Palaeogenomes of Eurasian straight-tusked elephants...

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This is a repository copy of Palaeogenomes of Eurasian straight-tusked elephants challenge the current view of elephant evolution. White Rose Research Online URL for this paper: http://eprints.whiterose.ac.uk/116979/ Version: Accepted Version Article: Meyer, Matthias, Palkopoulou, Eleftheria, Baleka, Sina et al. (18 more authors) (2017) Palaeogenomes of Eurasian straight-tusked elephants challenge the current view of elephant evolution. eLife. e25413. pp. 1-14. ISSN 2050-084X https://doi.org/10.7554/eLife.25413 [email protected] https://eprints.whiterose.ac.uk/ Reuse This article is distributed under the terms of the Creative Commons Attribution (CC BY) licence. This licence allows you to distribute, remix, tweak, and build upon the work, even commercially, as long as you credit the authors for the original work. More information and the full terms of the licence here: https://creativecommons.org/licenses/ Takedown If you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing [email protected] including the URL of the record and the reason for the withdrawal request.

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This is a repository copy of Palaeogenomes of Eurasian straight-tusked elephants challenge the current view of elephant evolution.

White Rose Research Online URL for this paper:http://eprints.whiterose.ac.uk/116979/

Version: Accepted Version

Article:

Meyer, Matthias, Palkopoulou, Eleftheria, Baleka, Sina et al. (18 more authors) (2017) Palaeogenomes of Eurasian straight-tusked elephants challenge the current view of elephant evolution. eLife. e25413. pp. 1-14. ISSN 2050-084X

https://doi.org/10.7554/eLife.25413

[email protected]://eprints.whiterose.ac.uk/

Reuse

This article is distributed under the terms of the Creative Commons Attribution (CC BY) licence. This licence allows you to distribute, remix, tweak, and build upon the work, even commercially, as long as you credit the authors for the original work. More information and the full terms of the licence here: https://creativecommons.org/licenses/

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Palaeogenomes of Eurasian straight-tusked elephants challenge the current view of elephantevolution

Tracking no: 24-01-2017-SR-eLife-25413R1

Matthias Meyer (Max Planck Institute for Evolutionary Anthropolgy), Eleftheria Palkopoulou (Harvard Medical School), Sina Baleka(University of Potsdam), Mathias Stiller (Max Planck Institute for Evolutionary Anthropology), Kirsty Penkman (University of York), Kurt Alt(Basel University), Yasuko Ishida (University of Illinois at Urbana-Champaign), Dietrich Mania (State Office for Heritage Management andArchaeology Saxony-Anhalt with State Museum of Prehistory), Swapan Mallick (Harvard Medical School), Tom Meijer (Naturalis BiodiversityCenter), Harald Meller (State Office for Heritage Management and Archaeology Saxony-Anhalt with State Museum of Prehistory), SarahNagel (Max Planck Institute for Evolutionary Anthropology), Birgit Nicker (Max Planck Institute for Evolutionary Anthropology), Sven Ostritz(Thüringisches Landesamt für Denkmalpflege und Archäologie), Nadin Rohland (Harvard Medical School), Karol Schauer (State Office forHeritage Management and Archaeology Saxony-Anhalt with State Museum of Prehistory), Tim Schüler (Thüringisches Landesamt fürDenkmalpflege und Archäologie), Alfred Roca (University of Illinois at Urbana-Champaign), David Reich (Broad Institute of Harvard andMIT), Beth Shapiro (University of California, Santa Cruz), and Michael Hofreiter (University of Potsdam)

Abstract:The straight-tusked elephants Palaeoloxodon spp. were widespread across Eurasia during the Pleistocene. Phylogenetic reconstructionsusing morphological traits have grouped them with Asian elephants (Elephas maximus), and many paleontologists place Palaeoloxodonwithin Elephas. Here we report the recovery of full mitochondrial genomes from four and partial nuclear genomes from two P. antiquusfossils. These fossils were collected at two sites in Germany, Neumark-Nord and Weimar-Ehringsdorf, and likely date to interglacial periods~120 and ~244 thousand years ago, respectively. Unexpectedly, nuclear and mitochondrial DNA analyses suggest that P. antiquus was aclose relative of extant African forest elephants (Loxodonta cyclotis). Species previously referred to Palaeoloxodon are thus mostparsimoniously explained as having diverged from the lineage of Loxodonta, indicating that Loxodonta has not been constrained to Africa.Our results demonstrate that the current picture of elephant evolution is in need of substantial revision.

Impact statement: DNA sequences from the Middle Pleistocene reveal that the extinct Eurasian straight-tusked elephants were closelyrelated to today's African forest elephants (Loxodonta cyclotis) in Africa.

Competing interests: No competing interests declared

Author contributions:Matthias Meyer: Conceptualization; Data curation; Formal analysis; Supervision; Validation; Investigation; Visualization; Methodology; Writing—original draft; Project administration; Writing—review and editing Eleftheria Palkopoulou: Data curation; Formal analysis; Validation;Investigation; Methodology; Writing—original draft; Writing—review and editing Sina Baleka: Conceptualization; Data curation; Validation;Investigation; Methodology; Writing—review and editing Mathias Stiller: Formal analysis; Validation; Investigation; Writing—review and editingKirsty Penkman: Resources; Formal analysis; Funding acquisition; Validation; Investigation; Methodology; Writing—original draft; Writing—review and editing Kurt Alt: Resources; Investigation; Writing—review and editing Yasuko Ishida: Resources; Data curation; Formal analysis;Validation; Investigation; Writing—review and editing Dietrich Mania: Resources; Investigation; Writing—review and editing Swapan Mallick:Data curation; Validation; Writing—review and editing Tom Meijer: Resources; Investigation; Writing—review and editing Harald Meller:Resources; Investigation; Writing—review and editing Sarah Nagel: Investigation; Methodology; Writing—review and editing Birgit Nicker:Investigation; Methodology; Writing—review and editing Sven Ostritz: Resources; Investigation; Writing—review and editing Nadin Rohland:Investigation; Methodology; Writing—review and editing Karol Schauer: Investigation; Visualization; Writing—review and editing Tim Schüler:Conceptualization; Resources; Investigation; Writing—review and editing Alfred Roca: Conceptualization; Resources; Data curation;Supervision; Validation; Project administration; Writing—review and editing David Reich: Conceptualization; Data curation; Supervision;Validation; Investigation; Methodology; Writing—review and editing Beth Shapiro: Conceptualization; Data curation; Formal analysis;Validation; Investigation; Methodology; Writing—review and editing Michael Hofreiter: Conceptualization; Data curation; Formal analysis;Supervision; Validation; Investigation; Visualization; Writing—original draft; Project administration; Writing—review and editing

Funding:Max Planck Society: Matthias Meyer, Mathias Stiller, Sarah Nagel, Open-access funding; Gordon and Betty Moore Foundation (Gordon E.and Betty I. Moore Foundation): Beth Shapiro; US Fish and Wildlife Service: Yasuko Ishida, Alfred Roca; Wellcome Trust: Kirsty E. H.Penkman; Leverhulme Trust: Kirsty E. H. Penkman; European Research Council: Michael Hofreiter, 310763 GeneFlow The funders had norole in study design, data collection and interpretation, or the decision to submit the work for publication.

Datasets:Datasets Generated: Nuclear DNA sequences from two Palaeoloxodon antiquus fossils: Palkopoulou E, Baleka S, Mallick S, Rohland N, Reich

eLife Sciences Publications, Ltd is a limited liability non-profit nonstock corporation incorporated in the State of Delaware, USA with company number 5030732, and is registered in the UK withcompany number FC030576 and branch number BR015634 at the address 24 Hills Road, Cambridge, CB2 1JP.

D, Hofreiter M, 2017, http://www.ebi.ac.uk/ena, PRJEB18563; Mitochondrial DNA sequences from 4 Palaeoloxodon antiquus fossils: MeyerM, Baleka S, Stiller M, Nagel S, Nickel B, Schüler T, Hofreiter M, 2017, www.ncbi.nlm.nih.gov/genbank/, KY499555 - KY499558 ReportingStandards: N/A

Ethics:Human Subjects: No Animal Subjects: No

Author Affiliation:Matthias Meyer(Evolutionary Genetics,Max Planck Institute for Evolutionary Anthropolgy,Germany) Eleftheria Palkopoulou(Department ofGenetics,Harvard Medical School,United States) Sina Baleka(Institute for Biochemistry and Biology,University of Potsdam,Germany)Mathias Stiller(Evolutionary Genetics,Max Planck Institute for Evolutionary Anthropology,Germany) Kirsty Penkman(Chemistry,University ofYork,United Kingdom) Kurt Alt(Department of Biomedical Engineering and Integrative Prehistory and Archaeological Science,BaselUniversity,Switzerland) Yasuko Ishida(Department of Animal Sciences,University of Illinois at Urbana-Champaign,United States) DietrichMania(N/A,State Office for Heritage Management and Archaeology Saxony-Anhalt with State Museum of Prehistory,Germany) SwapanMallick(Department of Genetics,Harvard Medical School,United States) Tom Meijer(-,Naturalis Biodiversity Center,Netherlands) HaraldMeller(-,State Office for Heritage Management and Archaeology Saxony-Anhalt with State Museum of Prehistory,Germany) SarahNagel(Evolutionary Genetics,Max Planck Institute for Evolutionary Anthropology,Germany) Birgit Nicker(Evolutionary Genetics,Max PlanckInstitute for Evolutionary Anthropology,Germany) Sven Ostritz(-,Thüringisches Landesamt für Denkmalpflege und Archäologie,Germany)Nadin Rohland(Department of Genetics,Harvard Medical School,United States) Karol Schauer(-,State Office for Heritage Management andArchaeology Saxony-Anhalt with State Museum of Prehistory,Germany) Tim Schüler(-,Thüringisches Landesamt für Denkmalpflege undArchäologie,Germany) Alfred Roca(Department of Animal Sciences,University of Illinois at Urbana-Champaign,United States) DavidReich(Program in Medical and Population Genetics,Broad Institute of Harvard and MIT,United States) Beth Shapiro(Department of Ecologyand Evolutionary Biology,University of California, Santa Cruz,United States) Michael Hofreiter(Department for Mathematics and NaturalSciences,University of Potsdam,Germany)

Dual-use research: No

Permissions: Have you reproduced or modified any part of an article that has been previously published or submitted to another journal?No

eLife Sciences Publications, Ltd is a limited liability non-profit nonstock corporation incorporated in the State of Delaware, USA with company number 5030732, and is registered in the UK withcompany number FC030576 and branch number BR015634 at the address 24 Hills Road, Cambridge, CB2 1JP.

Palaeogenomes of Eurasian straight-tusked elephants challenge the ヱ

current view of elephant evolution ヲ Matthias Meyerな┸ Eleftheria Palkopoulouに┸ Sina Balekaぬ┸ Mathias Stillerな┸ Kirsty Penkmanね┸ ン Kurt W┻ Altの┸ Yasuko )shidaは┸ Dietrich Maniaば┸ Swapan Mallickに┸ Tom Meijerぱ┸ (arald Mellerば┸ ヴ Sarah Nagelな┸ Birgit Nickelな┸ Sven Ostritzひ┸ Nadin Rohlandに┸ Karol Schauerば┸ Tim Schülerひ┸ ヵ Alfred L┻ Rocaは┸ David Reichに┸ Beth Shapiroなど┸ Michael (ofreiterぬ ヶ Α

1Max Planck Institute for Evolutionary Anthropology, Deutscher Platz 6, D-04103 Leipzig, Β Germany. Γ

2 Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA. ヱヰ

3Evolutionary Adaptive Genomics, Institute for Biochemistry and Biology, Department for ヱヱ Mathematics and Natural Sciences, University of Potsdam, Karl-Liebknechtstr. 24-25, 14476 ヱヲ Potsdam, Germany. ヱン

4Department of Chemistry, University of York, York YO10 5DD, York, UK. ヱヴ

5Center of Natural and Cultural History of Man, Danube Private University, Steiner ヱヵ Landstrasse 124, A � 3500 Krems-Stein, Austria, and Department of Biomedical Engineering ヱヶ and Integrative Prehistory and Archaeological Science, Basel University, Gewerbestrasse 14-ヱΑ 18, CH-4123 Basel-Allschwill and Spalenring 145, CH-4055 Basel, Switzerland. ヱΒ

6Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, IL ヱΓ 61801 USA. ヲヰ

7 State Office for Heritage Management and Archaeology Saxony-Anhalt with State Museum ヲヱ of Prehistory, Richard-Wagner-Strasse 9, 06114 Halle (Saale), Germany. ヲヲ

8Naturalis Biodiversity Center, P.O. Box 9517, NL-2300 RA Leiden, The Netherlands. ヲン

9Thüringisches Landesamt für Denkmalpflege und Archäologie, Humboldtstr. 11, D-99423 ヲヴ Weimar, Germany. ヲヵ

10 Department of Ecology and Evolutionary Biology, University of California, Santa Cruz, ヲヶ Santa Cruz, CA 95064, USA. ヲΑ ヲΒ

Abstract ヲΓ The straight┽tusked elephants Palaeoloxodon spp. were widespread across Eurasia ンヰ during the Pleistocene┻ Phylogenetic reconstructions using morphological traits have ンヱ grouped them with Asian elephants ゅElephas maximusょ┸ and many paleontologists place ンヲ

