11. The fate of European Neanderthals: results and perspectives from ancient DNA analyses

D. SERRE Max-Planck-Institute for Evolutionary Anthropology, Leipzig D-04103, Germany Current address: Genome Quebec Innovation Center and McGill University, Montreal, Canada [email protected]

S. PÄÄBO Max-Planck-Institut für Evolutionäre Anthropologie, Leipzig D04103, Germany [email protected]

Keywords: mtDNA, genetic diversity, population history, bottleneck, gene flow, coalescence, glaciation

Abstract

Analyses of mitochondrial DNA sequences extracted from several Neanderthal remains have provided new information on their genetic relationship with modern human individuals. However, these results have been inter- preted very differently among anthropologists. Here we review these results and present additional data directly addressing the question of genetic continuity among human populations during the Late Pleistocene. An analysis of additional Neanderthal and early modern human remains from Western and Central Europe do not provide any evidence of gene flow between the two groups. We also show that under reasonable assumptions of human demog- raphy, these data rule out a major genetic contribution by Neanderthals to the modern human gene pool. Finally, we present preliminary results showing that ancient DNA studies can also contribute to unraveling aspects of Neanderthal demography. Promising avenues of research, such as the investigation of Neanderthal population genetic diversity and organization, as well as analyses of mammal populations contemporary with Neanderthals, could allow us to better understand the dynamics, and perhaps causes, of the demographic changes that occurred in Eurasia during the Late Pleistocene.

Introduction Africa around two million years ago to colonize Europe and Asia as far as Indonesia Most researchers agree that the first hominids (e.g., Wolpoff and Caspari, 1997; Gabunia evolved in Africa (e.g., Campbell, 1988; Klein, et al., 2000; Oms et al., 2000; Wood and 1989; Lewin, 1999) and that Homo erectus left Richmond, 2000; Roebroeks, 2001; Balter and 211 K. Harvati and T. Harrison (eds.), Neanderthals Revisited: New Approaches and Perspectives, 211–220. © 2008 Springer. 212 D. SERRE & S. PÄÄBO Gibbons, 2002; Vekua et al., 2002). However, Two problems limit the range of the con- the fate of archaic human populations that clusions drawn from these studies: first, due evolved regionally from this ancestral stock is to the impossibility of differentiating modern much debated, especially with regards to a sec- contamination from endogenous DNA ond wave of colonization from Africa around sequences, a sample from a Neanderthal indi- 100 ka. Most notably, attention has been vidual carrying a sequence similar to that of a focused on the fate of the Neanderthals,1 the current human could be discarded as putative archaic humans that inhabited Europe and contamination (Nordborg, 1998; Trinkaus, Western Asia during the later part of the 2001). Second, the absence of early modern Pleistocene (i.e., between 300 ka and 30 ka). human DNA sequences leaves a long time Recent 14C-dating confirms that the last span during which simple demographic Neanderthals could have co-existed with the processes can lead to the loss of Neanderthal first modern humans in Europe (Bocquet- sequences even with a substantial amount of Appel and Demars, 2000). However, it is still admixture in the past (e.g., Relethford, 1998, unclear whether this possible cohabitation 1999, 2001). Thus, the Neanderthal mtDNA influenced the gene pool of the newcomers or could have been swamped by a continuous if, on the contrary, the Neanderthals went influx of modern human mtDNA into the extinct without contributing to the gene pool Neanderthal gene pool (Enflo et al., 2001), of early modern humans in Europe. lost by genetic drift (Nordborg, 1998), or by a In 1997, Krings extracted DNA from a population replacement much later than the humerus of the Neanderthal holotype (Krings Paleolithic transition, for example during the et al., 1997). The 379 base pairs (bp) ampli- Neolithic expansion (e.g., Cavalli-Sforza fied from the hypervariable region of the et al., 1993). Here we summarize results that mitochondrial (mt) genome were different overcome these problems. The paper from all modern human DNA sequences. addresses the question of continuity or Furthermore, this DNA sequence was too dif- replacement between Neanderthals and early ferent from the current human sequences modern humans, as well as, more generally, observed in the gene pool to be likely to be aspects of what happened to the human popu- found in an individual that has not been ana- lations during the transition from the Middle lyzed yet. Tree reconstructions confirmed to Upper Paleolithic in Europe. these analyses: while all human mtDNA sequences group together with a recent com- Looking for Gene Flow Between mon ancestor,2 the sequence retrieved from Neanderthals and Early the type specimen of Neanderthal shows a Modern Humans much deeper separation with strong statistical support. This result is often interpreted as Contamination is the major problem of compelling evidence for the absence of inter- ancient DNA studies dealing with human breeding between Neanderthals and modern remains because it is currently impossible to humans, or even as proof that Neanderthals differentiate endogenous DNA sequences and modern humans were two different from modern contaminants