephant, L. cyclotis and the Asian elephant, Elephas maximus), represent the last remnants of this family and of the formerly much more widely distributed and species-rich order Proboscidea. Apart from mammoths, the elephant genus with the most abundant fossil record in Eurasia is Palaeoloxodon (straight-tusked elephants; Figure 1), which appears in Eurasia around 0.75 million years ago (Ma) (Lister, 2016). Based on morphological analyses, Palaeoloxodon is widely accepted as being more closely related to the extant Asian elephant than to mammoths or extant African elephants (Shoshani et al., 2007; Todd, 2010) and is often subsumed into the genus Elephas (Maglio, 1973; Sanders et al., 2010). Across its range from Western Europe to Japan, Palaeoloxodon probably comprised several species (Shoshani et al., 2007), and, based on morphological comparisons, all of them are considered to be derived from the African Palaeoloxodon (or Elephas) recki (Maglio, 1973; Saegusa and Gilbert, 2008), which was the predominant proboscidean lineage in Africa during the Pliocene and Pleistocene but went extinct around 100 thousand years ago (ka) (Owen-Smith, 2013). Straight-tusked elephants may have survived in mainland Eurasia until around 35 ka, although the youngest reliably dated remains are from the last interglacial, 115–130 ka (Stuart, 2005).

Figure 1

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Recent technological progress has pushed back the temporal limit of ancient DNA research, enabling, for example, recovery of a low coverage genome of a ~700,000 year-old horse preserved in permafrost (Orlando et al., 2013). For more temperate regions, however, evidence of DNA preservation reaching far beyond the last glacial period is still limited to a single locality, Sima de los Huesos in Spain, where DNA has been recovered from ~430 ka old hominin and bear remains (Dabney et al., 2013; Meyer et al., 2016). While genetic analyses of the extinct interglacial fauna remain a challenging undertaking, recent advances in ancient DNA extraction (Dabney et al., 2013) and sequencing library construction (Meyer et al., 2012) have improved access to highly degraded DNA.

To better understand the evolutionary relationships between the extinct straight-tusked elephants and other elephant species, we attempted DNA extraction and sequencing from several P. antiquus fossils, four of which we investigated in depth. Three of these, which were all unambiguously assigned to P. antiquus based on their morphology, were from Neumark-Nord (NN) 1 in Germany, a fossil-rich site that has been proposed to date to MIS 5e (~120 ka) or MIS 7 (~244 ka) or both (Mania, 2010; Schüler, 2010; Penkman, 2010). This site has yielded one of the largest collections of P. antiquus remains known to date. The fourth fossil was recovered during recent active mining in the travertine deposits of Weimar-Ehringsdorf (WE), Germany, a quarry that has for more than a century yielded a rich collection of fossils representing a typical European interglacial fauna (Kahlke, 1975). Weimar-Ehringsdorf is best known for the discovery of Neanderthal remains in the early 20th century, and the assemblage is dated to MIS 7 (Mallick and Frank, 2002). The Paleoloxodon bone fragment from Weimar-Ehringsdorf is morphologically undiagnostic with respect to species. However, it was found in the Lower Travertine, which was dated to ~233 ka (Schüler, 2003) and where P. antiquus is the only elephantid found so far. We performed DNA extraction, library preparation, hybridization capture and high-throughput sequencing on all four fossils (Figure 2—source data 1) and obtained full mitochondrial genome sequences for all of them (Figure 2—figure supplement 1). All sequences show short fragment lengths (Figure 2—figure supplement 2) and signals of cytosine deamination compatible with the old age of the specimens (Figure 2—figure supplement 3).

We inferred a phylogeny using the four Paleoloxodon mitochondrial genomes and mitochondrial genomes from 16 M. primigenius, 2 E. maximus and 13 Loxodonta individuals. The latter were chosen for a diversity of haplotypes, including forest elephant derived (‘F-clade’) haplotypes as well as ‘S-clade’ haplotypes found only among savanna elephants (Debruyne, 2005). For calibration, we used an estimated divergence of the African elephant lineage from that of Asian elephants and mammoths of 6.6–8.6 Ma (Rohland et al., 2007). Surprisingly, P. antiquus did not cluster with E. maximus, as hypothesized from morphological analyses. Instead, it fell within the mito-genetic diversity of extant L. cyclotis, with very high statistical support (Figure 2). The four straight-tusked elephants did not cluster together within this mitochondrial clade, but formed two separate lineages that share a common ancestor with an extant L. cyclotis lineage 0.7–1.6 Ma (NN) and 1.5–3.0 Ma (WE) ago, respectively. However, mitochondrial DNA represents a single, maternally inherited locus and does not reflect the full evolutionary history of populations or species. Furthermore, the transfer of mitochondrial DNA between hybridizing species is not unusual when gene flow is strongly male-mediated (Petit and Excoffier, 2009; Li et al., 2016; Cahill et al., 2013), as is the case with elephants. For example, mitochondrial sequences of the F-clade have also been found in some L. africana individuals (Debruyne, 2005) despite the very substantial divergence of their nuclear genomes (Roca et al., 2005; Rohland et al., 2010), a pattern that has been attributed to mitochondrial gene flow from forest to savanna elephants (Roca et al., 2005).

Figure 2 with 5 supplements see all

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Figure 2—source data 1

This spreadsheet contains summary statistics of all sequence data generated in this study, the sequences of PCR primers used for reconstructing mtDNA sequences of extant elephants, as well as amino acid racemization data on opercula of Bithynia tentaculata from Amersfoort.

https://doi.org/10.7554/eLife.25413.005

Download elife-25413-fig2-data1-v1.xls

We therefore performed shotgun sequencing of DNA libraries prepared from the two best-preserved NN individuals (a petrous bone and a molar) to recover nuclear DNA sequences. When mapped to the L. africana reference genome, 39% and 28% of the sequence reads generated from these specimens were identified as elephant, respectively (Figure 2—source data 1). A neighbor-joining phylogenetic tree based on ~770M (petrous) and ~210M (molar) base pairs of P. antiquus nuclear DNA placed P. antiquus and L. cyclotis as sister taxa to the exclusion of L. africana (Figure 2). A tree with identical topology was obtained using coding sequences only and a maximum likelihood approach (Figure 2—figure supplement 4). Despite the high sequence error rates associated with the low-coverage genomes generated from the P. antiquus specimens, all nodes in the nuclear trees show maximal bootstrap support. The mitochondrial and nuclear phylogenies thus support a sister group relationship between P. antiquus and L. cyclotis.