Palaeoloxodon within Elephas┻ (ere we report the recovery of full mitochondrial ンン genomes from four and partial nuclear genomes from two P. antiquus fossils┻ These ンヴ fossils were collected at two sites in Germany┸ Neumark┽Nord and Weimar┽Ehringsdorf┸ ンヵ and likely date to interglacial periods bなにど and bにねね thousand years ago┸ respectively┻ ンヶ Unexpectedly┸ nuclear and mitochondrial DNA analyses suggest that P. antiquus was a ンΑ close relative of extant African forest elephants ゅLoxodonta cyclotisょ┻ Species previously ンΒ referred to Palaeoloxodon are thus most parsimoniously explained as having diverged ンΓ from the lineage of Loxodonta┸ indicating that Loxodonta has not been constrained to ヴヰ Africa┻ Our results demonstrate that the current picture of elephant evolution is in need ヴヱ of substantial revision┻ ヴヲ

M;キミ デW┝デ ヴン

Introduction ヴヴ )n the late Miocene in Africa┸ the last of several major radiations within Proboscidea ヴヵ gave rise to the family Elephantidae┸ which comprises living elephants and their extinct ヴヶ relatives including mammoths ゅgenus Mammuthusょ and various dwarf elephant species ヴΑ from Mediterranean islands┻ The three living elephant species ゅthe African savanna ヴΒ elephant┸ Loxodonta africana┸ the African forest elephant┸ L. cyclotis and the Asian ヴΓ elephant┸ Elephas maximusょ┸ represent the last remnants of this family and of the ヵヰ formerly much more widely distributed and species┽rich order Proboscidea┻ Apart from ヵヱ mammoths┸ the elephant genus with the most abundant fossil record in Eurasia is ヵヲ

Palaeoloxodon ゅstraight┽tusked elephants┹ Figure なょ┸ which appears in Eurasia around ヵン ど┻ばの million years ago ゅMaょ ゅLister にどなのょ┻ Based on morphological analyses┸ ヵヴ

Palaeoloxodon is widely accepted as being more closely related to the extant Asian ヵヵ elephant than to mammoths or extant African elephants ゅShoshani et al┻ にどどば┸ Todd ヵヶ にどなどょ┸ and is often subsumed into the genus Elephas ゅMaglio なひばぬ┸ Sanders et al┻ にどなどょ┻ ヵΑ Across its range from Western Europe to Japan┸ Palaeoloxodon probably comprised ヵΒ several species ゅShoshani et al┻ にどどばょ┸ and based on morphological comparisons all of ヵΓ them are considered to be derived from the African Palaeoloxodon ゅor Elephasょ recki ヶヰ ゅMaglio なひばぬ┸ Saegusa and Gilbert にどどぱょ┸ which was the predominant proboscidean ヶヱ lineage in Africa during the Pliocene and Pleistocene but went extinct around などど ヶヲ thousand years ago ゅkaょ ゅOwen┽Smith にどなぬょ┻ Straight┽tusked elephants may have ヶン survived in mainland Eurasia until around ぬの ka┸ although the youngest reliably dated ヶヴ remains are from the last interglacial┸ ななの┽なぬど ka ゅStuart にどどのょ┻ ヶヵ Recent technological progress has pushed back the temporal limit of ancient DNA ヶヶ research┸ enabling┸ for example┸ recovery of a low coverage genome of a bばどど┸どどど year┽ヶΑ old horse preserved in permafrost ゅOrlando et al┻ にどなぬょ┻ For more temperate regions┸ ヶΒ however┸ evidence of DNA preservation reaching far beyond the last glacial period is still ヶΓ limited to a single locality┸ Sima de los (uesos in Spain┸ where DNA has been recovered Αヰ from bねぬど ka old hominin and bear remains ゅDabney et al┻ にどなぬ┸ Meyer et al┻ にどなはょ┻ Αヱ While genetic analyses of the extinct interglacial fauna remain a challenging Αヲ undertaking┸ recent advances in ancient DNA extraction ゅDabney et al┻ にどなぬょ and Αン

sequencing library construction ゅMeyer et al┻ にどなにょ have improved access to highly Αヴ degraded DNA┻ Αヵ

Results and Discussion Αヶ To better understand the evolutionary relationships between the extinct straight┽tusked ΑΑ elephants and other elephant species┸ we attempted DNA extraction and sequencing ΑΒ from several P. antiquus fossils┸ four of which we investigated in┽depth┻ Three of these┸ ΑΓ which were all unambiguously assigned to P. antiquus based on their morphology┸ were Βヰ from Neumark┽Nord ゅNNょ な in Germany┸ a fossil┽rich site that has been proposed to date Βヱ to M)S のe ゅb なにど kaょ or M)S ば ゅb にねね kaょ or both ゅMania にどなど┸ Schüler にどなど┸ Penkman Βヲ にどなどょ┻ This site has yielded one of the largest collections of P. antiquus remains known Βン to date┻ The fourth fossil was recovered during recent active mining in the travertine Βヴ deposits of Weimar┽Ehringsdorf ゅWEょ┸ Germany┸ a quarry that has for more than a Βヵ century yielded a rich collection of fossils representing a typical European interglacial Βヶ fauna ゅKahlke なひばのょ┻ Weimar┽Ehringsdorf is best known for the discovery of ΒΑ Neanderthal remains in the early にどth century┸ and the assemblage is dated to M)S ば ΒΒ ゅMallick and Frank にどどにょ┻ The Paleoloxodon bone fragment from Weimar┽Ehringsdorf is ΒΓ morphologically undiagnostic with respect to species┻ (owever┸ it was found in the Γヰ Lower Travertine┸ which was dated to b にぬぬ ka ゅSchüler にどどぬょ and where P. antiquus is Γヱ the only elephantid found so far┻ We performed DNA extraction┸ library preparation┸ Γヲ hybridization capture and high┽throughput sequencing on all four fossils ゅFigure に ‒ Γン source data なょ and obtained full mitochondrial genome sequences for all of them ゅFigure Γヴ に ‒ figure supplement なょ┻ All sequences show short fragment lengths ゅFigure に ‒ figure Γヵ supplement にょ and signals of cytosine deamination compatible with the old age of the Γヶ specimens ゅFigure に ‒ figure supplement ぬょ┻ ΓΑ We inferred a phylogeny using the four Paleoloxodon mitochondrial genomes and ΓΒ mitochondrial genomes from なは M. primigenius┸ two E. maximus and なぬ Loxodonta ΓΓ individuals┻ The latter were chosen for a diversity of haplotypes┸ including forest ヱヰヰ elephant derived ゅ╅F┽clade╆ょ haplotypes as well as ╅S┽clade╆ haplotypes found only among ヱヰヱ savanna elephants ゅDebruyne にどどのょ┻ For calibration┸ we used an estimated divergence ヱヰヲ of the African elephant lineage from that of Asian elephants and mammoths of は┻は┽ぱ┻は ヱヰン Ma ゅRohland et al┻ にどどばょ. Surprisingly┸ P. antiquus did not cluster with E. maximus┸ as ヱヰヴ hypothesized from morphological analyses┻ )nstead┸ it fell within the mito┽genetic ヱヰヵ

diversity of extant L. cyclotis┸ with very high statistical support ゅFigure にょ┻ The four ヱヰヶ straight┽tusked elephants did not cluster together within this mitochondrial clade┸ but ヱヰΑ formed two separate lineages that share a common ancestor with an extant L. cyclotis ヱヰΒ lineage ど┻ば┽な┻は Ma ゅNNょ and な┻の┽ぬ┻ど Ma ゅWEょ ago┸ respectively┻ (owever┸ mitochondrial ヱヰΓ DNA represents a single┸ maternally inherited locus and does not reflect the full ヱヱヰ evolutionary history of populations or species┻ Furthermore┸ the transfer of ヱヱヱ mitochondrial DNA between hybridizing species is not unusual when gene flow is ヱヱヲ strongly male┽mediated ゅPetit and Excoffier にどどひ┸ Li et al┻ にどなは┸ Cahill et al┻ にどなぬょ┸ as is ヱヱン the case with elephants┻ For example┸ mitochondrial sequences of the F┽clade have also ヱヱヴ been found in some L. africana individuals ゅDebruyne にどどのょ despite the very substantial ヱヱヵ divergence of their nuclear genomes ゅRoca┸ Georgiadis┸ and OfBrien にどどの┸ Rohland et al┻ ヱヱヶ にどなどょ┸ a pattern that has been attributed to mitochondrial gene flow from forest to ヱヱΑ savanna elephants ゅRoca┸ Georgiadis┸ and OfBrien にどどのょ┻ ヱヱΒ We therefore performed shotgun sequencing of DNA libraries prepared from the ヱヱΓ two best┽preserved NN individuals ゅa petrous bone and a molarょ to recover nuclear DNA ヱヲヰ sequences┻ When mapped to the L. africana reference genome┸ ぬひガ and にぱガ of the ヱヲヱ sequence reads generated from these specimens were identified as elephant┸ ヱヲヲ respectively ゅFigure に ‒ source data なょ┻ A neighbor┽joining phylogenetic tree based on ヱヲン bばばどM ゅpetrousょ and bになどM ゅmolarょ base pairs of P. antiquus nuclear DNA placed P. ヱヲヴ

antiquus and L. cyclotis as sister taxa to the exclusion of L. africana ゅFigure にょ┻ A tree ヱヲヵ with identical topology was obtained using coding sequences only and a maximum ヱヲヶ likelihood approach ゅFigure に ‒ figure supplement ねょ┻ Despite the high sequence error ヱヲΑ rates associated with the low coverage genomes generated from the P. antiquus ヱヲΒ specimens┸ all nodes in the nuclear trees show maximal bootstrap support┻ The ヱヲΓ mitochondrial and nuclear phylogenies thus support a sister group relationship between ヱンヰ

P. antiquus and L. cyclotis┻ ヱンヱ Despite their geographical proximity┸ the WE and NN specimens are found in ヱンヲ different positions in the mitochondrial tree┻ Given that the three NN specimens show ヱンン highly similar mitogenome sequences┸ we considered whether the sites date to different ヱンヴ interglacials┻ Electron Spin Resonance ゅESRょ dating of tooth enamel has been applied to ヱンヵ both sites┸ suggesting an age of b ななば ka ゅrange ひば ‒ なねに ka ゅSchüler にどなどょょ for the NNな ヱンヶ layers from which our samples originate and of にぬぬ ka ゅrange になは ‒ にのど ka ゅSchüler ヱンΑ にどどぬょょ for the WE specimen┻ )n order to better estimate the age of the NNな site┸ we used ヱンΒ

amino acid racemization of snail opercula ゅPenkman et al┻ にどななょ┸ including samples ヱンΓ from the continental Eemian type┽site of Amersfoort ゅZagwijn なひはなょ ゅFigure に ‒ source ヱヴヰ data なょ┸ which is correlated with M)S のe ゅCleveringa et al┻ にどどどょ┻ The NNな opercula show ヱヴヱ similar ゅperhaps slightly lowerょ levels of biomolecular degradation compared to ヱヴヲ Amersfoort┸ suggesting an Eemian age for NNな ゅFigure に ‒ figure supplement のょ┻ ヱヴン )mportantly┸ NNな shows very similar levels of amino acid racemization in intra┽ヱヴヴ crystalline protein decomposition as a second site at Neumark┽Nord ゅNNにょ┸ indicating ヱヴヵ that both sites are of the same age┻ Considerable evidence supports an Eemian age for ヱヴヶ NNに┸ including palaeomagnetic data that shows a correlation with the M)S のe Blake ヱヴΑ event and thermoluminescence dating of flint to bなには グ は ka ゅSier et al┻ にどななょ┻ These ヱヴΒ results therefore indicate an Eemian age also for NNな┻ Since the WE specimen likely ヱヴΓ dates to the previous interglacial┸ this suggests that the very different mitogenomes ヱヵヰ between WE and NNな may reflect the contraction and re┽expansion of the range of P. ヱヵヱ

antiquus across glacial cycles┻ ヱヵヲ Our results have implications both for understanding elephant evolutionary ヱヵン history and for the use of morphological data to decipher phylogenetic relationships ヱヵヴ among elephants┻ The combination of strongly supported mitochondrial and nuclear ヱヵヵ DNA phylogenies clearly demonstrate that Palaeoloxodon antiquus is more closely ヱヵヶ related to Loxodonta than to Elephas ゅFigure ぬょ┸ suggesting that Elephas antiquus should ヱヵΑ not be used synonymously for Paleoloxodon antiquus when referring to the taxon┻ The ヱヵΒ new phylogeny suggests a remarkable degree of evolutionary transformation┸ from an ヱヵΓ ancestor that possessed the features of the cranium and dentition of Loxodonta┸ shared ヱヶヰ by both L. africana and L. cyclotis ゅMaglio なひばぬ┸ Sanders et al┻ にどなどょ┸ to a descendant ヱヶヱ that is highly similar to Elephas ゅsensu stricto┸ the lineage of the Asian elephantょ in many ヱヶヲ morphological features┻ (owever┸ it should be noted that currently available genomic ヱヶン data from elephantids only allow for reconstructing the broad picture of elephant ヱヶヴ evolution┻ More complex evolutionary scenarios are conceivable┸ which might explain ヱヶヵ the presence of some Elephas┽like traits in P. antiquus┻ These could for example involve ヱヶヶ gene flow┸ as has been shown for L. africana and L. cyclotis based on mitochondrial ヱヶΑ evidence ゅRoca┸ Georgiadis┸ and OfBrien にどどのょ┻ )n addition┸ the very large effective ヱヶΒ population size of the forest elephants ゅRohland et al┻ にどなどょ could have allowed the ヱヶΓ retention of ancestral traits by incomplete lineage sorting┻ ヱΑヰ