present on the species (e.g., Lindahl, 2000). However, even bones and those potentially left by excavators, after the publication of two additional mtDNA curators and scientists that handled the bones. sequences, very similar to that of the first It has been shown that most ancient animal individual (Krings et al., 2000; Ovchinnikov remains yield human DNA sequences when et al., 2000), many scenarios are still consis- sensitive enough amplifications are used tent with the data. (Hofreiter et al., 2001; Wandeler et al., 2003). ANCIENT DNA ANALYSES 213 This hampers the range of conclusions that the chemical preservation of a particular can be drawn from studies of Neanderthal amino acid) we could define strict criteria by mtDNA, since (1) possible evidence of gene which endogenous DNA from animal remains flow from modern humans to Neanderthals, could always be successfully retrieved and such as a Neanderthal specimen yielding only amplified (Serre et al., 2004). This method a modern mtDNA sequence, could be consid- also offers the advantage of being quick and ered a contamination artifact and therefore largely non-destructive (less than 10 mg of discarded, and (2) there are no conclusive bone powder is required), thus allowing mtDNA sequences from early modern screening of a large collection of material from humans that can be compared with which one can later choose only the most Neanderthals. These DNA sequences would promising ones. We screened more than be especially informative due to their closer 25 Neanderthal and 40 early modern human proximity in time to that of Neanderthals than remains for amino acid preservation. Five current genetic diversity (Relethford, 1998, Neanderthal bones and five early modern 1999, 2001). humans (Table 1) fulfilled our criteria of However, one can investigate the genetic preservation and therefore must contain relationship between Neanderthals and early retrievable endogenous DNA sequences (Serre modern humans by making use of the fact that et al., 2004; Beauval et al., 2005). the Neanderthal mtDNA sequences retrieved We extracted DNA from each of the ten so far are distinguishable from all current remains and amplified it under two different mtDNA sequences found in the human popu- conditions: lation. Thus, one way to look for gene flow 1. an amplification of mtDNA was per- between Neanderthals and early modern formed under conditions where mod- humans is to ask two questions: (1) Do all ern human and Neanderthal, as well as Neanderthal remains yield a “Neanderthal- chimpanzee and gorilla, DNA were like” mtDNA sequence? (2) Do any early successfully amplified. This amplifica- modern human remains yield a “Neanderthal- tion allowed a wide screening of possi- like” mtDNA sequence? ble molecules present in the bones. For As this approach relies on the presence/ example, if a bone contained an absence of a Neanderthal mtDNA sequence it mtDNA sequence different both from requires some independent criteria to validate Neanderthal and from modern human that any non-retrieval of Neanderthal mtDNA is effectively due to its absence and not to a Table 1. Specimen included in the gene flow study lack of preservation of the biomolecules. We used animal remains, for which contamination Specimen is easily differentiable from endogenous DNA, Neanderthal remains to determine which state of biomolecular Vindija 77 (Vi-77) (Croatia) preservation is correlated with successful Vindija 80 (Vi-80) (Croatia) retrieval of endogenous DNA. We looked at Engis 2 (Belgium) the preservation of amino acids, the building La-Chapelle-aux-Saints (France) Les-Rochers-de-Villeneuve (RdV 1) (France) blocks of the proteins that represent the major biomolecular component of the bone. Early modern human remains Mladecˇ 25c (Czech Republic) Analyses of numerous faunal remains showed Mladecˇ 2 (Czech Republic) that using three independent measurements of Cro-Magnon (France) amino acids preservation (i.e., the total amount Abri Pataud (France) of molecules, the ratio of two amino acids, and La Madeleine (France) 214 D. SERRE & S. PÄÄBO sequences, this “unspecific” amplifi- product. Interestingly, all five Neanderthal cation could likely detect it. remains did yield an amplification product 2. a “Neanderthal-specific” amplification and, after sequencing, a short mtDNA was performed. Under the conditions sequence fragment was identified that was used, only mtDNA sequences similar to identical to the corresponding region of one of those retrieved from the previously ana- the four Neanderthals already sequenced lyzed Neanderthal remains could be (Krings et al., 1997, 2000; Ovchinnikov et al., amplified while the amplification did 2000; Schmitz et al., 2002). Given that the not work on modern human mtDNA overall state of preservation of the biomole- sequences. This procedure allowed us to cules is similar, this shows that the “fish out” a Neanderthal mtDNA Neanderthals formed a homogenous genetic sequence, even if it was in the presence population different from that of early modern of a much larger amount of contaminant humans (Serre et al., 2004; Beauval et al., sequences. 