No Evidence of Neandertal mtDNA Contributionto Early Modern HumansDavid Serre1, Andre´Langaney2,3, Mario Chech2, Maria Teschler-Nicola4, Maja Paunovic5z, Philippe Mennecier2,Michael Hofreiter1,Go¨ran Possnert6, Svante Pa¨a¨bo1*1 Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany, 2 Laboratoire d’Anthropologie Biologique, Muse´e de l’Homme, Paris, France, 3 Laboratoire deGe´ne´tique et Biome´trie, Universite´de Gene`ve, Gene`ve, Switzerland, 4 Department of Anthropology, Natural History Museum, Vienna, Austria, 5 Institute of QuaternaryPaleontology and Geology, Croatian Academy of Sciences and Arts, Zagreb, Croatia, 6 A˚ngstro¨m Laboratory, Uppsala University, Uppsala, SwedenThe retrieval of mitochondrial DNA (mtDNA) sequences from four Neandertal fossils from Germany, Russia, and Croatiahas demonstrated that these individuals carried closely related mtDNAs that are not found among current humans.However, these results do not definitively resolve the question of a possible Neandertal contribution to the gene poolof modern humans since such a contribution might have been erased by genetic drift or by the continuous influx ofmodern human DNA into the Neandertal gene pool. A further concern is that if some Neandertals carried mtDNAsequences similar to contemporaneous humans, such sequences may be erroneously rega rded as moderncontaminations when retrieved from fossils. Here we address these issues by the analysis of 24 Neandertal and 40early modern human remains. The biomolecular preservation of four Neandertals and of five early modern humanswas good enough to suggest the preservation of DNA. All four Neandertals yielded mtDNA sequences similar to thosepreviously determined from Neandertal individuals, whereas none of the five early modern humans contained suchmtDNA sequences. In combination with current mtDNA data, this excludes any large genetic contribution byNeandertals to early modern humans, but does not rule out the possibility of a smaller contribution.IntroductionDespite intense research efforts, no consensus has beenreached about the genetic relationship between early modernhumans and archaic human forms such as the Neandertals.While supporters of ‘‘ multiregional evolution’’ argue forgenetic exchange or even continuity between archaic andmodern humans (Weidenreich 1943; Wolpoff et al. 1984,2000; Duarte et al. 1999; Hawks and Wolpoff 2001),proponents of a ‘‘ single African origin’’ of contemporaryhumans claim that negligible genetic interaction took place(Cann et al. 1987; Stringer and Andrews 1988; Ingman et al.2000; Underhill et al. 2000; Stringer 2002). MitochondrialDNA (mtDNA) sequences from early modern humans wouldin principle be able to resolve the question of a contributionof Neandertal mtDNA to modern humans. However, humanDNA is pervasive in palaeontological and archaeologicalremains as well as in most laboratory environments (e.g.,Krings et al. 2000; Hofreiter et al. 2001b; Wandeler et al.2003). It is therefore currently impossible to differentiatecontaminating modern DNA sequences from endogenoushuman DNA in human remains. Thus, although mtDNAsequences have been reported from remains of early modernhumans (Adcock et al. 2001; Caramelli et al. 2003), it is notpossible to determine whether such DNA sequences indeedrepresent endogenous DNA sequences (Abbott 2003). Arelated problem is that if a Neandertal fossil yields modernhuman-like DNA sequences, those might be discarded asputative contaminations (Nordborg 1998; Trinkaus 2001),even if they may be endogenous and represent evidence for aclose genetic relationship or interbreeding between the twogroups.To explore the genetic relationship between early modernhumans and Neandertals in spite of these difficulties, wemade use of the fact that the four Neandertal mtDNAsequences determined to date can easily be distinguishedfrom those of modern humans (Krings et al. 1997, 2000;Ovchinnikov et al. 2000; Schmitz et al. 2002; Knight 2003).This allowed us to ask whether all well-preserved Neandertalremains contain Neandertal-like mtDNA and whether allwell-preserved early modern human remains fail to containsuch DNA sequences. Thus, we did not attempt to determineDNA sequences that are similar to present-day humanmtDNA. Instead, we determined whether Neandertal-likemtDNA sequences were present or absent in well-preservedremains of Neandertals and of early modern humans.Results and DiscussionThe preservation of endogenous DNA in fossils is corre-lated with the amount, composition, and chemical preserva-tion of amino acids (Poinar et al. 1996). We find thatendogenous DNA can be amplified from Pleistocene remainswhen the amino acid content is more than 30,000 parts permillion (ppm), the ratio of glycine to aspartic acid betweentwo and ten, and the aspartic acid racemization (i.e., thestereoisomeric D/L ratio) less than 0.10 (Poinar et al. 1996;Received November 2, 2003; Accepted December 18, 2003; Published March 16,2004DOI: 10.1371/journal.pbio.0020057Copyright: Ó 2004 Serre et al. This is an open-access article distributed underthe terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided theoriginal work is properly cited.Abbreviations: BP, before present; mtDNA, mitochondrial DNA; PCR, polymerasechain reaction; ppm, parts per millionAcademic Editor: David Penny, Massey University* To whom correspondence should be addressed. E-mail: [email protected] Deceased.PLoS Biology | http://biology.plosjournals.org March 2004 | Volume 2 | Issue 3 | Page 0313PLoSBIOLOGYKrings et al. 1997, 2000; Schmitz et al. 2002; data not shown).We analyzed the amino acid preservation of 24 Neandertaland 40 early modern human fossils (Table S1). Severalimportant Neandertal fossils, such as La Ferrassie andKrapina, as well as important modern human fossils, suchas Veternica, proved to be too poorly preserved to be likely toallow DNA retrieval. Thus, further destructive sampling ofthese specimens was not considered justified. However, fourNeandertal and five early modern human fossils fulfilled theabove criteria for amino acid preservation and were thusexpected to contain endogenous DNA (Figure 1; Table 1).These samples were geographically well distributed acrossEurope (Figure 2) and included remains whose morphology istypical of Neandertals (e.g., La Chapelle-aux-Saints) and ofmodern humans (La Madeleine,