Despite their geographical proximity, the WE and NN specimens are found in different positions in the mitochondrial tree. Given that the three NN specimens show highly similar mitogenome sequences, we considered whether the sites date to different interglacials. Electron Spin Resonance (ESR) dating of tooth enamel has been applied to both sites, suggesting an age of ~117 ka (range 97–142 ka [Schüler, 2010]) for the NN1 layers from which our samples originate and of 233 ka (range 216–250 ka [Schüler, 2003]) for the WE specimen. In order to better estimate the age of the NN1 site, we used amino acid racemization of snail opercula (Penkman et al., 2011), including samples from the continental Eemian type-site of Amersfoort (Zagwijn, 1961) (Figure 2—source data 1), which is correlated with MIS 5e (Cleveringa et al., 2000). The NN1 opercula show similar (perhaps slightly lower) levels of biomolecular degradation compared to Amersfoort, suggesting an Eemian age for NN1 (Figure 2—figure supplement 5). Importantly, NN1 shows very similar levels of amino acid racemization in intra-crystalline protein decomposition as a second site at Neumark-Nord (NN2), indicating that both sites are of the same age. Considerable evidence supports an Eemian age for NN2, including palaeomagnetic data that shows a correlation with the MIS 5e Blake event and thermoluminescence dating of flint to ~126 ± 6 ka (Sier et al., 2011). These results therefore indicate an Eemian age also for NN1. Since the WE specimen likely dates to the previous interglacial, this suggests that the very different mitogenomes between WE and NN1 may reflect the contraction and re-expansion of the range of P. antiquus across glacial cycles.

Our results have implications both for understanding elephant evolutionary history and for the use of morphological data to decipher phylogenetic relationships among elephants. The strongly supported mitochondrial and nuclear DNA phylogenies clearly demonstrate that Palaeoloxodon antiquus is more closely related to Loxodonta than to Elephas (Figure 3), suggesting that Elephas antiquus should not be used synonymously for Paleoloxodon antiquus when referring to the taxon. The new phylogeny suggests a remarkable degree of evolutionary transformation, from an ancestor that possessed the features of the cranium and dentition of Loxodonta, shared by both L. africana and L. cyclotis (Maglio, 1973; Sanders et al., 2010), to a descendant that is highly similar to Elephas (sensu stricto, the lineage of the Asian elephant) in many morphological features. However, it should be noted that currently available genomic data from elephantids only allow for reconstructing the broad picture of elephant evolution. More complex evolutionary scenarios are conceivable, which might explain the presence of some Elephas-like traits in P. antiquus. These could for example involve gene flow, as has been shown for L. africana and L. cyclotis based on mitochondrial evidence (Roca et al., 2005). In addition, the very large effective population size of the forest elephants (Rohland et al., 2010) could have allowed the retention of ancestral traits by incomplete lineage sorting.

Figure 3

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In summary, the molecular results presented here urge for a re-examination of morphology across the Elephantidae. This is especially important as the fossil record for elephants dates back several million years, well beyond the survival of ancient DNA. If, for example, P. recki, which was the most abundant Pleistocene elephant species in Africa, is indeed ancestral to P. antiquus and thus also represents a member of the Loxodonta lineage, the interpretation of the fossil record of elephantids in Africa is in strong need of revision. Furthermore, in contrast to the genera Mammuthus and Elephas, which also had their origin in Africa, the lineage of Loxodonta is generally assumed never to have left Africa. Although Osborn, 1942 placed Palaeoloxodon in the Loxodontinae on the basis of several cranial characters, later authors (Shoshani et al., 2007; Todd, 2010) have rejected this placement in favor of a placement in Elephantinae, restricting Loxodonta (and Loxodontinae) geographically to Africa. However, our data reveal that the Loxodonta lineage (as Paleoloxodon) also colonized the Eurasian continent. Last, the finding that L. africana is genetically more distant from L. cyclotis than is P. antiquus strongly supports previous evidence that urged recognition of L. cyclotis and L. africana as distinct species and underlines the importance of conservation efforts directed toward African forest elephants.

1. 1. Palkopoulou E
   2. Baleka S
   3. Mallick S
   4. Rohland N
   5. Reich D
   6. Hofreiter M

   (2017) Nuclear DNA sequences from two Palaeoloxodon antiquus fossils

   Publicly available at EBI European Nucleotide Archive (accession no: PRJEB18563).

   http://www.ebi.ac.uk/ena/data/search?query=PRJEB18563
2. 1. Ishida I
   2. Roca AL

   (2017) Mitochondrial DNA sequences from 3 Loxodonta cyclotis individuals

   Publicly available at NCBI GenBank (accession no: KY616976).

   https://www.ncbi.nlm.nih.gov/nuccore/KY616976
3. 1. Ishida I
   2. Roca AL

   (2017) Mitochondrial DNA sequences from 3 Loxodonta cyclotis individuals

   Publicly available at NCBI GenBank (accession no: KY616978).

   https://www.ncbi.nlm.nih.gov/nuccore/KY616978
4. 1. Ishida I
   2. Roca AL

   (2017) Mitochondrial DNA sequences from 3 Loxodonta cyclotis individuals

   Publicly available at NCBI GenBank (accession no: KY616979).

   https://www.ncbi.nlm.nih.gov/nuccore/KY616979
5. 1. Meyer M
   2. Baleka S
   3. Stiller M
   4. Nagel S
   5. Nickel B
   6. Schüler T
   7. Hofreiter M

   (2017) Mitochondrial DNA sequences from 4 Palaeoloxodon antiquus fossils

   Publicly available at NCBI GenBank (accession no: KY499555).

   https://www.ncbi.nlm.nih.gov/nuccore/KY499555
6. 1. Meyer M
   2. Baleka S
   3. Stiller M
   4. Nagel S
   5. Nickel B
   6. Schüler T
   7. Hofreiter M

   (2017) Mitochondrial DNA sequences from 4 Palaeoloxodon antiquus fossils

   Publicly available at NCBI GenBank (accession no: KY499556).

   https://www.ncbi.nlm.nih.gov/nuccore/KY499556
7. 1. Meyer M
   2. Baleka S
   3. Stiller M
   4. Nagel S
   5. Nickel B
   6. Schüler T
   7. Hofreiter M

   (2017) Mitochondrial DNA sequences from 4 Palaeoloxodon antiquus fossils

   Publicly available at NCBI GenBank (accession no: KY499557).

   https://www.ncbi.nlm.nih.gov/nuccore/KY499557
8. 1. Meyer M
   2. Baleka S
   3. Stiller M
   4. Nagel S
   5. Nickel B
   6. Schüler T
   7. Hofreiter M

   (2017) Mitochondrial DNA sequences from 4 Palaeoloxodon antiquus fossils

   Publicly available at NCBI GenBank (accession no: KY499558).