Α

)n summary┸ the molecular results presented here urge for a re┽examination of ヱΑヱ morphology across the Elephantidae┻ This is especially important as the fossil record for ヱΑヲ

elephants dates back several million years, well beyond the survival of ancient DNA. )f┸ for ヱΑン example┸ P. recki┸ which was the most abundant Pleistocene elephant species in Africa┸ is ヱΑヴ indeed ancestral to P. antiquus and thus also represents a member of the Loxodonta ヱΑヵ lineage┸ the interpretation of the fossil record of elephantids in Africa is in strong need of ヱΑヶ revision┻ Furthermore┸ in contrast to the genera Mammuthus and Elephas┸ which also ヱΑΑ had their origin in Africa┸ the lineage of Loxodonta is generally assumed never to have ヱΑΒ left Africa┻ Although Osborn ゅなひねにょ placed Palaeoloxodon in the Loxodontinae on the ヱΑΓ basis of several cranial characters┸ later authors ゅShoshani et al┻ にどどば┸ Todd にどなどょ have ヱΒヰ rejected this placement in favour of a placement in Elephantinae┸ restricting Loxodonta ヱΒヱ ゅand Loxodontinaeょ geographically to Africa┻ (owever┸ our data reveal that the ヱΒヲ

Loxodonta lineage ゅas Paleoloxodonょ also colonized the Eurasian continent┻ Last┸ the ヱΒン finding that L. africana is genetically more distant from L. cyclotis than is P. antiquus ヱΒヴ strongly supports previous evidence that urged recognition of L. cyclotis and L. africana ヱΒヵ as distinct species and underlines the importance of conservation efforts directed ヱΒヶ towards African forest elephants┻ ヱΒΑ

Β

Material and Methods ヱΒΒ

Sampling, DNA extraction and library preparation ヱΒΓ )n January にどなね┸ a fragment of an elephant long bone was discovered during work ヱΓヰ at the Ehringsdorf quarries┻ The specimen was removed from the lower travertine ゅbぬm ヱΓヱ above the basis and に┻の m below the Pariser horizonょ┸ which has been dated by micro ヱΓヲ probe U【Th┽series dating of primary travertine ゅなはょ and ESR dating of tooth enamel ヱΓン ゅSchüler にどどぬょ to b にぬぬ ka┻ A piece of the bone ゅinventory number なね【なぱ┽なょ was ヱΓヴ transferred to the ancient DNA laboratory at the MP)┽EVAN in Leipzig┻ Ten extracts were ヱΓヵ prepared using between ぬは and のぬ mg of bone ゅtotaling ねにの mgょ material following the ヱΓヶ method of Dabney et al┻ にどなぬ ゅDabney et al┻ にどなぬょ┻ From these extracts┸ ぬど libraries ヱΓΑ were prepared using single┽stranded library preparation ゅGansauge and Meyer にどなぬょ ヱΓΒ with input volumes of ね┸ ぱ and なに ヅl DNA extract ゅof にの ヅl extract volumeょ┸ respectively┻ ヱΓΓ The number of library molecules was determined by digital droplet PCR using the ヲヰヰ QXにどど system ゅBio┽Radょ with EvaGreen chemistry ゅQXにどど ddPCR EvaGreen Supermix┸ ヲヰヱ Bio┽Radょ and primers )Sば and )Sぱ ゅMeyer and Kircher にどなどょ following the ヲヰヲ manufacturer╆s instructions ゅFigure に ‒ source data なょ┻ Libraries were then amplified ヲヰン using AccuPrime Pfx DNA polymerase ゅThermo Fisher Scientificょ ゅDabney and Meyer ヲヰヴ にどなにょ and labeled with two sample┽specific indices ゅKircher┸ Sawyer┸ and Meyer にどなにょ┻ ヲヰヵ Ancient DNA work on the Neumark┽Nord specimens was carried out in the ancient ヲヰヶ DNA laboratory at the University of Potsdam┻ )nitially┸ ぱ specimens were screened for ヲヰΑ the presence of elephant DNA of which the two best preserved ones ゅindividual にぬ┸ ヲヰΒ Landesmuseum (alle museum inventory number (K にどどば┺にの┻にぱの┸ななば┹ a tusk fragment ヲヰΓ いNEUにAう and a fragmentary upper jaw いNEUぱBう┹ inventory number (K ひに┺ひひどょ were ヲヱヰ selected for further analyses┻ )n addition┸ in にどなぬ┸ the petrous bone of individual ぬど ヲヱヱ ゅNEPEC┹ inventory number (K にどどば┺にの┺にぱど サ Eなの┻な┻ひはょ was sampled and also used in ヲヱヲ the analysis┻ Fourteen DNA extracts were prepared from individual ぬど┸ and は from each ヲヱン of the two other specimens┸ using approximately のど mg of bone powder in each ヲヱヴ extraction┻ DNA extraction and library preparation were performed as described above ヲヱヵ but including Archaeoglobus fulgidus uracil┽DNA glycosylase in library preparation ヲヱヶ ゅGansauge and Meyer にどなぬょ┸ which removes the majority of uracils that are typically ヲヱΑ present in ancient DNA fragments┻ )n addition┸ to maximize yields in library preparation┸ ヲヱΒ two extracts ゅにの ヅl eachょ from each specimen were combined and ねど ヅl were used as ヲヱΓ input for library preparation┻ Reaction volumes in steps な┽ぬ of the protocol were ヲヲヰ

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doubled to accommodate larger input volumes of extract┻ Optimal amplification cycle ヲヲヱ numbers were established using qPCR ゅPikoReal Real┽Time PCR system┸ Thermo Fisher ヲヲヲ Scientificょ with primers )Sば and )Sぱ ゅGansauge and Meyer にどなぬょ┻ Libraries were then ヲヲン amplified and labeled with one sample┽specific index┻ After purification ゅMinElute PCR ヲヲヴ purification kit┸ Qiagenょ┸ the different libraries for each sample were pooled┻ ヲヲヵ

Enrichment and sequencing of mtDNA ヲヲヶ のに┽mer capture probes for the enrichment of mtDNA sequences from elephants ヲヲΑ were designed using the published mtDNA genome sequences of African forest elephant ヲヲΒ ゅNCeどにどばのひょ┸ Asian elephant ゅNCeどどのなにひょ┸ African savanna elephant ゅNCeどどどひぬねょ and ヲヲΓ the mastodon ゅNCeどどひのばねょ┸ with one probe starting at each position in these genomes┻ ヲンヰ Probes containing simple repeats longer than にねbp ゅrepetition of the same な┽ぱ bp ヲンヱ sequence motifょ were removed┻ Single┽stranded biotinylated DNA probes were ヲンヲ generated as described elsewhere ゅFu et al┻ にどなぬょ and used for two successive rounds of ヲンン hybridization capture following a bead┽based protocol ゅMaricic┸ Whitten┸ and Pääbo ヲンヴ にどなどょ┻ Enriched libraries were pooled and sequenced on one lane of an )llumina ヲンヵ (iSeqにのどど in paired┽end mode ゅにx ばは cycles plus にx ば cycles index reads┹ Weimar┽ヲンヶ Ehringsdorf librariesょ or on an )llumina NextSeq のどど ゅにx ばは cycles plus なx ぱ cycles index ヲンΑ read┹ Neumark┽Nord librariesょ┻ ヲンΒ

Mitochondrial sequence data processing and consensus calling ヲンΓ Sequences were assigned to their source library requiring perfect matches to one ヲヴヰ of the expected indices or index pairs and overlap┽merged to reconstruct full┽length ヲヴヱ molecule sequences ゅRenaud┸ Stenzel┸ and Kelso にどなねょ┻ Due to the different properties ヲヴヲ of the data obtained from Weimar┽Ehringsdorf and Neumark┽Nord with regard to ヲヴン sequence length distribution and damage patterns ゅFigure に ‒ figure supplements に and ヲヴヴ ぬょ┸ two different strategies were used for mapping and consensus calling┻ To minimize ヲヴヵ the loss of alignments due to the high frequencies of damaged┽induced substitutions in ヲヴヶ the Weimar┽Ehringsdorf data┸ mapping to the L. cyclotis mtDNA genome ゅJNはばぬにはねょ ヲヴΑ was performed as previously described for the Sima de los (uesos mtDNA assemblies ヲヴΒ ゅDabney et al┻ にどなぬょ┸ using BWA and allowing up to five C to T substitutions but not ヲヴΓ more than three of other types┻ The sequences from Neumark┽Nord were mapped with ヲヵヰ ╅ancient╆ parameters as described elsewhere ゅMeyer et al┻ にどなにょ┻ PCR duplicates were ヲヵヱ removed with bam┽rmdup ゅStenzel ゅにどなねょ Biohazard┸ available from ヲヵヲ

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https┺【【bitbucket┻org【ustenzel【biohazardょ by calling a consensus from sequences with ヲヵン identical alignment start and end coordinates┻ Sequences shorter than ぬど bp were ヲヵヴ discarded┻ An overview of the DNA extracts┸ libraries and sequences generated in this ヲヵヵ study is provided in Figure に ‒ source data な┻ ヲヵヶ When visually inspecting the Weimar┽Ehringsdorf sequence alignments┸ we ヲヵΑ identified several regions in the mitochondrial genome where more than one sequence ヲヵΒ variant was present┻ Based on BLAST searches on a subset of these sequences┸ we found ヲヵΓ that they derived from present┽day human or microbial contamination┻ We thus aligned ヲヶヰ all sequences to the identified contaminant genomes ゅGenBank accession nos┻ ヲヶヱ NCeどなにひにど┸ AFぬはのはぬの and CPどどぱぱぱひょ and removed sequences that showed a greater ヲヶヲ similarity to one of the contaminants than to the African forest elephant mtDNA┻ No ヲヶン removal of contaminant sequences was necessary for the Neumark┽Nord samples┻ To ヲヶヴ minimize the impact of damage┽derived C to T substitutions on consensus calling┸ all T ヲヶヵ occurring in the first and last three positions of the Weimar┽Ehringsdorf sequences were ヲヶヶ substituted by N┻ Next┸ a position┽based tabular output was generated from the ヲヶΑ alignment files using the ╅mpileup╆ function of SAMtools ゅLi et al┻ にどどひょ┻ This file was ヲヶΒ used to call the consensus at positions with minimum sequence coverage of ぬ if the ヲヶΓ sequences were in at least はば┻どガ agreement┻ At three positions in the mtDNA genome ヲΑヰ ゅpositions ぬぱね┸ ぱねはば┸ ぱねはひょ with low consensus support┸ we spotted obvious alignment ヲΑヱ errors in one or all specimens and determined the consensus base manually┻ Apart from ヲΑヲ a bのどど bp stretch of repetitive sequence in the D┽loop┸ which cannot be reconstructed ヲΑン with short DNA fragments┸ only ね positions remain undetermined in the Weimar┽ヲΑヴ Ehringsdorf sequence and even fewer ゅbetween none and ぬょ in the Neumark┽Nord ヲΑヵ sequences┻ ヲΑヶ

MtDNA phylogenetic reconstructions ヲΑΑ We estimated mitochondrial phylogenies using the software BEAST ゅDrummond ヲΑΒ et al┻ にどなにょ v な┻ぱ┻に and a data set including ぬな complete mitochondrial genomes ヲΑΓ ゅGenBank accession nos┻┹ L. cyclotis┺ JNはばぬにはね┸ JNはばぬにはぬ┸ KJののばねにね┸ KJののばねにぬ┸ ヲΒヰ KYはなはひばは┸ KYはなはひばひ┸ KYはなはひばぱ┹ L. africana: WAねどにど┸ KRどどなね┸ KRどなぬぱ┸ NCどどどひぬね┸ ヲΒヱ DQぬなはどはひ┸ ABねねぬぱばひ┹ E. maximus┺ NCどどのなにひ┸ DQぬなはどはぱ┹ M. primigenius┺ DQぬなはどはば┸ ヲΒヲ NCどどばのひは┸ EUなののになど┸ EUなのぬねねひ┸ EUなのぬねのの┸ EUなのぬねのは┸ EUなのぬねのぱ┸ EUなのぬねねの┸ ヲΒン EUなのぬねねは┸ EUなのぬねねば┸ EUなのぬねねぱ┸ EUなのぬねのに┸ EUなのぬねのぬ┸ EUなのぬねのね┸ JFひなににどど┹ M. ヲΒヴ