2005). This result is supported by the mtDNA sequence of a fragment of 47bp recently All remains (the five Neanderthals and the retrieved from a Neanderthal from El Sidrón five early modern humans) analyzed yielded Cave, Spain, that is identical to the sequences DNA sequences identical to contemporary from Vindija and Feldhofer 1 (Lalueza-Fox human DNA sequences when amplified using et al., 2005). the “unspecific” conditions. In 75% of the Thus, while we applied an unbiased cases, more than one human mtDNA methodology that can detect gene flow sequence was amplified from a single bone between populations, we did not find any evi- (Serre et al., 2004). This confirmed previous dence of gene flow in either direction. It is results that most ancient remains yield human important to stress here that some of the sam- DNA sequences when sensitive enough ples analyzed in this study have been amplifications are used (Hofreiter et al., 2001; described as “transitional” between “classi- Wandeler et al., 2003). Additionally, all DNA cal” Neanderthals and early modern humans, sequences retrieved from the early modern such as the Vindija Neanderthals (Smith and human remains were identical to modern Spencer, 1984; Wolpoff, 1999) and the human mtDNA sequences present in DNA Mladecˇ individuals (Frayer, 1992; Wolpoff, sequence database (http://www.ncbi. 1999), so they represent good candidates to nlm.nih.gov/Genbank/). Due to ubiquitous reveal potential gene flow. contamination in four samples (i.e., those yielding each more than one sequence) and the fact that any DNA sequence amplified can What is the maximum genetic potentially be a contaminant, it is impossible contribution that might have occurred? to identify the endogenous mtDNA sequence for any of the early modern human remains. In Our analysis of five Neanderthal remains and our view, this shows that it is currently impos- five early modern humans did not detect any sible to trust the veracity of any ancient DNA evidence of gene flow. However, given the sequence similar to the one found in the mod- small sample size one might question the ern human gene pool. power of this study to detect genetic contribu- By contrast, when the DNA amplification tion. In other words, one might want to esti- was performed under “Neanderthal-specific” mate the level of genetic contribution that can conditions, none of the five early modern be statistically ruled out given the data. It is human remains yielded an amplification important to note that, while the former ANCIENT DNA ANALYSES 215 results were obtained by straight-forward considered alone, only a scenario of random- analyses of the data, estimation of the maxi- mating population comprising both mum genetic contribution relies on a theoretical Neanderthals and modern humans can be model of what we think is a fair representation excluded (Nordborg, 1998). Thus, even using a of human demographic history: what were the conservative model of population history we population sizes of Neandertals and early can exclude a large Neanderthal contribution to modern humans, when did they meet each the modern human gene pool. other, how long did they interact for, when and If we consider a more realistic scenario how quickly did the modern human popula- where the spread of modern humans (before tion expanded? All these parameters need to and during their migration out of Africa and be estimated in the model. Therefore, one subsequent colonization of western Eurasia) should keep in mind that any results obtained was accompanied by a population growth, we using this approach are dependant on the can exclude a smaller Neanderthal contribution. assumptions made. However, the importance of the contribution We decided to use the simplest model possi- that can be excluded depends critically on when ble (to account for the small data set we have) and how the expansion occurred. For example, and to work under the assumption that the Currat and Excoffier (2004) recently estimated human population is panmictic (i.e., random that under a much more complex scenario, in mating) and of constant size through time. We which an expanding modern human population estimated, using this model (Tavare, 1984), that spread progressively in Europe and competed the current mtDNA gene pool had only between with the less numerous Neanderthals, the maxi- four and seven ancestors at 20–30 ka. This mal genetic contribution compatible with the shows the limitations of using only current data is smaller than 0.1%. diversity to obtain insights about the mtDNA gene pool in the late Pleistocene. In fact, the five Can Ancient DNA Studies Tell us early modern human individuals analyzed here What Happened to the Neanderthals provide almost as much information about the During the Middle to Upper mtDNA gene pool of modern humans in the late Paleolithic Transition? Pleistocene as would the sequencing of mtDNA sequence from all now-living humans. They The genetic data collected so far support a also add information that could not be obtained scenario of no major interbreeding between by studying additional now-living individuals. the two human populations in the Late The mtDNA ancestry of current humans is Pleistocene. Leaving aside discussions of already intensively explored with respect to species/sub-species status and interbreeding deep divergences, so that additional major line- capacity/incapacity, we can still try to under- ages are unlikely to be discovered (Sykes, stand why Neanderthals disappeared during 2001). Given that all Neanderthal bones ana- the transition from Middle to Upper lyzed yield mtDNA sequences that are similar to Paleolithic. Two avenues of research are each other and absent in the five early modern promising for this purpose: (1) the analyses of humans analyzed, as well as in all modern genetic diversity within Neanderthals that can humans, we can exclude (at 95% confidence) lead to a greater understanding of their demo- any Neanderthal contribution to the modern graphic history; and (2) the investigations of human gene pool greater than 25% (Serre et al., potential demographic changes in animal pop- 2004). This might seem a rather uninformative ulations contemporary with the Neanderthals result, but it is in fact a major improvement. to obtain a more global understanding of the When Neanderthal mtDNA sequences are environment and its influences. 