   https://www.ncbi.nlm.nih.gov/nuccore/KY499558

1. 1. Brandt AL
   2. Ishida Y
   3. Georgiadis NJ
   4. Roca AL

   (2012) Forest elephant mitochondrial genomes reveal that elephantid diversification in Africa tracked climate transitions

   Molecular Ecology 21:1175–1189.

   https://doi.org/10.1111/j.1365-294X.2012.05461.x

   • PubMed
   • Google Scholar
2. 1. Cahill JA
   2. Green RE
   3. Fulton TL
   4. Stiller M
   5. Jay F
   6. Ovsyanikov N
   7. Salamzade R
   8. St John J
   9. Stirling I
   10. Slatkin M
   11. Shapiro B

   (2013) Genomic evidence for island population conversion resolves conflicting theories of polar bear evolution

   PLoS Genetics 9:e1003345.

   https://doi.org/10.1371/journal.pgen.1003345

   • PubMed
   • Google Scholar
3. 1. Cleveringa P
   2. Meijer T
   3. van Leeuwen RJW
   4. de Wolf H
   5. Pouwer R
   6. Lissenberg T
   7. Burger AW

   (2000) The Eemian stratotype locality at Amersfoort in the central Netherlands: a re-evaluation of old and new data

   Netherlands Journal of Geosciences 79:197–216.

   https://doi.org/10.1017/S0016774600023659

   • Google Scholar
4. 1. Dabney J
   2. Meyer M

   (2012) Length and GC-biases during sequencing library amplification: a comparison of various polymerase-buffer systems with ancient and modern DNA sequencing libraries

   BioTechniques 52:87–94.

   https://doi.org/10.2144/000113809

   • PubMed
   • Google Scholar
5. 1. Dabney J
   2. Knapp M
   3. Glocke I
   4. Gansauge MT
   5. Weihmann A
   6. Nickel B
   7. Valdiosera C
   8. García N
   9. Pääbo S
   10. Arsuaga JL
   11. Meyer M

   (2013) Complete mitochondrial genome sequence of a Middle Pleistocene cave bear reconstructed from ultrashort DNA fragments

   PNAS 110:15758–15763.

   https://doi.org/10.1073/pnas.1314445110

   • PubMed
   • Google Scholar
6. 1. Debruyne R

   (2005) A case study of apparent conflict between molecular phylogenies: the interrelationships of African elephants

   Cladistics : The International Journal of the Willi Hennig Society 21:31–50.

   https://doi.org/10.1111/j.1096-0031.2004.00044.x

   • Google Scholar
7. 1. Drummond AJ
   2. Ho SY
   3. Phillips MJ
   4. Rambaut A

   (2006) Relaxed phylogenetics and dating with confidence

   PLoS Biology 4:e88.

   https://doi.org/10.1371/journal.pbio.0040088

   • PubMed
   • Google Scholar
8. 1. Drummond AJ
   2. Suchard MA
   3. Xie D
   4. Rambaut A

   (2012) Bayesian phylogenetics with BEAUti and the BEAST 1.7

   Molecular Biology and Evolution 29:1969–1973.

   https://doi.org/10.1093/molbev/mss075

   • PubMed
   • Google Scholar
9. Software

   1. Felsenstein J

   (2005)

   PHYLIP (Phylogeny Inference Package), version Version 3.6

   Distributed by the Author. Department of Genome Sciences, University of Washington, Seattle.
10. 1. Fu Q
    2. Meyer M
    3. Gao X
    4. Stenzel U
    5. Burbano HA
    6. Kelso J
    7. Pääbo S

    (2013) DNA analysis of an early modern human from Tianyuan cave, China

    PNAS 110:2223–2227.

    https://doi.org/10.1073/pnas.1221359110

    • PubMed
    • Google Scholar
11. 1. Gamba C
    2. Jones ER
    3. Teasdale MD
    4. McLaughlin RL
    5. Gonzalez-Fortes G
    6. Mattiangeli V
    7. Domboróczki L
    8. Kővári I
    9. Pap I
    10. Anders A
    11. Whittle A
    12. Dani J
    13. Raczky P
    14. Higham TF
    15. Hofreiter M
    16. Bradley DG
    17. Pinhasi R

    (2014) Genome flux and stasis in a five millennium transect of European prehistory

    Nature Communications 5:5257.

    https://doi.org/10.1038/ncomms6257

    • PubMed
    • Google Scholar
12. 1. Gansauge MT
    2. Meyer M

    (2013) Single-stranded DNA library preparation for the sequencing of ancient or damaged DNA

    Nature Protocols 8:737–748.

    https://doi.org/10.1038/nprot.2013.038

    • PubMed
    • Google Scholar
13. 1. Gill MS
    2. Lemey P
    3. Faria NR
    4. Rambaut A
    5. Shapiro B
    6. Suchard MA

    (2013) Improving Bayesian population dynamics inference: a coalescent-based model for multiple loci

    Molecular Biology and Evolution 30:713–724.

    https://doi.org/10.1093/molbev/mss265

    • PubMed
    • Google Scholar
14. 1. Hill RL

    (1965)

    Hydrolysis of proteins

    Advances in Protein Chemistry 20:37–107.

    • PubMed
    • Google Scholar
15. 1. Ishida Y
    2. Georgiadis NJ
    3. Hondo T
    4. Roca AL

    (2013) Triangulating the provenance of African elephants using mitochondrial DNA

    Evolutionary Applications 6:253–265.

    https://doi.org/10.1111/j.1752-4571.2012.00286.x

    • PubMed
    • Google Scholar
16. 1. Kahlke HD

    (1975)

    Das Pleistozän von Weimar-Ehringsdorf, Teil 2

    Abhandl. Zentr. Geol. Inst 23:1–596.

    • Google Scholar
17. 1. Katoh K
    2. Misawa K
    3. Kuma K
    4. Miyata T

    (2002) MAFFT: a novel method for rapid multiple sequence alignment based on fast fourier transform

    Nucleic Acids Research 30:3059–3066.

    https://doi.org/10.1093/nar/gkf436

    • PubMed
    • Google Scholar
18. 1. Katoh K
    2. Standley DM

    (2013) MAFFT multiple sequence alignment software version 7: improvements in performance and usability

    Molecular Biology and Evolution 30:772–780.

    https://doi.org/10.1093/molbev/mst010

    • PubMed
    • Google Scholar
19. 1. Kircher M
    2. Sawyer S
    3. Meyer M

    (2012) Double indexing overcomes inaccuracies in multiplex sequencing on the Illumina platform

    Nucleic Acids Research 40:e3.

    https://doi.org/10.1093/nar/gkr771

    • PubMed
    • Google Scholar
20. 1. Li H
    2. Durbin R

    (2009) Fast and accurate short read alignment with Burrows-Wheeler transform

    Bioinformatics 25:1754–1760.