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columbi┺ JFひなになひひょ┻ For three of the L. cyclotis mitogenomes ゅKYはなはひばは┸ KYはなはひばひ and ヲΒヵ KYはなはひばぱょ┸ only partial mitochondrial sequences were previously published┻ Full ヲΒヶ genome sequences were obtained using previously collected samples ゅ)shida et al┻ にどなぬょ ヲΒΑ and the amplification and sequencing strategy detailed by ゅBrandt et al┻ にどなにょ┸ except ヲΒΒ that additional primers were used in sequencing ゅFigure に ┽ source data なょ┻ The ヲΒΓ complete mitochondrial genome sequences were partitioned prior to analysis into four ヲΓヰ partitions┸ representing concatenated genes ゅwith NDは reversedょ┸ tRNAs┸ rRNAs┸ and the ヲΓヱ control region┸ and analyses were performed with and without the control region ヲΓヲ fragment┻ All BEAST analyses were performed assuming the flexible skygrid coalescent ヲΓン model ゅGill et al┻ にどなぬょ and the uncorrelated lognormal relaxed molecular clock ヲΓヴ ゅDrummond et al┻ にどどはょ┻ We calibrated the molecular clock using the ages of ancient tips ヲΓヵ and a lognormal prior with a mean of ば┻は million years and standard deviation of ヲΓヶ のどど┸どどど years for the divergence of the Loxodonta and Elephas【Mammuthus lineages ヲΓΑ ゅRohland et al┻ にどどばょ┻ Ages of the ancient samples were sampled from normal ヲΓΒ distributions derived from stratigraphic and previously estimated radiometric dates┺ ヲΓΓ Neumark┽Nord┺ なねに┽ひに ka ゅSchüler にどなどょ┹ Ehringsdorf にのど┽になは ka ゅMallick and Frank ンヰヰ にどどにょ┻ Separate evolutionary rates and models of nucleotide substitution┸ as estimated ンヰヱ using jModelTest ゅPosada にどどぱょ┸ were estimated for each partition in the alignment┻ We ンヰヲ ran two MCMC chains for はど million iterations each┸ with trees and model parameters ンヰン sampled every は thousand iterations┻ Chain convergence and parameter sampling were ンヰヴ examined by eye using Tracer v な┻は ゅRambaut A┸ Suchard MA┸ Xie D ┃ Drummond AJ ンヰヵ ゅにどなねょ Tracer vな┻は┸ available from http┺【【beast┻bio┻ed┻ac┻uk【Tracerょ┻ The first などガ of ンヰヶ samples were discarded from each run after which the two runs were combined┻ Trees ンヰΑ were summarized and maximum clade credibility ゅMCCょ trees identified using ンヰΒ TreeAnnotator v な┻ぱ┻に┸ which is distributed as part of the BEAST package┻ MCC trees ンヰΓ were edited and annotated using FigTree vな┻ね┻に ンヱヰ ゅhttp┺【【tree┻bio┻ed┻ac┻uk【software【figtree【ょ┻ ンヱヱ

Shotgun sequencing of nuclear DNA ンヱヲ Libraries from the three Neumark┽Nord samples and another sample ゅnot ンヱン included in this studyょ were pooled in equimolar concentrations and shotgun┽sequenced ンヱヴ on a NextSeq のどど ゅにx ばはbp cyclesょ at (arvard Medical School┻ Following determination ンヱヵ of endogenous content and complexity in each library┸ two of them ゅNEPEC from the ンヱヶ petrous bone and NEUにA from the molar fragmentょ were chosen for additional ンヱΑ

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sequencing and were pooled together with another sample ゅnot included in this studyょ ンヱΒ for one additional run on )llumina╆s NextSeq のどど┻ ンヱΓ

Nuclear sequence data processing ンヲヰ Sequences were assigned to their source library according to their index allowing ンヲヱ for one mismatch┻ Adapters were trimmed and paired┽end sequences were merged with ンヲヲ SeqPrep な┻な ゅhttps┺【【github┻com【jstjohn【SeqPrepょ using default parameters but with a ンヲン modification in the source code to retain the best base quality scores in the merged ンヲヴ region┻ Merged sequences shorter than ぬどbp were discarded┻ Alignment to the African ンヲヵ savanna elephant reference genome ゅloxAfrね┹ downloaded from ンヲヶ ftp┺【【ftp┻broadinstitute┻org【distribution【assemblies【mammals【elephant【loxAfrね【ょ was ンヲΑ performed with BWA╆s version ど┻ば┻ぱ ゅLi and Durbin にどどひょ using ╅ancient╆ parameters ンヲΒ and SAMtools╆ v┻ど┻な┻なひ ╅samse╆ command ゅLi et al┻ にどどひょ┻ A custom script was used to ンヲΓ remove duplicates┸ which takes into account the alignment coordinates of both ends of ンンヰ the sorted sequences and their orientation┻ ンンヱ From the first sequencing run┸ ねばガ and ぬのガ of the sequences from the libraries ンンヲ from the petrous bone and the molar fragment ゅNEPEC and NEUにA┸ respectivelyょ ンンン aligned to the reference genome while only ど┻のガ of the sequences from the third library ンンヴ ゅNEUぱBょ aligned to the reference genome┻ The high percentage of mapped sequences in ンンヵ the petrous sample is consistent with previous reports on the superior DNA ンンヶ preservation in this part of the skeleton ゅGamba et al┻ にどなねょ┻ Following the second ンンΑ sequencing run┸ the total endogenous content of the first two libraries was estimated to ンンΒ ぬひガ and にぱガ┸ with an average sequence length of ぬひbp and ぬぱbp┸ respectively ゅFigure ンンΓ に ‒ source data なょ┻ The average depth of coverage was ど┻はの┽fold for NEPEC and ど┻なね┽fold ンヴヰ for NEUにA┻ Both of them showed low frequencies of C to T to substitutions at the の╆ and ンヴヱ ぬ╆ end┸ which are characteristic for Afu UDG┽treated single┽stranded libraries ゅGansauge ンヴヲ and Meyer にどなぬょ┸ except for in CpG context┸ where deamination of の┽methylcytosine ンヴン leaves thymine and not uracil ゅFigure に ‒ figure supplement ぬょ┻ ンヴヴ We also processed sequencing data from an African forest elephant ゅSLどどどなょ that ンヴヵ was sequenced to high┽coverage at the Broad )nstitute and re┽processed sequencing ンヴヶ data of an Asian elephant from ゅLynch et al┻ にどなのょ┻ We trimmed adapters with SeqPrep ンヴΑ な┻な using default parameters and aligned paired┽end reads to loxAfrね using BWA╆s ╅aln╆ ンヴΒ algorithm and SAMtools╆ ╅sampe╆ command┻ Duplicate reads were removed with ンヴΓ

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SAMtools╆s ╅rmdup╆┻ Moreover┸ we used the high┽coverage genome of a woolly mammoth ンヵヰ ゅWrangelょ from ゅPalkopoulou et al┻ にどなのょ┻ The woolly mammoth alignments were re┽ンヵヱ processed for removal of duplicate reads with the custom script mentioned above┻ ンヵヲ

Nuclear DNA phylogenetic reconstruction ンヵン To determine the phylogenetic relationships between the two P. antiquus ンヵヴ specimens and other members of the Elephantidae family┸ we called pseudo┽haploid ンヵヵ consensus sequences for all autosomes of the two P. antiquus samples ゅbばばど and bになど ンヵヶ million sites┸ respectivelyょ┻ Sites with base quality below ぬど and reads with mapping ンヵΑ quality below ぬど were filtered out┻ To exclude post┽mortem damage┽derived C to T ンヵΒ substitutions┸ we trimmed にbp from the ends of all reads┻ We included regions of the ンヵΓ loxAfrね genome for which at least ひどガ of all possible ぬの┽mers do not find a match at ンヶヰ another position allowing for up to one mismatch┸ similar to the mappability filter ンヶヱ described in ゅPrüfer et al┻ にどなねょ. We used a majority┽allele calling rule that required at ンヶヲ least one read aligned at each position of the genome┻ Using the same approach┸ we ンヶン called sequences for an Asian elephant ゅUno ゅLynch et al┻ にどなのょょ┸ a woolly mammoth ンヶヴ ゅWrangel ゅPalkopoulou et al┻ にどなのょょ and an African forest elephant ゅSLどどどな┹ Broad ンヶヵ )nstituteょ┻ We also used the reference sequence loxAfrね┸ as an African savanna elephant┻ ンヶヶ We estimated the number of differences per base┽pair for pairwise comparisons of all ンヶΑ sequences and constructed a distance matrix┸ from which we built a Neighbor┽joining ンヶΒ ゅNJょ tree using P(YL)P version ぬ┻はひは ゅFelsenstein にどどのょ┻ To obtain support values for ンヶΓ the nodes of the tree┸ we performed a bootstrap analysis ゅなどど replicatesょ by splitting all ンΑヰ autosomes in blocks of のMb and randomly sampling blocks with replacement and built a ンΑヱ majority┽rule consensus tree┻ ンΑヲ We also extracted coding DNA sites ゅCDSょ of protein┽coding genes using the ンΑン Ensembl ぱば release for the loxAfrぬ genome ゅdownloaded from ンΑヴ http┺【【www┻ensembl┻org【ょ from each elephant genome sequence┻ CDS mapping to ンΑヵ unknown chromosomes as well as CDS containing partial codons were excluded┸ ンΑヶ resulting in a total of ぱは┸になに CDS┻ Multiple sequence alignments were generated for each ンΑΑ gene using MAFFT ‒ginsi ゅKatoh et al┻ にどどに┸ Katoh and Standley にどなぬょ with な┸どどど ンΑΒ iterations┸ which were concatenated into a single fasta file┻ Maximum likelihood ンΑΓ phylogenetic analysis was performed with RAxML vぱ┻に ゅStamatakis にどなねょ with the ンΒヰ GTRGAMMA model of nucleotide substitutions and などど bootstrap trees┻ The resulting ンΒヱ

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phylogeny is identical in its topology to that of the NJ tree with などどガ bootstrap support ンΒヲ ゅFigure に ‒ figure supplement ねょ┻ ンΒン

Dating the specimens ンΒヴ Amino acid racemization ゅAARょ analyses were undertaken on the intra┽ンΒヵ crystalline protein from four individual Bithynia tentaculata opercula from the Eemian ンΒヶ type┽site┸ Amersfoort ゅCleveringa et al┻ にどどどょ┺ Amersfoort┽な┸ upper depth にば┻ばな┸ lower ンΒΑ depth にぱ┻のど ゅNEaar にひぱに┽ぬ┸ ぬひばに ┃ ねはぱなょ and compared with previously published ンΒΒ data from a single horizon at Neumark┽Nord な ゅなの┻の┻ぱば【に┸ Schluffmudde┸ にの cm under ンΒΓ Anmoor サ surface of the lower shore area┹ NEaar のはひぱ┽のばどぬ ゅPenkman にどなどょょ and ンΓヰ several horizons from Neumark┽Nord に ゅSier et al┻ にどななょ┻ All samples were prepared ンΓヱ using procedures of isolating the intra┽crystalline protein by bleaching ゅPenkman et al┻ ンΓヲ にどどぱょ┻ Two subsamples were then taken from each shell┹ one fraction was directly ンΓン demineralised and the free amino acids analysed ゅreferred to as the ffreef amino acids┸ ンΓヴ FAA┸ Fょ┸ and the second was treated to release the peptide┽bound amino acids┸ thus ンΓヵ yielding the ftotalf amino acid concentration┸ referred to as the ╅total hydrolysable amino ンΓヶ acid fraction ゅT(AA┸ (こょ┻ Samples were analysed in duplicate by RP┽(PLC┻ During ンΓΑ preparative hydrolysis┸ both asparagine and glutamine undergo rapid irreversible ンΓΒ deamination to aspartic acid and glutamic acid respectively ゅ(ill なひはのょ┻ )t is therefore ンΓΓ not possible to distinguish between the acidic amino acids and their derivatives and they ヴヰヰ are reported together as Asx and Glx respectively┻ The D【L values of aspartic ヴヰヱ acid【asparagine┸ glutamic acid【glutamine┸ alanine and valine ゅD【L Asx┸ Glx┸ Ala┸ Valょ are ヴヰヲ then assessed to provide an overall estimate of intra┽crystalline protein decomposition ヴヰン ゅPenkman et al┻ にどななょ┻ ヴヰヴ

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Figure and data legends ヴヰΑ

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Figure 1: Palaeoloxodon antiquus┸ geographic range based on fossil finds ゅafter Pushkina ヴヰΓ にどどばょ┻ White dots indicate the locations of Weimar┽Ehringsdorf and Neumark┽Nord┻ ヴヱヰ ヴヱヱ

Figure 2┺ Phylogenetic trees relating the mitochondrial and nuclear sequences of P. ヴヱヲ

antiquus ゅNN and WEょ to other elephantids┻ (A) Maximum clade credibility ゅMCCょ tree ヴヱン resulting from a BEAST ゅDrummond et al┻ にどなにょ analysis of ぬの complete mitochondrial ヴヱヴ genomes using なの┸ねねば sites┻ Node bars and numbers show the ひのガ highest posterior ヴヱヵ density estimates for node ages and clade support┸ respectively┻ Mitochondrial ヴヱヶ partitioning scheme and molecular and coalescent models are described in ╅Materials ヴヱΑ and methods╆┻ ゅB) Pairwise┽distance Neighbor┽joining tree from between になど million ヴヱΒ and に┻の billion base pairs of nuclear shotgun sequence data┻ Bootstrap support values ヴヱΓ from などど replicates are shown inside nodes┻ Summary statistics of the underlying ヴヲヰ sequence data are available in Figure に ‒ source data な┻ ヴヲヱ

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Figure 3: A revised tree of phylogenetic relationships among elephantids┸ color┽coded ヴヲン by their presumed geographical range┻ ヴヲヴ

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Figure 2 -figure supplement 1: Sequence coverage of the NN and WE mitochondrial ヴヲヶ genomes┻ ヴヲΑ ヴヲΒ

Figure 2 � figure supplement 2: DNA fragment size distribution inferred from full┽ヴヲΓ length mtDNA sequences┻ ヴンヰ ヴンヱ

