Cell, Vol. 90, 19–30, July 11, 1997, Copyright 1997 by Cell PressNeandertal DNA Sequencesand the Origin of Modern HumansMatthias Krings,* Anne Stone,†Ralf W. Schmitz,‡these analyses rely on assumptions, such as the ab-sence of selection and a clock-like rate of molecularHeikeKrainitzki,§MarkStoneking,†andSvantePa¨a¨bo**Zoological Institute evolution in the DNA sequences under study, whosevalidity hasbeenquestioned (Wolpoff,1989;Templeton,University of MunichPO Box 202136 1992). An additional and more directway to address thequestion of the relationship between modern humansD-80021 MunichGermany and Neandertals would be to analyze DNA sequencesfrom the remains of Neandertals.†Department of AnthropologyPennsylvania State University The reproducible retrieval of ancient DNA sequencesbecame possible with the invention of the polymeraseState College, Pennsylvania 16802‡Rheinisches Amt fu¨r Bodendenkmalpflege chain reaction (Mullis and Faloona, 1987; Pa¨a¨bo et al.,1989). However, theoretical considerations,(Pa¨a¨bo andEndenicher Strasse 133D-53115 Bonn Wilson, 1991;Lindahl1993a)aswellas empiricalstudies(Pa¨a¨bo, 1989; Ho¨ss et al., 1996a), show that DNA inGermany§Ho¨here Berufsfachschule fu¨r fossil remains is highly affected by hydrolytic as wellas oxidative damage. Therefore, the retrieval of DNApra¨parationstechnische AssistentenMarkstrasse 185 sequences older than about 100,000 years is expectedto be difficult, if not impossible, to achieve (Pa¨a¨bo andD-44799 BochumGermany Wilson, 1991). Fortunately, Neandertal remains fallwithin the age range that in principle allows DNA se-quences to survive. It is noteworthy, though, that evenamong remains that are younger than 100,000 yearsSummarymost failto yieldamplifiableDNAsequences(Ho¨ssetal.,1996b). Inaddition, contamination ofancientspecimensDNA was extracted from the Neandertal-type speci-and extractswithmodern DNA posesa seriousproblemmenfound in1856in westernGermany. Bysequencing(Handt etal.,1994a) thatrequiresnumerous precautionsclones from short overlapping PCR products, a hith-and controls. This is particularly the case when humanerto unknown mitochondrial (mt) DNA sequence wasremains arestudied, sincehuman DNA is themost com-determined. Multiple controls indicate that this se-mon source of contamination. Therefore, a number ofquence is endogenous to the fossil. Sequence com-criterianeedto befulfilled beforeaDNA sequencedeter-parisons with human mtDNA sequences, as well asmined from extracts of an ancient specimen can bephylogenetic analyses, show that the Neandertal se-taken to be genuine (Pa¨a¨bo et al., 1989;Lindahl, 1993b;quence falls outside the variation of modern humans.Handt et al., 1994a; Handt et al., 1996).Furthermore, the age of the common ancestor of theSince 1991, the Neandertal-type specimen, found inNeandertal and modern human mtDNAs is estimated1856 near Du¨sseldorf, Germany, has been the subjectto be four times greater than that of the common an-of an interdisciplinary project of the RheinischescestorofhumanmtDNAs. Thissuggests thatNeander-Landesmuseum Bonn, initiated and led by R. W. S.tals went extinct without contributing mtDNA to mod-(Schmitz et al., 1995; Schmitz, 1996). As a part of thisern humans.project, a sample was removed from the Neandertalspecimen for DNA analysis. Here, we present the se-quence of a hypervariable part of the mtDNA controlIntroductionregion derived from this sample. We describe the evi-dence in supportofits authenticityand analyze therela-Neandertals are a group of extinct hominids that inhab-tionship of this sequence to the contemporary humanited Europe and western Asia from about 300,000 tomtDNA gene pool.30,000years ago. Duringpart of thistime theycoexistedwithmodernhumans. Basedonmorphological compari-sons, it has been variously claimed that Neandertals:(1) were the direct ancestors of modern Europeans; (2) Resultscontributed some genes to modern humans; or (3) werecompletely replaced by modern humans without con- Amino Acid RacemizationA 3.5 g section of the right humerus was removed fromtributing any genes (reviewed in Stringer and Gamble,1993; Trinkaus and Shipman 1993; Bra¨uer and Stringer, the Neandertal fossil (Figure 1). It has previously beenshown that ancient specimens exhibiting high levels of1997). Analyses of molecular genetic variation in themitochondrial and nuclear genomes of contemporary amino acid racemization do not contain sufficient DNAfor analysis (Poinar et al., 1996). To investigate whetherhuman populations have generally supported the thirdview, i.e.,that Neandertals were aseparate species that the state of preservation of the fossil is compatible withDNAretrieval, wethereforeanalyzed theextent ofaminowent extinct without contributing genes to modern hu-mans (Cann et al., 1987; Vigilant et al., 1991; Hammer, acid racemization. Samples of 10 mg were removedfrom theperiostal surfaceofthe bone,fromthecompact1995;Armour etal.,1996; Tishkoff etal.,1996).However,Cell20tends to be degraded and damaged to an extent thatmakes amplification of segments of mtDNA longer than100–200 bp difficult (Pa¨a¨bo, 1989). Therefore, two prim-ers (L16,209, H16,271) that amplify a 105-bp-segmentof the human mtDNA control region (including primers)were used to perform amplifications from the bone ex-tract as well asfrom an extractioncontrol. Anamplifica-tion product was obtained in the bone extract but notin the control (data not shown). In asubsequent experi-ment, this was repeated and the same results were ob-tained.Sequence Variation of the Amplification ProductThetwo amplificationproductswereclonedina plasmidFigure 1. SampleRemovedfromtheRight Humerus oftheNeander-tal-Type Specimenvector and 18 and 12 clones, respectively, were se-quenced (Figure 2, extract A). Twenty-two of the 30clones contained seven nucleotide substitutions andcortical bone, and from theendostal surfaceof the mar-one insertion of an adenine residue, when compared torow cavity. Samples were also removed from remnantsthe standard human reference sequence (Anderson etof a varnish, with which the specimen has been treatedal., 1981). Three of these eight differences to the refer-at least twice. The samples were hydrolyzed under acidence sequencewere individuallylacking ina total offiveconditions,andthe releasedaminoacids wereanalyzedof the clones. In addition, among the 27 clones wereusing highperformanceliquidchromatographyand fluo-nine differences that each occurred