    https://doi.org/10.1093/bioinformatics/btp324

    • PubMed
    • Google Scholar
21. 1. Li H
    2. Handsaker B
    3. Wysoker A
    4. Fennell T
    5. Ruan J
    6. Homer N
    7. Marth G
    8. Abecasis G
    9. Durbin R
    10. Subgroup Genome Project Data Processing

    (2009) The sequence alignment/Map format and SAMtools

    Bioinformatics 25:2078–2079.

    https://doi.org/10.1093/bioinformatics/btp352

    • Google Scholar
22. 1. Li G
    2. Davis BW
    3. Eizirik E
    4. Murphy WJ

    (2016) Phylogenomic evidence for ancient hybridization in the genomes of living cats (Felidae)

    Genome Research 26:1–11.

    https://doi.org/10.1101/gr.186668.114

    • PubMed
    • Google Scholar
23. 1. Lister AM

    (2016)

    Dating the arrival of straight-tusked elephant (Palaeoloxodon spp.) in Eurasia

    Bulletin Du Musée d'anthropologie Préhistorique De Monaco. Supplément N° 6:123–128.

    • Google Scholar
24. 1. Lynch VJ
    2. Bedoya-Reina OC
    3. Ratan A
    4. Sulak M
    5. Drautz-Moses DI
    6. Perry GH
    7. Miller W
    8. Schuster SC

    (2015) Elephantid genomes reveal the molecular bases of woolly mammoth adaptations to the Arctic

    Cell Reports 12:217–228.

    https://doi.org/10.1016/j.celrep.2015.06.027

    • PubMed
    • Google Scholar
25. 1. Maglio VJ

    (1973) Origin and evolution of the Elephantidae

    Transactions of the American Philosophical Society 63:1–149.

    https://doi.org/10.2307/1006229

    • Google Scholar
26. 1. Mallick R
    2. Frank N

    (2002) A new technique for precise uranium-series dating of travertine micro-samples

    Geochimica Et Cosmochimica Acta 66:4261–4272.

    https://doi.org/10.1016/S0016-7037(02)00999-7

    • Google Scholar
27. Book

    1. Mania D

    (2010)

    The positioning of the warm period of Neumark-Nord and its elephant-fauna in the context of the history of the earth

    In: Meller H, editors. Elefantenreich – Eine Fossilwelt in Europa. Landesamt für Denkmalpflege und Archäologie Sachsen-Anhalt – Landesmuseum für Vorgeschichte Halle. pp. 65–69.

    • Google Scholar
28. 1. Maricic T
    2. Whitten M
    3. Pääbo S

    (2010) Multiplexed DNA sequence capture of mitochondrial genomes using PCR products

    PLoS One 5:e14004.

    https://doi.org/10.1371/journal.pone.0014004

    • PubMed
    • Google Scholar
29. 1. Meyer M
    2. Kircher M

    (2010) Illumina sequencing library preparation for highly multiplexed target capture and sequencing

    Cold Spring Harbor Protocols 2010:pdb.prot5448.

    https://doi.org/10.1101/pdb.prot5448

    • PubMed
    • Google Scholar
30. 1. Meyer M
    2. Kircher M
    3. Gansauge MT
    4. Li H
    5. Racimo F
    6. Mallick S
    7. Schraiber JG
    8. Jay F
    9. Prüfer K
    10. de Filippo C
    11. Sudmant PH
    12. Alkan C
    13. Fu Q
    14. Do R
    15. Rohland N
    16. Tandon A
    17. Siebauer M
    18. Green RE
    19. Bryc K
    20. Briggs AW
    21. Stenzel U
    22. Dabney J
    23. Shendure J
    24. Kitzman J
    25. Hammer MF
    26. Shunkov MV
    27. Derevianko AP
    28. Patterson N
    29. Andrés AM
    30. Eichler EE
    31. Slatkin M
    32. Reich D
    33. Kelso J
    34. Pääbo S

    (2012) A high-coverage genome sequence from an archaic Denisovan individual

    Science 338:222–226.

    https://doi.org/10.1126/science.1224344

    • PubMed
    • Google Scholar
31. 1. Meyer M
    2. Arsuaga JL
    3. de Filippo C
    4. Nagel S
    5. Aximu-Petri A
    6. Nickel B
    7. Martínez I
    8. Gracia A
    9. Bermúdez de Castro JM
    10. Carbonell E
    11. Viola B
    12. Kelso J
    13. Prüfer K
    14. Pääbo S

    (2016) Nuclear DNA sequences from the Middle Pleistocene Sima de los Huesos hominins

    Nature 531:504–507.

    https://doi.org/10.1038/nature17405

    • PubMed
    • Google Scholar
32. 1. Orlando L
    2. Ginolhac A
    3. Zhang G
    4. Froese D
    5. Albrechtsen A
    6. Stiller M
    7. Schubert M
    8. Cappellini E
    9. Petersen B
    10. Moltke I
    11. Johnson PL
    12. Fumagalli M
    13. Vilstrup JT
    14. Raghavan M
    15. Korneliussen T
    16. Malaspinas AS
    17. Vogt J
    18. Szklarczyk D
    19. Kelstrup CD
    20. Vinther J
    21. Dolocan A
    22. Stenderup J
    23. Velazquez AM
    24. Cahill J
    25. Rasmussen M
    26. Wang X
    27. Min J
    28. Zazula GD
    29. Seguin-Orlando A
    30. Mortensen C
    31. Magnussen K
    32. Thompson JF
    33. Weinstock J
    34. Gregersen K
    35. Røed KH
    36. Eisenmann V
    37. Rubin CJ
    38. Miller DC
    39. Antczak DF
    40. Bertelsen MF
    41. Brunak S
    42. Al-Rasheid KA
    43. Ryder O
    44. Andersson L
    45. Mundy J
    46. Krogh A
    47. Gilbert MT
    48. Kjær K
    49. Sicheritz-Ponten T
    50. Jensen LJ
    51. Olsen JV
    52. Hofreiter M
    53. Nielsen R
    54. Shapiro B
    55. Wang J
    56. Willerslev E

    (2013) Recalibrating Equus evolution using the genome sequence of an early Middle Pleistocene horse

    Nature 499:74.

    https://doi.org/10.1038/nature12323

    • PubMed
    • Google Scholar
33. 1. Osborn HF

    (1942)

    Stegodontoidea, Elephantoidea

    Proboscidea: A Monograph of the Discovery, Evolution, Migration and Extinction of the Mastodonts and Elephants of the World, Stegodontoidea, Elephantoidea, II, New York, The American Museum Press.