Figure 2 � figure supplement 3┺ Frequency of C to T substitutions for each position in ヴンヲ the sequence alignments┻ (A) Substitution frequencies in mitochondrial alignments┻ ヴンン Substitution frequencies are depressed in the Neumark┽Nord libraries due treatment ヴンヴ with uracil┽DNA┽glycosylase ゅUDGょ┻ (B) )n nuclear sequence alignments┸ the ヴンヵ

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deamination signal could be partly restored by limiting analysis to cytosines in CpG ヴンヶ content┻ Since the majority of cytosines in CpG dinucleotides are methylated in ヴンΑ mammalian genomes┸ deamination leaves thymines┸ which are not excised by UDG┻ ヴンΒ

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Figure 2 � figure supplement 4: Maximum likelihood tree from concatenated nuclear ヴヴヰ protein┽coding sequences with bootstrap support values shown inside nodes┻ ヴヴヱ

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Figure 2 - figure supplement 5: Amino acid racemization data┻ D【L values of Asx┸ Glx┸ ヴヴン Ala and Val for the free amino acid ゅFAA┸ panels on the leftょ and total hydrolysable ヴヴヴ amino acid ゅT(AA┸ panels on the rightょ fraction of bleached Bithynia tentaculata ヴヴヵ opercula from Amersfoort┸ Neumark┽Nord な and に┻ Ranges for samples from UK sites ヴヴヶ correlated with M)S のe and M)S ば are indicative only┸ as effective diagenetic ヴヴΑ temperatures are likely to have differed significantly between Britain and continental ヴヴΒ Europe┻ The boundary of the box closest to zero indicates the にのth percentile┸ the ヴヴΓ dashed line within the box marks the mean and the boundary of the box farthest from ヴヵヰ zero indicates the ばのth percentile┻ The などth and ひどth percentiles are represented by ヴヵヱ lines above and below the boxes┻ The results of each duplicate analysis are included in ヴヵヲ order to provide a statistically significant sample size┻ ヴヵン

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Figure 2 � source data 1 ヴヵヵ This spreadsheet contains summary statistics of all sequence data generated in this ヴヵヶ study┸ the sequences of PCR primers used for reconstructing mtDNA sequences of extant ヴヵΑ elephants┸ as well as amino acid racemization data on opercula of Bithynia tentaculata ヴヵΒ from Amersfoort┻ ヴヵΓ

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Acknowledgments ヴヶヰ We thank the Elephant Genome Sequencing Consortium┸ especially Elinor Karlsson and ヴヶヱ Jessica Alfoldi┸ for permission to use the African forest elephant genome data┻ We thank ヴヶヲ Chloé Piot for help with preparing the figures┸ A┻ Brandt and T┻ Perrin┽Stowe for ヴヶン sequence information and Adrian Lister for comments on the manuscript┻ Mitochondrial ヴヶヴ genome sequences were deposited in GenBank under accession nos┻ KY499555 ┽ ヴヶヵ

KY499558┸ nuclear sequences alignments are available through the European ヴヶヶ Nucleotide Archive ゅENAょ project no┻ PRJEBなぱのはぬ┻ ヴヶΑ ヴヶΒ

Funding � separate metainformation ヴヶΓ MM is supported by the Max Planck Society┻ BS was supported by the Gordon and Betty ヴΑヰ Moore Foundation┻ AR and Y) were supported by the US Fish and Wildlife Service┻ The ヴΑヱ amino acid analyses were supported by Wellcome Trust and Leverhulme Trust funding ヴΑヲ to KP┸ with thanks to Sheila Taylor for technical support┻ M( is supported by ERC ヴΑン consolidator grant ぬなどばはぬ GeneFlow┻ ヴΑヴ

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References ヴΑヶ

Bヴ;ミSデが Aく Lくが Yく IゲエキS;が Nく Jく GWラヴェキ;Sキゲが ;ミS Aく Lく RラI;く ヲヰヱヲく ゎFラヴWゲデ WノWヮエ;ミデ マキデラIエラミSヴキ;ノ ヴΑΑ ェWミラマWゲ ヴW┗W;ノ デエ;デ WノWヮエ;ミデキS Sキ┗WヴゲキaキI;デキラミ キミ AaヴキI; デヴ;IニWS Iノキマ;デW デヴ;ミゲキデキラミゲくゎ ヴΑΒ MラノWI┌ノ;ヴ EIラノラェ┞ ヲヱ ふヵぶぎヱヱΑヵどヱヱΒΓく Sラキぎ ヱヰくヱヱヱヱっテくヱンヶヵどヲΓヴXくヲヰヱヲくヰヵヴヶヱく┝く ヴΑΓ

C;エキノノが Jく Aくが Rく Eく GヴWWミが Tく Lく F┌ノデラミが Mく “デキノノWヴが Fく J;┞が Nく O┗ゲ┞;ミキニラ┗が Rく “;ノ;マ┣;SWが Jく “デ Jラエミが Iく ヴΒヰ “デキヴノキミェが Mく “ノ;デニキミが ;ミS Bく “エ;ヮキヴラく ヲヰヱンく ゎGWミラマキI W┗キSWミIW aラヴ キゲノ;ミS ヮラヮ┌ノ;デキラミ ヴΒヱ Iラミ┗Wヴゲキラミ ヴWゲラノ┗Wゲ IラミaノキIデキミェ デエWラヴキWゲ ラa ヮラノ;ヴ HW;ヴ W┗ラノ┌デキラミくゎ PLラS GWミWデ Γ ヴΒヲ ふンぶぎWヱヰヰンンヴヵく Sラキぎ ヱヰくヱンΑヱっテラ┌ヴミ;ノくヮェWミくヱヰヰンンヴヵく ヴΒン

CノW┗Wヴキミェ;が Pくが Tく MWキテWヴが Rく Jく Wく ┗;ミ LWW┌┘Wミが Hく SW Wラノaが Rく Pラ┌┘Wヴが Tく LキゲゲWミHWヴェが ;ミS Aく Wく ヴΒヴ B┌ヴェWヴく ヲヰヰヰく ゎTエW EWマキ;ミ ゲデヴ;デラデ┞ヮW ノラI;ノキデ┞ ;デ AマWヴゲaララヴデ キミ デエW IWミデヴ;ノ ヴΒヵ NWデエWヴノ;ミSゲぎ ; ヴWどW┗;ノ┌;デキラミ ラa ラノS ;ミS ミW┘ S;デ;くゎ GWラノラェキW Eミ MキテミHラ┌┘どヴΒヶ NWデエWヴノ;ミSゲ Jラ┌ヴミ;ノ ラa GWラゲIキWミIWゲ ΑΓ ふヲどンぶぎヱΓΑどヲヱヶく ヴΒΑ

D;HミW┞が Jくが Mく Kミ;ヮヮが Iく GノラIニWが Mく Tく G;ミゲ;┌ェWが Aく WWキエマ;ミミが Bく NキIニWノが Cく V;ノSキラゲWヴ;が Nく ヴΒΒ G;ヴIキ;が “く P@@Hラが Jく Lく Aヴゲ┌;ェ;が ;ミS Mく MW┞Wヴく ヲヰヱンく ゎCラマヮノWデW マキデラIエラミSヴキ;ノ ェWミラマW ヴΒΓ ゲWケ┌WミIW ラa ; MキSSノW PノWキゲデラIWミW I;┗W HW;ヴ ヴWIラミゲデヴ┌IデWS aヴラマ ┌ノデヴ;ゲエラヴデ DNA ヴΓヰ aヴ;ェマWミデゲくゎ PヴラI N;デノ AI;S SIキ U S A ヱヱヰ ふンΓぶぎヱヵΑヵΒどヶンく Sラキぎ ヴΓヱ ヱヰくヱヰΑンっヮミ;ゲくヱンヱヴヴヴヵヱヱヰく ヴΓヲ

D;HミW┞が Jくが ;ミS Mく MW┞Wヴく ヲヰヱヲく ゎLWミェデエ ;ミS GCどHキ;ゲWゲ S┌ヴキミェ ゲWケ┌WミIキミェ ノキHヴ;ヴ┞ ヴΓン ;マヮノキaキI;デキラミぎ A Iラマヮ;ヴキゲラミ ラa ┗;ヴキラ┌ゲ ヮラノ┞マWヴ;ゲWどH┌aaWヴ ゲ┞ゲデWマゲ ┘キデエ ;ミIキWミデ ;ミS ヴΓヴ マラSWヴミ DNA ゲWケ┌WミIキミェ ノキHヴ;ヴキWゲくゎ BキラデWIエミキケ┌Wゲ ヵヲ ふヲぶぎΒΑどЩく Sラキぎ ヴΓヵ ヱヰくヲヱヴヴっヰヰヰヱヱンΒヰΓく ヴΓヶ

DWHヴ┌┞ミWが Rく ヲヰヰヵく ゎA I;ゲW ゲデ┌S┞ ラa ;ヮヮ;ヴWミデ IラミaノキIデ HWデ┘WWミ マラノWI┌ノ;ヴ ヮエ┞ノラェWミキWゲぎ デエW ヴΓΑ キミデWヴヴWノ;デキラミゲエキヮゲ ラa AaヴキI;ミ WノWヮエ;ミデゲくゎ Cノ;SキゲデキIゲ ヲヱ ふヱぶぎンヱどヵヰく ヴΓΒ

Dヴ┌ママラミSが Aく Jくが “く Yく Hラが Mく Jく Pエキノノキヮゲが ;ミS Aく R;マH;┌デく ヲヰヰヶく ゎRWノ;┝WS ヮエ┞ノラェWミWデキIゲ ;ミS ヴΓΓ S;デキミェ ┘キデエ IラミaキSWミIWくゎ PLラS Bキラノ ヴ ふヵぶぎWΒΒく Sラキぎ ヱヰくヱンΑヱっテラ┌ヴミ;ノくヮHキラくヰヰヴヰヰΒΒく ヵヰヰ

Dヴ┌ママラミSが Aく Jくが Mく Aく “┌Iエ;ヴSが Dく XキWが ;ミS Aく R;マH;┌デく ヲヰヱヲく ゎB;┞Wゲキ;ミ ヮエ┞ノラェWミWデキIゲ ┘キデエ ヵヰヱ BEAUデキ ;ミS デエW BEA“T ヱくΑくゎ Mラノ Bキラノ E┗ラノ ヲΓ ふΒぶぎヱΓヶΓどΑンく Sラキぎ ヵヰヲ ヱヰくヱヰΓンっマラノHW┗っマゲゲヰΑヵく ヵヰン

FWノゲWミゲデWキミが Jく ヲヰヰヵく ゎPHYLIP ふPエ┞ノラェWミ┞ IミaWヴWミIW P;Iニ;ェWぶ ┗Wヴゲキラミ ンくヶくゎ DキゲデヴキH┌デWS H┞ デエW ヵヰヴ ;┌デエラヴく DWヮ;ヴデマWミデ ラa GWミラマW SIキWミIWゲが Uミキ┗Wヴゲキデ┞ ラa W;ゲエキミェデラミが SW;デデノWく ヵヰヵ

F┌が Qくが Mく MW┞Wヴが Xく G;ラが Uく “デWミ┣Wノが Hく Aく B┌ヴH;ミラが Jく KWノゲラが ;ミS “く P@@Hラく ヲヰヱンく ゎDNA ヵヰヶ ;ミ;ノ┞ゲキゲ ラa ;ミ W;ヴノ┞ マラSWヴミ エ┌マ;ミ aヴラマ Tキ;ミ┞┌;ミ C;┗Wが Cエキミ;くゎ PヴラI N;デノ AI;S SIキ U ヵヰΑ S A ヱヱヰ ふヶぶぎヲヲヲンどΑく Sラキぎ ヱヰくヱヰΑンっヮミ;ゲくヱヲヲヱンヵΓヱヱヰく ヵヰΒ

G;マH;が Cくが Eく Rく JラミWゲが Mく Dく TW;ゲS;ノWが Rく Lく MIL;┌ェエノキミが Gく Gラミ┣;ノW┣どFラヴデWゲが Vく M;デデキ;ミェWノキが Lく ヵヰΓ DラマHラヴラI┣ニキが Iく Kラ┗;ヴキが Iく P;ヮが Aく AミSWヴゲが Aく WエキデデノWが Jく D;ミキが Pく R;I┣ニ┞が Tく Fく Hキェエ;マが Mく ヵヱヰ HラaヴWキデWヴが Dく Gく Bヴ;SノW┞が ;ミS Rく Pキミエ;ゲキく ヲヰヱヴく ゎGWミラマW aノ┌┝ ;ミS ゲデ;ゲキゲ キミ ; aキ┗W ヵヱヱ マキノノWミミキ┌マ デヴ;ミゲWIデ ラa E┌ヴラヮW;ミ ヮヴWエキゲデラヴ┞くゎ N;デ Cラママ┌ミ ヵぎヵヲヵΑく Sラキぎ ヵヱヲ ヱヰくヱヰンΒっミIラママゲヶヲヵΑく ヵヱン

G;ミゲ;┌ェWが Mく Tくが ;ミS Mく MW┞Wヴく ヲヰヱンく ゎ“キミェノWどゲデヴ;ミSWS DNA ノキHヴ;ヴ┞ ヮヴWヮ;ヴ;デキラミ aラヴ デエW ヵヱヴ ゲWケ┌WミIキミェ ラa ;ミIキWミデ ラヴ S;マ;ェWS DNAくゎ N;デ PヴラデラI Β ふヴぶぎΑンΑどヴΒく Sラキぎ ヵヱヵ ヱヰくヱヰンΒっミヮヴラデくヲヰヱンくヰンΒく ヵヱヶ

Gキノノが Mく “くが Pく LWマW┞が Nく Rく F;ヴキ;が Aく R;マH;┌デが Bく “エ;ヮキヴラが ;ミS Mく Aく “┌Iエ;ヴSく ヲヰヱンく ゎIマヮヴラ┗キミェ ヵヱΑ B;┞Wゲキ;ミ ヮラヮ┌ノ;デキラミ S┞ミ;マキIゲ キミaWヴWミIWぎ ; Iラ;ノWゲIWミデどH;ゲWS マラSWノ aラヴ マ┌ノデキヮノW ノラIキくゎ ヵヱΒ Mラノ Bキラノ E┗ラノ ンヰ ふンぶぎΑヱンどヲヴく Sラキぎ ヱヰくヱヰΓンっマラノHW┗っマゲゲヲヶヵく ヵヱΓ

Hキノノが Rく Lく ヱΓヶヵく ゎH┞Sヴラノ┞ゲキゲ ラa ヮヴラデWキミゲくゎ AS┗ PヴラデWキミ CエWマ ヲヰぎンΑどヱヰΑく ヵヲヰ IゲエキS;が Yくが Nく Jく GWラヴェキ;Sキゲが Tく HラミSラが ;ミS Aく Lく RラI;く ヲヰヱンく ゎTヴキ;ミェ┌ノ;デキミェ デエW ヮヴラ┗Wミ;ミIW ラa ヵヲヱ

AaヴキI;ミ WノWヮエ;ミデゲ ┌ゲキミェ マキデラIエラミSヴキ;ノ DNAくゎ E┗ラノ┌デキラミ;ヴ┞ AヮヮノキI;デキラミゲ ヶ ふヲぶぎヲヵンどヲヶヵく ヵヲヲ Sラキぎ ヱヰくヱヱヱヱっテくヱΑヵヲどヴヵΑヱくヲヰヱヲくヰヰヲΒヶく┝く ヵヲン

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K;エノニWが Hく Dく ふWSぶく ヱΓΑヵく ゎD;ゲ PノWキゲデラ┣@ミ ┗ラミ WWキマ;ヴどEエヴキミェゲSラヴaが TWキノ ヲくゎ AHエ;ミSノく )Wミデヴく ヵヲヴ GWラノく Iミゲデく ヲンぎヱどヵΓヶく ヵヲヵ

K;デラエが Kくが Mキゲ;┘;が Kくが K┌マ;が Kく わ Mキ┞;デ;が Tく ヲヰヰヲく ゎMAFFTぎ ; ミラ┗Wノ マWデエラS aラヴ ヴ;ヮキS マ┌ノデキヮノW ヵヲヶ ゲWケ┌WミIW ;ノキェミマWミデ H;ゲWS ラミ a;ゲデ Fラ┌ヴキWヴ デヴ;ミゲaラヴマくゎ N┌IノWキI AIキSゲ RWゲ ンヰぎ ンヰヵΓどヵヲΑ ンヰヶヶく ヵヲΒ

K;デラエが Kく わ “デ;ミSノW┞が DくMく ヲヰヱンく ゎMAFFT マ┌ノデキヮノW ゲWケ┌WミIW ;ノキェミマWミデ ゲラaデ┘;ヴW ┗Wヴゲキラミ Αぎ ヵヲΓ キマヮヴラ┗WマWミデゲ キミ ヮWヴaラヴマ;ミIW ;ミS ┌ゲ;Hキノキデ┞くゎ Mラノ Bキラノ E┗ラノ ンヰぎ ΑΑヲどΑΒヰく ヵンヰ

KキヴIエWヴが Mくが “く “;┘┞Wヴが ;ミS Mく MW┞Wヴく ヲヰヱヲく ゎDラ┌HノW キミSW┝キミェ ラ┗WヴIラマWゲ キミ;II┌ヴ;IキWゲ キミ ヵンヱ マ┌ノデキヮノW┝ ゲWケ┌WミIキミェ ラミ デエW Iノノ┌マキミ; ヮノ;デaラヴマくゎ N┌IノWキI AIキSゲ RWゲ ヴヰ ふヱぶぎWンく Sラキぎ ヵンヲ ヱヰくヱヰΓンっミ;ヴっェニヴΑΑヱく ヵンン

Lキが Gくが Bく Wく D;┗キゲが Eく Eキ┣キヴキニが ;ミS Wく Jく M┌ヴヮエ┞く ヲヰヱヶく ゎPエ┞ノラェWミラマキI W┗キSWミIW aラヴ ;ミIキWミデ ヵンヴ エ┞HヴキSキ┣;デキラミ キミ デエW ェWミラマWゲ ラa ノキ┗キミェ I;デゲ ふFWノキS;Wぶくゎ GWミラマW RWゲ ヲヶ ふヱぶぎヱどヱヱく Sラキぎ ヵンヵ ヱヰくヱヱヰヱっェヴくヱΒヶヶヶΒくヱヱヴく ヵンヶ

Lキが Hくが ;ミS Rく D┌ヴHキミく ヲヰヰΓく ゎF;ゲデ ;ミS ;II┌ヴ;デW ゲエラヴデ ヴW;S ;ノキェミマWミデ ┘キデエ B┌ヴヴラ┘ゲどWエWWノWヴ ヵンΑ デヴ;ミゲaラヴマくゎ Bキラキミaラヴマ;デキIゲ ヲヵ ふヱヴぶぎヱΑヵヴどヶヰく Sラキぎ ヱヰくヱヰΓンっHキラキミaラヴマ;デキIゲっHデヮンヲヴく ヵンΒ

Lキが Hくが Bく H;ミSゲ;ニWヴが Aく W┞ゲラニWヴが Tく FWミミWノノが Jく R┌;ミが Nく HラマWヴが Gく M;ヴデエが Gく AHWI;ゲキゲが Rく ヵンΓ D┌ヴHキミが ;ミS “┌Hェヴラ┌ヮ GWミラマW PヴラテWIデ D;デ; PヴラIWゲゲキミェく ヲヰヰΓく ゎTエW “Wケ┌WミIW ヵヴヰ AノキェミマWミデっM;ヮ aラヴマ;デ ;ミS “AMデララノゲくゎ Bキラキミaラヴマ;デキIゲ ヲヵ ふヱヶぶぎヲヰΑΒどΓく Sラキぎ ヵヴヱ ヱヰくヱヰΓンっHキラキミaラヴマ;デキIゲっHデヮンヵヲく ヵヴヲ

LキゲデWヴが Aく Mく ヲヰヱヵく ゎD;デキミェ デエW ;ヴヴキ┗;ノ ラa ゲデヴ;キェエデどデ┌ゲニWS WノWヮエ;ミデ ふP;ノ;Wラノラ┝ラSラミ ゲヮヮくぶ キミ ヵヴン E┌ヴ;ゲキ;くゎ B┌ノノく M┌ゲく Aミデエヴラヮラノく ヮヴYエキゲデく Mラミ;Iラ ゲ┌ヮヮノく ミェ ヶぎヱンどヱΒく ヵヴヴ

L┞ミIエが Vく Jくが Oく Cく BWSラ┞;どRWキミ;が Aく R;デ;ミが Mく “┌ノ;ニが Dく Iく Dヴ;┌デ┣どMラゲWゲが Gく Hく PWヴヴ┞が Wく MキノノWヴが ヵヴヵ ;ミS “く Cく “Iエ┌ゲデWヴく ヲヰヱヵく ゎEノWヮエ;ミデキS GWミラマWゲ RW┗W;ノ デエW MラノWI┌ノ;ヴ B;ゲWゲ ラa Wララノノ┞ ヵヴヶ M;ママラデエ AS;ヮデ;デキラミゲ デラ デエW AヴIデキIくゎ CWノノ RWヮ ヱヲ ふヲぶぎヲヱΑどヲΒく Sラキぎ ヵヴΑ ヱヰくヱヰヱヶっテくIWノヴWヮくヲヰヱヵくヰヶくヰヲΑく ヵヴΒ

M;ェノキラが Vく Jく ヱΓΑンく ゎOヴキェキミ ;ミS W┗ラノ┌デキラミ ラa デエW WノWヮエ;ミデキS;Wくゎ Tヴ;ミゲ;Iデキラミゲ ラa デエW AマWヴキI;ミ ヵヴΓ PエキノラゲラヮエキI;ノ SラIキWデ┞ ヶンンぎヱどヱヴΓく ヵヵヰ

M;ノノキIニが Rくが ;ミS Nく Fヴ;ミニく ヲヰヰヲく ゎA ミW┘ デWIエミキケ┌W aラヴ ヮヴWIキゲW ┌ヴ;ミキ┌マどゲWヴキWゲ S;デキミェ ラa ヵヵヱ デヴ;┗WヴデキミW マキIヴラどゲ;マヮノWゲくゎ GWラIエキマキI; Eデ CラゲマラIエキマキI; AIデ; ヶヶ ふヲヴぶぎヴヲヶヱどヴヲΑヲく ヵヵヲ

M;ミキ;が Dく ヲヰヱヰく ゎTエW ヮラゲキデキラミキミェ ラa デエW ┘;ヴマ ヮWヴキラS ラa NW┌マ;ヴニどNラヴS ;ミS キデゲ WノWヮエ;ミデどa;┌ミ; ヵヵン キミ デエW IラミデW┝デ ラa デエW エキゲデラヴ┞ ラa デエW W;ヴデエくゎ EノWa;ミデWミヴWキIエ に WキミW Fラゲゲキノ┘Wノデ キミ E┌ヴラヮ; ヵヵヴ ふWS MWノノWヴが Hくぶが L;ミSWゲ;マデ a┑ヴ SWミニマ;ノヮaノWェW ┌ミS AヴIエ@ラノラェキW S;IエゲWミどAミエ;ノデ に ヵヵヵ L;ミSWゲマ┌ゲW┌マ a┑ヴ VラヴェWゲIエキIエデW H;ノノW ふS;;ノWぶぎヶヵどヶΓく ヵヵヶ

M;ヴキIキIが Tくが Mく WエキデデWミが ;ミS “く P@@Hラく ヲヰヱヰく ゎM┌ノデキヮノW┝WS DNA ゲWケ┌WミIW I;ヮデ┌ヴW ラa ヵヵΑ マキデラIエラミSヴキ;ノ ェWミラマWゲ ┌ゲキミェ PCR ヮヴラS┌Iデゲくゎ PLラS OミW ヵ ふヱヱぶぎWヱヴヰヰヴく Sラキぎ ヵヵΒ ヱヰくヱンΑヱっテラ┌ヴミ;ノくヮラミWくヰヰヱヴヰヰヴく ヵヵΓ

MW┞Wヴが Mくが Jく Lく Aヴゲ┌;ェ;が Cく SW Fキノキヮヮラが “く N;ェWノが Aく A┝キマ┌どPWデヴキが Bく NキIニWノが Iく M;ヴデキミW┣が Aく Gヴ;Iキ;が ヵヶヰ Jく Mく Bく SW C;ゲデヴラが Eく C;ヴHラミWノノが Bく Vキラノ;が Jく KWノゲラが Kく Pヴ┌aaWヴが ;ミS “く P@@Hラく ヲヰヱヶく ヵヶヱ ゎN┌IノW;ヴ DNA ゲWケ┌WミIWゲ aヴラマ デエW MキSSノW PノWキゲデラIWミW “キマ; SW ノラゲ H┌Wゲラゲ エラマキミキミゲくゎ ヵヶヲ N;デ┌ヴW ヵンヱ ふΑヵΓヵぶぎヵヰヴどЩく Sラキぎ ヱヰくヱヰンΒっミ;デ┌ヴWヱΑヴヰヵく ヵヶン

MW┞Wヴが Mくが ;ミS Mく KキヴIエWヴく ヲヰヱヰく ゎIノノ┌マキミ; ゲWケ┌WミIキミェ ノキHヴ;ヴ┞ ヮヴWヮ;ヴ;デキラミ aラヴ エキェエノ┞ ヵヶヴ マ┌ノデキヮノW┝WS デ;ヴェWデ I;ヮデ┌ヴW ;ミS ゲWケ┌WミIキミェくゎ CラノS Sヮヴキミェ H;ヴH PヴラデラI ヲヰヱヰ ふヶぶぎヮSH ヵヶヵ ヮヴラデヵヴヴΒく Sラキぎ ヱヰくヱヱヰヱっヮSHくヮヴラデヵヴヴΒく ヵヶヶ

MW┞Wヴが Mくが Mく KキヴIエWヴが Mく Tく G;ミゲ;┌ェWが Hく Lキが Fく R;Iキマラが “く M;ノノキIニが Jく Gく “Iエヴ;キHWヴが Fく J;┞が Kく ヵヶΑ Pヴ┑aWヴが Cく SW Fキノキヮヮラが Pく Hく “┌Sマ;ミデが Cく Aノニ;ミが Qく F┌が Rく Dラが Nく Rラエノ;ミSが Aく T;ミSラミが Mく ヵヶΒ “キWH;┌Wヴが Rく Eく GヴWWミが Kく Bヴ┞Iが Aく Wく Bヴキェェゲが Uく “デWミ┣Wノが Jく D;HミW┞が Jく “エWミS┌ヴWが Jく ヵヶΓ Kキデ┣マ;ミが Mく Fく H;ママWヴが Mく Vく “エ┌ミニラ┗が Aく Pく DWヴW┗キ;ミニラが Nく P;デデWヴゲラミが Aく Mく AミSヴWゲが ヵΑヰ Eく Eく EキIエノWヴが Mく “ノ;デニキミが Dく RWキIエが Jく KWノゲラが ;ミS “く P@@Hラく ヲヰヱヲく ゎA エキェエどIラ┗Wヴ;ェW ヵΑヱ