    • Google Scholar
34. 1. Owen-Smith N

    (2013) Contrasts in the large herbivore faunas of the southern continents in the late Pleistocene and the ecological implications for human origins

    Journal of Biogeography 40:1215–1224.

    https://doi.org/10.1111/jbi.12100

    • Google Scholar
35. 1. Palkopoulou E
    2. Mallick S
    3. Skoglund P
    4. Enk J
    5. Rohland N
    6. Li H
    7. Omrak A
    8. Vartanyan S
    9. Poinar H
    10. Götherström A
    11. Reich D
    12. Dalén L

    (2015) Complete genomes reveal signatures of demographic and genetic declines in the woolly mammoth

    Current Biology 25:1395–1400.

    https://doi.org/10.1016/j.cub.2015.04.007

    • PubMed
    • Google Scholar
36. 1. Penkman KE
    2. Kaufman DS
    3. Maddy D
    4. Collins MJ

    (2008) Closed-system behaviour of the intra-crystalline fraction of amino acids in mollusc shells

    Quaternary Geochronology 3:2–25.

    https://doi.org/10.1016/j.quageo.2007.07.001

    • PubMed
    • Google Scholar
37. Book

    1. Penkman K

    (2010)

    Neumark-Nord 1: Preliminary results of the amino acid analysis

    In: Meller H, editors. Elefantenreich – Eine Fossilwelt in Europa. Landesamt für Denkmalpflege und Archäologie Sachsen-Anhalt – Landesmuseum für Vorgeschichte Halle. pp. 75–78.

    • Google Scholar
38. 1. Penkman KE
    2. Preece RC
    3. Bridgland DR
    4. Keen DH
    5. Meijer T
    6. Parfitt SA
    7. White TS
    8. Collins MJ

    (2011) A chronological framework for the British Quaternary based on Bithynia opercula

    Nature 476:446–449.

    https://doi.org/10.1038/nature10305

    • PubMed
    • Google Scholar
39. 1. Petit RJ
    2. Excoffier L

    (2009) Gene flow and species delimitation

    Trends in Ecology and Evolution 24:386–393.

    https://doi.org/10.1016/j.tree.2009.02.011

    • PubMed
    • Google Scholar
40. 1. Posada D

    (2008) jModelTest: phylogenetic model averaging

    Molecular Biology and Evolution 25:1253–1256.

    https://doi.org/10.1093/molbev/msn083

    • PubMed
    • Google Scholar
41. 1. Prüfer K
    2. Racimo F
    3. Patterson N
    4. Jay F
    5. Sankararaman S
    6. Sawyer S
    7. Heinze A
    8. Renaud G
    9. Sudmant PH
    10. de Filippo C
    11. Li H
    12. Mallick S
    13. Dannemann M
    14. Fu Q
    15. Kircher M
    16. Kuhlwilm M
    17. Lachmann M
    18. Meyer M
    19. Ongyerth M
    20. Siebauer M
    21. Theunert C
    22. Tandon A
    23. Moorjani P
    24. Pickrell J
    25. Mullikin JC
    26. Vohr SH
    27. Green RE
    28. Hellmann I
    29. Johnson PL
    30. Blanche H
    31. Cann H
    32. Kitzman JO
    33. Shendure J
    34. Eichler EE
    35. Lein ES
    36. Bakken TE
    37. Golovanova LV
    38. Doronichev VB
    39. Shunkov MV
    40. Derevianko AP
    41. Viola B
    42. Slatkin M
    43. Reich D
    44. Kelso J
    45. Pääbo S

    (2014) The complete genome sequence of a Neanderthal from the Altai Mountains

    Nature 505:43–49.

    https://doi.org/10.1038/nature12886

    • PubMed
    • Google Scholar
42. 1. Pushkina D

    (2007) The Pleistocene easternmost distribution in Eurasia of the species associated with the Eemian Palaeoloxodon antiquus assemblage

    Mammal Review 37:224–245.

    https://doi.org/10.1111/j.1365-2907.2007.00109.x

    • Google Scholar
43. 1. Renaud G
    2. Stenzel U
    3. Kelso J

    (2014) leeHom: adaptor trimming and merging for Illumina sequencing reads

    Nucleic Acids Research 42:e141.

    https://doi.org/10.1093/nar/gku699

    • PubMed
    • Google Scholar
44. 1. Roca AL
    2. Georgiadis N
    3. O'Brien SJ

    (2005) Cytonuclear genomic dissociation in African elephant species

    Nature Genetics 37:96–100.

    https://doi.org/10.1038/ng1485

    • Google Scholar
45. 1. Rohland N
    2. Malaspinas AS
    3. Pollack JL
    4. Slatkin M
    5. Matheus P
    6. Hofreiter M

    (2007) Proboscidean mitogenomics: chronology and mode of elephant evolution using mastodon as outgroup

    PLoS Biology 5:e207.

    https://doi.org/10.1371/journal.pbio.0050207

    • PubMed
    • Google Scholar
46. 1. Rohland N
    2. Reich D
    3. Mallick S
    4. Meyer M
    5. Green RE
    6. Georgiadis NJ
    7. Roca AL
    8. Hofreiter M

    (2010) Genomic DNA Sequences from mastodon and woolly mammoth reveal deep speciation of forest and savanna elephants

    PLoS Biology 8:e1000564.

    https://doi.org/10.1371/journal.pbio.1000564

    • Google Scholar
47. Book

    1. Saegusa H
    2. Gilbert WH

    (2008)

    Homo erectus in Africa, Pleistocene Evidence From the Middle Awash

    Henry W, Gilbert W. H, Asfaw B, editors. Univ. of California Press.

    • Google Scholar
48. Book

    1. Sanders WJ
    2. Gheerbrant E
    3. Harris JM
    4. Saegusa H
    5. Delmer C

    (2010)

    Cenozoic Mammals of Africa

    Werdelin L, Sanders W. J, editors. Univ. of California Press.

    • Google Scholar
49. 1. Schüler T

    (2003)

    ESR dating of a new Palaeolithic find layer of the travertine site of Weimar-Ehringsdorf (Central Germany)

    Terra Nostra 2:233–235.

    • Google Scholar
50. 1. Schüler T

    (2010)

    Elefantenreich – Eine Fossilwelt in Europa

    71–74, ESR dating of tooth enamel samples from the archaeological find horizons of Neumark-Nord, Elefantenreich – Eine Fossilwelt in Europa, Landesamt für Denkmalpflege und Archäologie Sachsen-Anhalt – Landesmuseum für Vorgeschichte Halle.