ヲヰ

ェWミラマW ゲWケ┌WミIW aヴラマ ;ミ ;ヴIエ;キI DWミキゲラ┗;ミ キミSキ┗キS┌;ノくゎ SIキWミIW ンンΒ ふヶヱヰヴぶぎヲヲヲどヶく ヵΑヲ Sラキぎ ヱヰくヱヱヲヶっゲIキWミIWくヱヲヲヴンヴヴく ヵΑン

Oヴノ;ミSラが Lくが Aく Gキミラノエ;Iが Gく Jく )エ;ミェが Dく FヴラWゲWが Aく AノHヴWIエデゲWミが Mく “デキノノWヴが Mく “Iエ┌HWヴデが Eく ヵΑヴ C;ヮヮWノノキミキが Bく PWデWヴゲWミが Iく MラノデニWが Pく Lく Fく Jラエミゲラミが Mく F┌マ;ェ;ノノキが Jく Tく Vキノゲデヴ┌ヮが Mく ヵΑヵ R;ェエ;┗;ミが Tく KラヴミWノキ┌ゲゲWミが Aく “く M;ノ;ゲヮキミ;ゲが Jく Vラェデが Dく “┣ニノ;ヴI┣┞ニが Cく Dく KWノゲデヴ┌ヮが Jく ヵΑヶ VキミデエWヴが Aく DラノラI;ミが Jく “デWミSWヴ┌ヮが Aく Mく Vく VWノ;┣ケ┌W┣が Jく C;エキノノが Mく R;ゲマ┌ゲゲWミが Xく Lく ヵΑΑ W;ミェが Jく Mく Mキミが Gく Dく );┣┌ノ;が Aく “Wェ┌キミどOヴノ;ミSラが Cく MラヴデWミゲWミが Kく M;ェミ┌ゲゲWミが Jく Fく ヵΑΒ Tエラマヮゲラミが Jく WWキミゲデラIニが Kく GヴWェWヴゲWミが Kく Hく RラWSが Vく EキゲWミマ;ミミが Cく Jく R┌Hキミが Dく Cく ヵΑΓ MキノノWヴが Dく Fく AミデI┣;ニが Mく Fく BWヴデWノゲWミが “く Bヴ┌ミ;ニが Kく Aく “く AノどR;ゲエWキSが Oく R┞SWヴが Lく ヵΒヰ AミSWヴゲゲラミが Jく M┌ミS┞が Aく Kヴラェエが Mく Tく Pく GキノHWヴデが Kく Kテ;Wヴが Tく “キIエWヴキデ┣どPラミデWミが Lく Jく ヵΒヱ JWミゲWミが Jく Vく OノゲWミが Mく HラaヴWキデWヴが Rく NキWノゲWミが Bく “エ;ヮキヴラが Jく W;ミェが ;ミS Eく WキノノWヴゲノW┗く ヵΒヲ ヲヰヱンく ゎRWI;ノキHヴ;デキミェ Eケ┌┌ゲ W┗ラノ┌デキラミ ┌ゲキミェ デエW ェWミラマW ゲWケ┌WミIW ラa ;ミ W;ヴノ┞ MキSSノW ヵΒン PノWキゲデラIWミW エラヴゲWくゎ N;デ┌ヴW ヴΓΓ ふΑヴヵヶぶぎΑヴどЩく Sラキぎ ヱヰくヱヰンΒっミ;デ┌ヴWヱヲンヲンく ヵΒヴ

OゲHラヴミが HくFくが ヱΓヴヲく PヴラHラゲIキSW;ぎ A Mラミラェヴ;ヮエ ラa デエW DキゲIラ┗Wヴ┞が E┗ラノ┌デキラミが Mキェヴ;デキラミ ;ミS ヵΒヵ E┝デキミIデキラミ ラa デエW M;ゲデラSラミデゲ ;ミS EノWヮエ;ミデゲ ラa デエW WラヴノSく Vラノく IIぎ “デWェラSラミデラキSW;が ヵΒヶ EノWヮエ;ミデラキSW;く TエW AマWヴキI;ミ M┌ゲW┌マ PヴWゲゲが NW┘ Yラヴニく ヵΒΑ

O┘Wミど“マキデエが Nく ヲヰヱンく ゎCラミデヴ;ゲデゲ キミ デエW ノ;ヴェW エWヴHキ┗ラヴW a;┌ミ;ゲ ラa デエW ゲラ┌デエWヴミ IラミデキミWミデゲ キミ ヵΒΒ デエW ノ;デW PノWキゲデラIWミW ;ミS デエW WIラノラェキI;ノ キマヮノキI;デキラミゲ aラヴ エ┌マ;ミ ラヴキェキミゲくゎ J BキラェWラェヴ ヴヰぎ ヵΒΓ ヱヲヱヵにヱヲヲヴく Dラキぎ ヱヰくヱヱヱヱっテHキくヱヲヱヰヰ ヵΓヰ

P;ノニラヮラ┌ノラ┌が Eくが “く M;ノノキIニが Pく “ニラェノ┌ミSが Jく Eミニが Nく Rラエノ;ミSが Hく Lキが Aく Oマヴ;ニが “く V;ヴデ;ミ┞;ミが Hく ヵΓヱ Pラキミ;ヴが Aく GラデエWヴゲデヴラマが Dく RWキIエが ;ミS Lく D;ノWミく ヲヰヱヵく ゎCラマヮノWデW ェWミラマWゲ ヴW┗W;ノ ヵΓヲ ゲキェミ;デ┌ヴWゲ ラa SWマラェヴ;ヮエキI ;ミS ェWミWデキI SWIノキミWゲ キミ デエW ┘ララノノ┞ マ;ママラデエくゎ C┌ヴヴ Bキラノ ヲヵ ヵΓン ふヱヰぶぎヱンΓヵどヴヰヰく Sラキぎ ヱヰくヱヰヱヶっテくI┌HくヲヰヱヵくヰヴくヰヰΑく ヵΓヴ

PWミニマ;ミが Kく ヲヰヱヰく ゎNW┌マ;ヴニどNラヴS ヱぎ PヴWノキマキミ;ヴ┞ ヴWゲ┌ノデゲ ラa デエW ;マキミラ ;IキS ;ミ;ノ┞ゲキゲくゎ ヵΓヵ EノWa;ミデWミヴWキIエ に WキミW Fラゲゲキノ┘Wノデ キミ E┌ヴラヮ; ふWS MWノノWヴが Hくぶが L;ミSWゲ;マデ a┑ヴ ヵΓヶ DWミニマ;ノヮaノWェW ┌ミS AヴIエ@ラノラェキW S;IエゲWミどAミエ;ノデ に L;ミSWゲマ┌ゲW┌マ a┑ヴ VラヴェWゲIエキIエデW ヵΓΑ H;ノノW ふS;;ノWぶぎΑヵどΑΒく ヵΓΒ

PWミニマ;ミが Kく Eく Hくが Dく “く K;┌aマ;ミが Dく M;SS┞が ;ミS Mく Jく Cラノノキミゲく ヲヰヰΒく ゎCノラゲWSどゲ┞ゲデWマ HWエ;┗キラ┌ヴ ヵΓΓ ラa デエW キミデヴ;どIヴ┞ゲデ;ノノキミW aヴ;Iデキラミ ラa ;マキミラ ;IキSゲ キミ マラノノ┌ゲI ゲエWノノゲくゎ Q┌;デWヴミ;ヴ┞ ヶヰヰ GWラIエヴラミラノラェ┞ ン ふヱどヲぶぎヲどヲヵく Sラキぎ ヱヰくヱヰヱヶっテくケ┌;ェWラくヲヰヰΑくヰΑくヰヰヱく ヶヰヱ

PWミニマ;ミが Kく Eくが Rく Cく PヴWWIWが Dく Rく BヴキSェノ;ミSが Dく Hく KWWミが Tく MWキテWヴが “く Aく P;ヴaキデデが Tく “く WエキデWが ヶヰヲ ;ミS Mく Jく Cラノノキミゲく ヲヰヱヱく ゎA IエヴラミラノラェキI;ノ aヴ;マW┘ラヴニ aラヴ デエW Bヴキデキゲエ Q┌;デWヴミ;ヴ┞ H;ゲWS ヶヰン ラミ Bキデエ┞ミキ; ラヮWヴI┌ノ;くゎ N;デ┌ヴW ヴΑヶ ふΑンヶヱぶぎヴヴヶどΓく Sラキぎ ヱヰくヱヰンΒっミ;デ┌ヴWヱヰンヰヵく ヶヰヴ

PWデキデが Rく Jくが ;ミS Lく E┝IラaaキWヴく ヲヰヰΓく ゎGWミW aノラ┘ ;ミS ゲヮWIキWゲ SWノキマキデ;デキラミくゎ TヴWミSゲ EIラノ E┗ラノ ヲヴ ヶヰヵ ふΑぶぎンΒヶどΓンく Sラキぎ ヱヰくヱヰヱヶっテくデヴWWくヲヰヰΓくヰヲくヰヱヱく ヶヰヶ

Pラゲ;S;が Dく ヲヰヰΒく ゎテMラSWノTWゲデぎ ヮエ┞ノラェWミWデキI マラSWノ ;┗Wヴ;ェキミェくゎ Mラノ Bキラノ E┗ラノ ヲヵ ふΑぶぎヱヲヵンどヶく ヶヰΑ Sラキぎ ヱヰくヱヰΓンっマラノHW┗っマゲミヰΒンく ヶヰΒ

Pヴ┑aWヴが Kくが Fく R;Iキマラが Nく P;デデWヴゲラミが Fく J;┞が “く “;ミニ;ヴ;ヴ;マ;ミが “く “;┘┞Wヴが Aく HWキミ┣Wが Gく RWミ;┌Sが Pく ヶヰΓ Hく “┌Sマ;ミデが Cく SW Fキノキヮヮラが Hく Lキが “く M;ノノキIニが Mく D;ミミWマ;ミミが Qく F┌が Mく KキヴIエWヴが Mく ヶヱヰ K┌エノ┘キノマが Mく L;Iエマ;ミミが Mく MW┞Wヴが Mく Oミェ┞Wヴデエが Mく “キWH;┌Wヴが Cく TエW┌ミWヴデが Aく T;ミSラミが ヶヱヱ Pく Mララヴテ;ミキが Jく PキIニヴWノノが Jく Cく M┌ノノキニキミが “く Hく Vラエヴが Rく Eく GヴWWミが Iく HWノノマ;ミミが Pく Lく Jラエミゲラミが ヶヱヲ Hく Bノ;ミIエWが Hく C;ミミが Jく Oく Kキデ┣マ;ミが Jく “エWミS┌ヴWが Eく Eく EキIエノWヴが Eく “く LWキミが Tく Eく B;ニニWミが Lく ヶヱン Vく Gラノラ┗;ミラ┗;が Vく Bく DラヴラミキIエW┗が Mく Vく “エ┌ミニラ┗が Aく Pく DWヴW┗キ;ミニラが Bく Vキラノ;が Mく “ノ;デニキミが ヶヱヴ Dく RWキIエが Jく KWノゲラが ;ミS “く P@@Hラく ヲヰヱヴく ゎTエW IラマヮノWデW ェWミラマW ゲWケ┌WミIW ラa ; ヶヱヵ NW;ミSWヴデエ;ノ aヴラマ デエW Aノデ;キ Mラ┌ミデ;キミゲくゎ N;デ┌ヴW ヵヰヵ ふΑヴΒヱぶぎヴンどΓく Sラキぎ ヶヱヶ ヱヰくヱヰンΒっミ;デ┌ヴWヱヲΒΒヶく ヶヱΑ

ヲヱ

P┌ゲエニキミ;が Dく ヲヰヰΑく ゎTエW PノWキゲデラIWミW W;ゲデWヴミマラゲデ SキゲデヴキH┌デキラミ キミ E┌ヴ;ゲキ; ラa デエW ゲヮWIキWゲ ヶヱΒ ;ゲゲラIキ;デWS ┘キデエ デエW EWマキ;ミ P;ノ;Wラノラ┝ラSラミ ;ミデキケ┌┌ゲ ;ゲゲWマHノ;ェWく ゎ M;ママ;ノ RW┗ ンΑぎ ヶヱΓ ヲヲヴどヲヴヵく Sラキぎ ヱヰくヱヱヱヱっテくヱンヶヵどヲΓヰΑくヲヰヰΑくヰヰヱヰΓく┝ ヶヲヰ

RWミ;┌Sが Gくが Uく “デWミ┣Wノが ;ミS Jく KWノゲラく ヲヰヱヴく ゎノWWHラマぎ ;S;ヮデラヴ デヴキママキミェ ;ミS マWヴェキミェ aラヴ ヶヲヱ Iノノ┌マキミ; ゲWケ┌WミIキミェ ヴW;Sゲくゎ N┌IノWキI AIキSゲ RWゲ ヴヲ ふヱΒぶぎWヱヴヱく Sラキぎ ヱヰくヱヰΓンっミ;ヴっェニ┌ヶΓΓく ヶヲヲ