    • Google Scholar
51. 1. Shoshani J
    2. Ferretti MP
    3. Lister AM
    4. Agenbroad LD
    5. Saegusa H
    6. Mol D
    7. Takahashi K

    (2007) Relationships within the Elephantinae using hyoid characters

    Quaternary International 169-170:174–185.

    https://doi.org/10.1016/j.quaint.2007.02.003

    • Google Scholar
52. 1. Sier MJ
    2. Roebroeks W
    3. Bakels CC
    4. Dekkers MJ
    5. Brühl E
    6. De Loecker D
    7. Gaudzinski-Windheuser S
    8. Hesse N
    9. Jagich A
    10. Kindler L
    11. Kuijper WJ
    12. Laurat T
    13. Mücher HJ
    14. Penkman KE
    15. Richter D
    16. van Hinsbergen DJ

    (2011) Direct terrestrial-marine correlation demonstrates surprisingly late onset of the last interglacial in central Europe

    Quaternary Research 75:213–218.

    https://doi.org/10.1016/j.yqres.2010.11.003

    • PubMed
    • Google Scholar
53. 1. Stamatakis A

    (2014) RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies

    Bioinformatics 30:1312–1313.

    https://doi.org/10.1093/bioinformatics/btu033

    • PubMed
    • Google Scholar
54. Website

    1. Stenzel U

    (2014) Biohazard

    Bitbucket.

    https://bitbucket.org/ustenzel/biohazard
55. 1. Stuart AJ

    (2005) The extinction of woolly mammoth (Mammuthus primigenius) and straight-tusked elephant (Palaeoloxodon antiquus) in Europe

    Quaternary International 126-128:171–177.

    https://doi.org/10.1016/j.quaint.2004.04.021

    • Google Scholar
56. 1. Todd NE

    (2010) New phylogenetic analysis of the family Elephantidae based on cranial-dental morphology

    The Anatomical Record: Advances in Integrative Anatomy and Evolutionary Biology 293:74–90.

    https://doi.org/10.1002/ar.21010

    • Google Scholar
57. 1. Zagwijn WH

    (1961)

    Vegetation, climate and radiocarbon datings in the Late Pleistocene of the Netherlands. Part I: Eemian and Early Weichselian

    Mededelingen Van De Geologische Stichting, Nieuwe Serie 14:15–45.

    • Google Scholar

Thank you for submitting your article "Palaeogenomes of Eurasian straight-tusked elephants challenge the current view of elephant evolution" for consideration by eLife. Your article has been favorably evaluated by Diethard Tautz (Senior Editor) and three reviewers, one of whom is a member of our Board of Reviewing Editors. The following individuals involved in review of your submission have agreed to reveal their identity: William Sanders (Reviewer #2); Thomas Gilbert (Reviewer #3).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

The compact manuscript by Meyer and colleagues reports the sequencing of four full mitochondrial genomes and two partial nuclear genomes from Palaeoloxodon antiquus fossils. P. antiquus is a species that is thought to have been quite widespread throughout Eurasia in the Pleistocene. Phylogenetic analyses of these mitochondrial and nuclear data yield the surprising conclusion that the closest extant relatives of P. antiquus are not the Asian elephants (genus Elephas), as suggested by morphology, but the African elephants (genus Loxodonta). If the view offered by the genetic data holds, the current interpretation of the evolution of elephant morphology would need to be substantially revised. In addition, the fossils from which ancient DNA was extracted are quite old (~120-244 thousand years ago), so it is remarkable that such old samples yielded so much data.

Essential revisions:

1) It is not true that no one has suggested that relatives of Loxodonta made it out of Africa; Kretzoi did so emphatically in 1950, and others (e.g., van den Bergh; Markov and Saegusa) have accepted the possibility without endorsing the notion. These studies should be discussed and cited in the revised manuscript.

2) The authors do not treat the paleontological/morphological hypothesis about the relationships of P. antiquus/Loxodonta/Elephas as a competing hypothesis – they dismiss it in a single sentence, saying morphological features showing P. antiquus to be closely related to Elephas (such as E. recki) are convergences. And then go on to act as if elephant systematics were in a shamble and need serious revision – this simply isn't the case. They should present their view as a competing hypothesis and say if further work supports their hypothesis, then it has serious implications for elephant systematics and here is what those implications are. But, morphology isn't as labile as the authors suggest, and they do not provide compelling selective reasons for why a forest elephant such as P. antiquus would converge morphologically on a savanna grazer such as E. recki.

For example, one good test would be to study the abundant postcrania of Loxodonta, P. antiquus, and E. recki – there are clear differences in many aspects of the postcranium, especially in manus and pes elements, between Loxodonta and Elephas – see where Palaeoloxodon falls out. Additionally, it will be essential for the authors to discuss how additional species that were not sampled could be critical in resolving the proposed conundrum between the morphological and genetic results. Examples of such species include Palaeoloxodon namadicus, a straight-tusked species from Asia, Elephas planifrons and E. hysudricus (and other fossil species of Elephas, such as E. playcephalus) or any fossil species of Loxodonta, such as the abundant material from Laetoli of L. exoptata. Finally, it would be interesting to know if the authors tried but failed to sample any of the Elephas ekorensis – E. recki – E. iolensis lineage.

3) Execution of the phylogenetic analyses. The authors used two different methods on the mtDNA (Bayesian inference) and nuclear (neighbor-joining) data. It is not clear to me why that was the case. Bayesian inference is quite well established. In contrast, neighbor-joining is great for generating quick-and-dirty trees during the data exploration phase of experiments, but not very good in exploring tree space (the model of sequence evolution employed is also not described). I would recommend the authors also use BEAST to analyze the nuclear data (and at the same time estimate divergence times on that data set as well). If the use of BEAST is problematic due to computational constraints (if that's the case, the authors should include a brief description of those constraints in the manuscript), then my suggestion would be to use maximum likelihood to infer the nuclear phylogeny (e.g., use RAxML with the PROTGAMMAAUTOF option) and then r8s to calculate the divergence times.

4) Interpretation of the phylogenetic results, which are shown in Figure 2. The branch lengths of the African forest elephant – Palaeoloxodon clade are rather different between the mitochondrial and the nuclear phylogenetic trees. For example, the three NN samples are nearly identical in the mtDNA phylogeny but quite divergent in the nuclear one even though in general the nuclear genes appear to be somewhat slower evolving than the mitochondrial ones. In contrast, the key internode supporting the monophyly of the African forest elephant-Palaeoloxodon clade is quite long in the mtDNA phylogeny but very short in the nuclear phylogeny. Please discuss these two "discrepancies" and provide potential explanations.

Essential revisions:

1) It is not true that no one has suggested that relatives of Loxodonta made it out of Africa; Kretzoi did so emphatically in 1950, and others (e.g., van den Bergh; Markov and Saegusa) have accepted the possibility without endorsing the notion. These studies should be discussed and cited in the revised manuscript.

We could not access Kretzoi 1950, but we were able to obtain copies of Markov and Saegusa 2008 and van der Bergh 1999. Both publications discuss Kretzoi 1950. However, van der Bergh concludes that Stegoloxodon represents "a dwarfed member of the Elephantinae" whereas Markov and Saegusa do not discuss the phylogenetic affiliation at all, but restrict their discussion to which taxonomic name should correctly be used for the sparse material available. Nevertheless, to acknowledge that an out-of-Africa migration of Loxodontinae has been proposed previously, even though this idea has been rejected by more recent cladistic studies, we have added a sentence to the Discussion: "Although Osborn (1942) placed Palaeoloxodon in the Loxodontinae on the basis of several cranial characters, later authors (Shoshani et al. 2007, Todd 2010) have rejected this placement in favour of a placement in Elephantinae, restricting (and Loxodontinae) geographically to Africa”. We also slightly modified the Abstract and the main text where we are discussing this issue.

2) The authors do not treat the paleontological/morphological hypothesis about the relationships of P. antiquus/Loxodonta/Elephas as a competing hypothesis – they dismiss it in a single sentence, saying morphological features showing P. antiquus to be closely related to Elephas (such as E. recki) are convergences. And then go on to act as if elephant systematics were in a shamble and need serious revision – this simply isn't the case. They should present their view as a competing hypothesis and say if further work supports their hypothesis, then it has serious implications for elephant systematics and here is what those implications are. But, morphology isn't as labile as the authors suggest, and they do not provide compelling selective reasons for why a forest elephant such as P. antiquus would converge morphologically on a savanna grazer such as E. recki.

For example, one good test would be to study the abundant postcrania of Loxodonta, P. antiquus, and E. recki – there are clear differences in many aspects of the postcranium, especially in manus and pes elements, between Loxodonta and Elephas – see where Palaeoloxodon falls out. Additionally, it will be essential for the authors to discuss how additional species that were not sampled could be critical in resolving the proposed conundrum between the morphological and genetic results. Examples of such species include Palaeoloxodon namadicus, a straight-tusked species from Asia, Elephas planifrons and E. hysudricus (and other fossil species of Elephas, such as E. playcephalus) or any fossil species of Loxodonta, such as the abundant material from Laetoli of L. exoptata. Finally, it would be interesting to know if the authors tried but failed to sample any of the Elephas ekorensis – E. recki – E. iolensis lineage.

The genetic evidence for the sister group relationship between P. antiquus and Loxodonta cyclotis is based on millions of mostly neutrally evolving markers. For this reason we do not agree that the morphological and genetic analyses should be given equal weight in the discussion of elephant systematics. We are however more cautious in our conclusions now. In addition to emphasizing that “[…] the currently available genomic data from elephantids only allow for reconstructing the broad picture of elephant evolution” we now state that “More complex evolutionary scenarios are conceivable, which might explain the presence of some Elephas-like traits in P. antiquus. These could for example involve gene flow, as has been shown for L. africana and L. cyclotis based on mitochondrial evidence (Roca, Georgiadis, and O'Brien 2005)”. We now also mention the possibility that “[…] the very large effective population size of the forest elephants (Rohland et al. 2010) could have allowed the retention of ancestral traits by incomplete lineage sorting.”

As in the previous version of the text, we remain cautious in our speculations about the phylogenetic position of P. recki: “If, for example, P. recki, which was the most abundant Pleistocene elephant species in Africa, is indeed ancestral to P. antiquus and thus also represents a member of the Loxodonta lineage, the interpretation of the fossil record of elephantids in Africa is in strong need of revision”. However, we have further toned down our conclusions in several places to acknowledge the concerns by the reviewer. While we agree that additional morphological and DNA sequence data would be interesting, the former is not our expertise (and also not feasible within a reasonable timeframe) and the latter is likely not possible as most fossil species mentioned exceed the age limits of ancient DNA preservation. We have now made this clear in Discussion: “[…] the fossil record for elephants dates back several million years, well beyond the survival of ancient DNA.”.

3) Execution of the phylogenetic analyses. The authors used two different methods on the mtDNA (Bayesian inference) and nuclear (neighbor-joining) data. It is not clear to me why that was the case. Bayesian inference is quite well established. In contrast, neighbor-joining is great for generating quick-and-dirty trees during the data exploration phase of experiments, but not very good in exploring tree space (the model of sequence evolution employed is also not described). I would recommend the authors also use BEAST to analyze the nuclear data (and at the same time estimate divergence times on that data set as well). If the use of BEAST is problematic due to computational constraints (if that's the case, the authors should include a brief description of those constraints in the manuscript), then my suggestion would be to use maximum likelihood to infer the nuclear phylogeny (e.g., use RAxML with the PROTGAMMAAUTOF option) and then r8s to calculate the divergence times.

Bayesian inference is indeed well established for phylogenetic analysis of single or few loci, such as mitochondrial DNA or unlinked noncoding loci, but is computationally not feasible for complete nuclear genome sequences. Apart from computational constraints, BEAST is also not appropriate for the analysis of nuclear genomes since it implements a model that assumes no recombination within loci. For these reasons, in the previous version of the manuscript we chose to construct a distance-based phylogeny using the neighbor-joining algorithm, which is practical and fast for analyzing large datasets and thus the most popular method for complete nuclear genomes. Given the amount of information in our nuclear dataset, NJ is expected to reconstruct the true tree with high confidence (100% bootstrap support). However, in the revised version of the manuscript, we followed the reviewers’ suggestion to use a maximum likelihood approach and have added a RAxML phylogenetic analysis of concatenated nuclear coding sequences from our genome-wide data (did not use the complete nuclear genomes due to computational constraints). The resulting topology is identical to that of the NJ tree, again with 100% bootstrap support. We added a description of this analysis to the Methods section and a brief statement to Results: “A tree with identical topology was obtained using coding sequences only and a maximum likelihood approach (Figure 2—figure supplement 7).” We refrain from estimating divergence times from the nuclear data since high coverage genomic data from all taxa, including P. antiquus would be required to obtain robust estimates. Errors in the P. antiquus sequence data, which is ancient and of relatively low quality (< 1x depth of coverage) could produce artifacts.

4) Interpretation of the phylogenetic results, which are shown in Figure 2. The branch lengths of the African forest elephant – Palaeoloxodon clade are rather different between the mitochondrial and the nuclear phylogenetic trees. For example, the three NN samples are nearly identical in the mtDNA phylogeny but quite divergent in the nuclear one even though in general the nuclear genes appear to be somewhat slower evolving than the mitochondrial ones. In contrast, the key internode supporting the monophyly of the African forest elephant-Palaeoloxodon clade is quite long in the mtDNA phylogeny but very short in the nuclear phylogeny. Please discuss these two "discrepancies" and provide potential explanations.