RラI;が Aく Lくが Nく GWラヴェキ;Sキゲが ;ミS “く Jく OろBヴキWミく ヲヰヰヵく ゎC┞デラミ┌IノW;ヴ ェWミラマキI SキゲゲラIキ;デキラミ キミ AaヴキI;ミ ヶヲン WノWヮエ;ミデ ゲヮWIキWゲくゎ N;デ┌ヴW GWミWデキIゲ ンΑ ふヱぶぎΓヶどヱヰヰく Sラキぎ ヱヰくヱヰンΒっミェヱヴΒヵく ヶヲヴ

Rラエノ;ミSが Nくが Aく “く M;ノ;ゲヮキミ;ゲが Jく Lく Pラノノ;Iニが Mく “ノ;デニキミが Pく M;デエW┌ゲが ;ミS Mく HラaヴWキデWヴく ヲヰヰΑく ヶヲヵ ゎPヴラHラゲIキSW;ミ マキデラェWミラマキIゲぎ Iエヴラミラノラェ┞ ;ミS マラSW ラa WノWヮエ;ミデ W┗ラノ┌デキラミ ┌ゲキミェ ヶヲヶ マ;ゲデラSラミ ;ゲ ラ┌デェヴラ┌ヮくゎ PLラS Bキラノ ヵ ふΒぶぎWヲヰΑく Sラキぎ ヱヰくヱンΑヱっテラ┌ヴミ;ノくヮHキラくヰヰヵヰヲヰΑく ヶヲΑ

Rラエノ;ミSが Nくが Dく RWキIエが “く M;ノノキIニが Mく MW┞Wヴが Rく Eく GヴWWミが Nく Jく GWラヴェキ;Sキゲが Aく Lく RラI;が ;ミS Mく ヶヲΒ HラaヴWキデWヴく ヲヰヱヰく ゎGWミラマキI DNA “Wケ┌WミIWゲ aヴラマ M;ゲデラSラミ ;ミS Wララノノ┞ M;ママラデエ ヶヲΓ RW┗W;ノ DWWヮ “ヮWIキ;デキラミ ラa FラヴWゲデ ;ミS “;┗;ミミ; EノWヮエ;ミデゲくゎ Pノラゲ Bキラノラェ┞ Β ふヱヲぶく Sラキぎ ヶンヰ ARTN WヱヰヰヰヵヶヴヱヰくヱンΑヱっテラ┌ヴミ;ノくヮHキラくヱヰヰヰヵヶヴく ヶンヱ

“;Wェ┌ゲ;が Hくが ;ミS Wく Hく GキノHWヴデく ヲヰヰΒく ゎキミ Hラマラ WヴWIデ┌ゲ キミ AaヴキI;が PノWキゲデラIWミW E┗キSWミIW aヴラマ ヶンヲ デエW MキSSノW A┘;ゲエ ふWSゲ HWミヴ┞が Wくが GキノHWヴデが Wく Hく わ Aゲa;┘が Bくぶくゎ Uミキ┗く ラa C;ノキaラヴミキ; ヶンン PヴWゲゲぎヱΓンにヲヲヶく ヶンヴ

“;ミSWヴゲが Wく Jくが Eく GエWWヴHヴ;ミデが Jく Mく H;ヴヴキゲが Hく “;Wェ┌ゲ;が ;ミS Cく DWノマWヴく ヲヰヱヰく ゎキミ CWミラ┣ラキI ヶンヵ M;ママ;ノゲ ラa AaヴキI; ふWSゲ WWヴSWノキミが Lく わ “;ミSWヴゲが Wく Jくぶくゎ Uミキ┗く ラa C;ノキaラヴミキ; PヴWゲゲぎヱヶヱにヶンヶ ヲヵヱく ヶンΑ

“Iエ┑ノWヴが Tく ヲヰヰンく ゎE“R S;デキミェ ラa ; ミW┘ ヮ;ノ;WラノキデエキI aキミS ノ;┞Wヴ ラa デエW デヴ;┗WヴデキミW ゲキデW ラa WWキマ;ヴどヶンΒ EエヴキミェゲSラヴa ふCWミデヴ;ノ GWヴマ;ミ┞ぶくゎ TWヴヴ; Nラゲデヴ; ヲぎヲンンどヲンヵく ヶンΓ

“Iエ┑ノWヴが Tく ヲヰヱヰく ゎER“ S;デキミェ ラa デララデエ Wミ;マWノ ゲ;マヮノWゲ aヴラマ デエW ;ヴIエ;WラノラェキI;ノ aキミS エラヴキ┣ラミゲ ヶヴヰ ラa NW┌マ;ヴニどNラヴSくゎ EノWa;ミデWミヴWキIエ に WキミW Fラゲゲキノ┘Wノデ キミ E┌ヴラヮ; ふWS MWノノWヴが Hくぶが ヶヴヱ L;ミSWゲ;マデ a┑ヴ DWミニマ;ノヮaノWェW ┌ミS AヴIエ@ラノラェキW S;IエゲWミどAミエ;ノデ に L;ミSWゲマ┌ゲW┌マ a┑ヴ ヶヴヲ VラヴェWゲIエキIエデW H;ノノW ふS;;ノWぶぎΑヱどΑヴく ヶヴン

“エラゲエ;ミキが Jくが Mく Pく FWヴヴWデデキが Aく Mく LキゲデWヴが Lく Dく AェWミHヴラ;Sが Hく “;Wェ┌ゲ;が Dく Mラノが ;ミS Kく T;ニ;エ;ゲエキく ヶヴヴ ヲヰヰΑく ゎRWノ;デキラミゲエキヮゲ ┘キデエキミ デエW EノWヮエ;ミデキミ;W ┌ゲキミェ エ┞ラキS Iエ;ヴ;IデWヴゲくゎ Q┌;デWヴミ;ヴ┞ ヶヴヵ IミデWヴミ;デキラミ;ノ ヱヶΓぎヱΑヴどヱΒヵく Sラキぎ ヱヰくヱヰヱヶっテくケ┌;キミデくヲヰヰΑくヰヲくヰヰンく ヶヴヶ

“キWヴが Mく Jくが Wく RラWHヴラWニゲが Cく Cく B;ニWノゲが Mく Jく DWニニWヴゲが Eく Bヴ┌エノが Dく DW LラWIニWヴが “く G;┌S┣キミゲニキどヶヴΑ WキミSエW┌ゲWヴが Nく HWゲゲWが Aく J;ェキIエが Lく KキミSノWヴが Wく Jく K┌キテヮWヴが Tく L;┌ヴ;デが Hく Jく M┌IエWヴが Kく Eく ヶヴΒ Hく PWミニマ;ミが Dく RキIエデWヴが ;ミS Dく Jく Jく ┗;ミ HキミゲHWヴェWミく ヲヰヱヱく ゎDキヴWIデ デWヴヴWゲデヴキ;ノどマ;ヴキミW ヶヴΓ IラヴヴWノ;デキラミ SWマラミゲデヴ;デWゲ ゲ┌ヴヮヴキゲキミェノ┞ ノ;デW ラミゲWデ ラa デエW ノ;ゲデ キミデWヴェノ;Iキ;ノ キミ IWミデヴ;ノ ヶヵヰ E┌ヴラヮWくゎ Q┌;デWヴミ;ヴ┞ RWゲW;ヴIエ Αヵ ふヱぶぎヲヱンどヲヱΒく Sラキぎ ヱヰくヱヰヱヶっテく┞ケヴWゲくヲヰヱヰくヱヱくヰヰンく ヶヵヱ

“デ;マ;デ;ニキゲが Aく ヲヰヱヴく さRA┝ML ┗Wヴゲキラミ Βぎ ; デララノ aラヴ ヮエ┞ノラェWミWデキI ;ミ;ノ┞ゲキゲ ;ミS ヮラゲデど;ミ;ノ┞ゲキゲ ラa ヶヵヲ ノ;ヴェW ヮエ┞ノラェWミキWゲくざ Bキラキミaラヴマ;デキIゲ ンヰぎ ヱンヱヲどヱンヱンく ヶヵン

“デWミ┣Wノが Uく ヲヰヱヴく Bキラエ;┣;ヴSく BキデH┌IニWデく エデデヮゲぎっっHキデH┌IニWデくラヴェっ┌ゲデWミ┣WノっHキラエ;┣;ヴSく ヴヲΑHaヱ;く ヶヵヴ “デ┌;ヴデが Aく Jく ヲヰヰヵく ゎTエW W┝デキミIデキラミ ラa ┘ララノノ┞ マ;ママラデエ ふM;ママ┌デエ┌ゲ ヮヴキマキェWミキ┌ゲぶ ;ミS ヶヵヵ

ゲデヴ;キェエデどデ┌ゲニWS WノWヮエ;ミデ ふP;ノ;Wラノラ┝ラSラミ ;ミデキケ┌┌ゲぶ キミ E┌ヴラヮWくゎ Q┌;デWヴミ;ヴ┞ ヶヵヶ IミデWヴミ;デキラミ;ノ ヱヲヶぎヱΑヱどヱΑΑく Sラキぎ ヱヰくヱヰヱヶっテくケ┌;キミデくヲヰヰヴくヰヴくヰヲヱく ヶヵΑ

TラSSが Nく Eく ヲヰヱヰく ゎNW┘ Pエ┞ノラェWミWデキI Aミ;ノ┞ゲキゲ ラa デエW F;マキノ┞ EノWヮエ;ミデキS;W B;ゲWS ラミ Cヴ;ミキ;ノどヶヵΒ DWミデ;ノ Mラヴヮエラノラェ┞くゎ Aミ;デラマキI;ノ RWIラヴSどAS┗;ミIWゲ キミ IミデWェヴ;デキ┗W Aミ;デラマ┞ ;ミS ヶヵΓ E┗ラノ┌デキラミ;ヴ┞ Bキラノラェ┞ ヲΓン ふヱぶぎΑヴどΓヰく Sラキぎ ヱヰくヱヰヰヲっ;ヴくヲヱヰヱヰく ヶヶヰ

);ェ┘キテミが Wく Hく ヱΓヶヱく ゎVWェWデ;デキラミが Iノキマ;デW ;ミS ヴ;SキラI;ヴHラミ S;デキミェゲ キミ デエW L;デW PノWキゲデラIWミW ラa ヶヶヱ デエW NWデエWヴノ;ミSゲく P;ヴデ Iぎ EWマキ;ミ ;ミS E;ヴノ┞ WWキIエゲWノキ;ミくゎ MWSWSWノキミェWミ ┗;ミ SW ヶヶヲ GWラノラェキゲIエW SデキIエデキミェが NキW┌┘W SWヴキW ヱヴぎヱヵどヴヵく ヶヶン

7 6 5 4 3 2 1 0

Million years ago

WE

NN

92

96

99

100

100

100

100

100

100

100

100

100

100

100

10095

99

100

100

8

NN

NN

African savannah

elephants

Loxodonta africana

Woolly mammoths

Mammuthus primigenius

Asian elephants

Elephas maximus

African forest

elephants

Loxodonta cyclotis

mtDNA nuDNA

1.0E-3

100

100

100

100

NN

NN

Eurasia

Africa

Loxodonta africana Loxodonta cyclotis Palaeoloxodon antiquus Elephas maximus Mammuthus primigenius

0 5000 10000 15000

Position in mitochondrial genome

1

10

100

1000

10000C

overa

ge

Neumark-Nord (NEPEC)

Neumark-Nord (NEU2A)

Neumark-Nord (NEU8B)

Weimar-Ehringsdorf

30 40 50 60 70 80 90 100

Fragment size [bp]

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08Fra

cti

on o

f sequences

Weimar-Ehringsdorf

Neumark-Nord

0 5 10 15

Distance to 5' end

0

0.2

0.4

0.6

0.8

C to T

substitu

tion fre

quency

15 10 5 0

Distance to 3' end

0

0.2

0.4

0.6

0.8 C to

T s

ubstitu

tion fre

quency

Weimar-Ehringsdorf

Neumark-Nord

0 5 10 15

Distance to 5' end

0

0.2

0.4

0.6

0.8

C to T

substitu

tion fre

quency

15 10 5 0

Distance to 3' end

0

0.2

0.4

0.6

0.8 C to

T s

ubstitu

tion fre

quency

NEPEC

NEU2A

A

B

6.0E-4

Loxodonta cyclotis

Paleoloxodon antiquus NEPEC

Mammuthus primigenius

Elephas maximus

Loxodonta africana

Paleoloxodon antiquus NEU2A

100

100

100

100

of each duplicate analysis are included in order to provide a statistically

size.

Asx bH

UK MIS 5e UK MIS 7 Amersfoort NN2 NN1

D/L

0.45

0.50

0.55

0.60

0.65

0.70

Asx bF

UK MIS 5e UK MIS 7 Amersfoort NN2 NN1

D/L

0.58

0.60

0.62

0.64

0.66

0.68

0.70

0.72

0.74

0.76

0.78

0.80

Glx bH

UK MIS 5e UK MIS 7 Amersfoort NN2 NN1

D/L

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.26

Glx bF

UK MIS 5e UK MIS 7 Amersfoort NN2 NN1

D/L

0.10

0.15

0.20

0.25

0.30

0.35

Ala bH

UK MIS 5e UK MIS 7 Amersfoort NN2 NN1

D/L

0.10

0.15

0.20

0.25

0.30

0.35

Ala bF

UK MIS 5e UK MIS 7 Amersfoort NN2 NN1

D/L

0.15

0.20

0.25

0.30

0.35

0.40

0.45

Val bH

UK MIS 5e UK MIS 7 Amersfoort NN2 NN1

D/L

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

Val bF

UK MIS 5e UK MIS 7 Amersfoort NN2 NN1

D/L

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.26