Branch lengths in the mitochondrial and nuclear trees are not expected to be the same. The mitochondrial tree depicts the coalescence of a single lineage while the nuclear tree depicts the coalescence of tens of thousands of nuclear loci across the genome. Hence the difference in the branch lengths of the NN samples. In addition, whereas the mitochondrial sequences are of high quality, sequencing error and DNA damage in the low coverage genomes contribute private mutations to the respective branches of the nuclear tree. We have added a statement to point to this issue: “Despite the high sequence error rates associated with the low coverage genomes generated from the P. antiquus specimens, all nodes in the nuclear trees show maximal bootstrap support.”

Regarding the node supporting the monophyly of the African forest elephant-Paleoloxodon clade, the mitochondrial tree suggests that forest elephants and our P. antiquus samples carry relatively similar mitochondrial genomes but are rather diverged in their nuclear genomes (as suggested by the nuclear phylogeny). This is not unusual in elephants, for instance as mentioned in the manuscript, some savanna elephants carry forest-type mtDNA although their nuclear genomes are deeply diverged.

Author details

1. Matthias Meyer

   Max Planck Institute for Evolutionary Anthropolgy, Leipzig, Germany

   Contribution

   MM, Conceptualization, Data curation, Formal analysis, Supervision, Validation, Investigation, Visualization, Methodology, Writing—original draft, Project administration, Writing—review and editing

   For correspondence

   mmeyer@eva.mpg.de

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0002-4760-558X

2. Eleftheria Palkopoulou

   Department of Genetics, Harvard Medical School, Boston, United States

   Contribution

   EP, Data curation, Formal analysis, Validation, Investigation, Methodology, Writing—original draft, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

3. Sina Baleka

   Evolutionary Adaptive Genomics, Institute for Biochemistry and Biology, Department for Mathematics and Natural Sciences, University of Potsdam, Potsdam, Germany

   Contribution

   SB, Conceptualization, Data curation, Validation, Investigation, Methodology, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

4. Mathias Stiller

   Max Planck Institute for Evolutionary Anthropolgy, Leipzig, Germany

   Contribution

   MS, Formal analysis, Validation, Investigation, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

5. Kirsty E H Penkman

   Department of Chemistry, University of York, York, United Kingdom

   Contribution

   KEHP, Resources, Formal analysis, Funding acquisition, Validation, Investigation, Methodology, Writing—original draft, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0002-6226-9799

6. Kurt W Alt

   1. Center of Natural and Cultural History of Man, Danube Private University, Krems-Stein, Austria
   2. Department of Biomedical Engineering and Integrative Prehistory and Archaeological Science, Basel University, Basel, Switzerland

   Contribution

   KWA, Resources, Investigation, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

7. Yasuko Ishida

   Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, United States

   Contribution

   YI, Resources, Data curation, Formal analysis, Validation, Investigation, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

8. Dietrich Mania

   State Office for Heritage Management and Archaeology Saxony-Anhalt with State Museum of Prehistory, Halle, Germany

   Contribution

   DM, Resources, Investigation, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

9. Swapan Mallick

   Department of Genetics, Harvard Medical School, Boston, United States

   Contribution

   SM, Data curation, Validation, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

10. Tom Meijer

    Naturalis Biodiversity Center, Leiden, Netherlands

    Contribution

    TM, Resources, Investigation, Writing—review and editing

    Competing interests

    The authors declare that no competing interests exist.

11. Harald Meller

    State Office for Heritage Management and Archaeology Saxony-Anhalt with State Museum of Prehistory, Halle, Germany

    Contribution

    HM, Resources, Investigation, Writing—review and editing

    Competing interests

    The authors declare that no competing interests exist.

12. Sarah Nagel

    Max Planck Institute for Evolutionary Anthropolgy, Leipzig, Germany

    Contribution

    SN, Investigation, Methodology, Writing—review and editing

    Competing interests

    The authors declare that no competing interests exist.

13. Birgit Nickel

    Max Planck Institute for Evolutionary Anthropolgy, Leipzig, Germany

    Contribution

    BN, Investigation, Methodology, Writing—review and editing

    Competing interests

    The authors declare that no competing interests exist.

14. Sven Ostritz

    Thüringisches Landesamt für Denkmalpflege und Archäologie, Weimar, Germany

    Contribution

    SO, Resources, Investigation, Writing—review and editing

    Competing interests

    The authors declare that no competing interests exist.

15. Nadin Rohland

    Department of Genetics, Harvard Medical School, Boston, United States

    Contribution

    NR, Investigation, Methodology, Writing—review and editing

    Competing interests

    The authors declare that no competing interests exist.

16. Karol Schauer

    State Office for Heritage Management and Archaeology Saxony-Anhalt with State Museum of Prehistory, Halle, Germany

    Contribution

    KS, Investigation, Visualization, Writing—review and editing

    Competing interests

    The authors declare that no competing interests exist.

17. Tim Schüler

    Thüringisches Landesamt für Denkmalpflege und Archäologie, Weimar, Germany

    Contribution

    TS, Conceptualization, Resources, Investigation, Writing—review and editing

    Competing interests

    The authors declare that no competing interests exist.

18. Alfred L Roca

    Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, United States

    Contribution

    ALR, Conceptualization, Resources, Data curation, Supervision, Validation, Project administration, Writing—review and editing

    Competing interests

    The authors declare that no competing interests exist.

19. David Reich

    1. Department of Genetics, Harvard Medical School, Boston, United States
    2. Broad Institute of Harvard and MIT, Cambridge, United States
    3. Howard Hughes Medical Institute, Harvard Medical School, Boston, United States

    Contribution

    DR, Conceptualization, Data curation, Supervision, Validation, Investigation, Methodology, Writing—review and editing

    Competing interests

    The authors declare that no competing interests exist.

20. Beth Shapiro

    Department of Ecology and Evolutionary Biology, University of California, Santa Cruz, United States

    Contribution

    BS, Conceptualization, Data curation, Formal analysis, Validation, Investigation, Methodology, Writing—review and editing

    Competing interests

    The authors declare that no competing interests exist.

21. Michael Hofreiter

    Evolutionary Adaptive Genomics, Institute for Biochemistry and Biology, Department for Mathematics and Natural Sciences, University of Potsdam, Potsdam, Germany

    Contribution

    MH, Conceptualization, Data curation, Formal analysis, Supervision, Validation, Investigation, Visualization, Writing—original draft, Project administration, Writing—review and editing

    For correspondence

    michael.hofreiter@uni-potsdam.de

    Competing interests

    The authors declare that no competing interests exist.

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