216 D. SERRE & S. PÄÄBO By comparing the Neanderthal mtDNA interesting that this preliminary observation sequences of the four individuals with the most contrasts with the picture given by some pale- complete genetic information, we find that the oanthropologists who present Neanderthals as Neanderthals carry a genetic diversity for the having strong cultural or behavioral differ- mtDNA similar to that of the current human ences correlated with their geographical ori- population and approximately 5 times smaller gins (e.g., Bahn, 1998; d’Errico et al., 1998; than that of the African great apes (Krings Stringer et al., 2000). In this context, one can et al., 2000; Schmitz et al., 2002). We have note that all Neanderthal DNA analyzed so far shown that this low diversity within dates from the early to middle part (~59–35 ka) Neanderthals is not an artifact, since all well- of the MIS 3 interstadial. An interesting work- enough preserved remains yield very similar ing hypothesis would be that the Neanderthals sequences (Serre et al., 2004). One commonly of the Saalian glaciation (MIS 6, ~195–128 ka) proposed explanation for the reduced genetic consisted of a metapopulation with strong phy- diversity in humans relative to our closest liv- logeographical structure, and that the MIS 3 ing relatives is that gorillas and chimpanzees Neanderthal population is the result of post- have always lived in the African rainforest, glacial expansion of only one, or a few, surviv- which was not drastically modified by climatic ing local population(s). changes (e.g., Lahr and Foley, 1998). The Another promising approach to better African great apes may, therefore, have main- understand the history of Neanderthal and tained a stable population over a long period of early modern human populations is to analyze time and accumulated a large genetic diversity. faunal remains contemporary with these pop- In contrast, human populations expanding in ulations. Ancient DNA analyses of animal open environments were more exposed to cli- remains are far easier and more efficient than matic fluctuations and likely underwent a those of human remains because: (1) many series of drastic reductions in population size more samples are available for analyses; and followed by expansions (e.g., Takahata, 1994; (2) contamination is not an issue.3 In a recent Lahr and Foley, 1998; Reich and Goldstein, pilot study we analyzed remains from cave 1998; Zietkiewicz et al., 1998; Adams et al., bears, cave hyenas, and brown bears across 2000). The preliminary data concerning the Europe, all dated to ~70–30 ka (Hofreiter Neanderthal population show the same general et al., 2004). In none of these data sets were trend, and suggest a rather unstable population we able to detect a correlation between the history. Additionally, it is interesting to note mtDNA sequence carried by an individual and that the mtDNA sequence retrieved from the its geographical origin (sometimes this is second individual of Feldhofer, Germany referred to as phylogeographic structure). This (Schmitz et al., 2002) carries three differences finding is striking when compared to current from the type specimen mtDNA sequence genetic diversity data: most species living (Krings et al., 1997) while carrying only one today in Europe show a strong correlation difference from the Croatian Neanderthal with the mtDNA gene pool organized in two mtDNA sequence (Krings et al., 2000). This or three clades found almost exclusively in suggests that no strong geographical clustering Western Europe, Eastern Europe or Southern of mtDNA sequences was present in Europe (e.g., Taberlet et al., 1998; Avise, Neanderthals, at least in western and central 2000; Hewitt, 2000). This organization of cur- Europe. It is clear that more individuals are rent genetic diversity is believed to be the needed in order to arrive at more definitive result of glacial periods when many species conclusions about the geographic organization survived only in a few ice-free refugia (the of the Neanderthal mtDNA gene pool, but it is Iberian Peninsula, the Balkans, and Italy) and ANCIENT DNA ANALYSES 217 spread from there across Europe at the end of the African great apes, and similar to that of the glaciation. Interestingly, while the time of current humans, suggests major demographic the setting of this phylogeographic structure is changes during the Late Pleistocene. The geo- believed to date to early in the Pleistocene graphic homogeneity of the gene pool of the (e.g., Hewitt, 2000), we find no evidence of Neanderthals investigated so far, strikingly such organization in the three species we contrasts with their apparent cultural diversity looked at. We concluded that the setting of and requires further investigation. Preliminary this phylogeographic structure possibly analyses of faunal remains contemporary with occurred just a couple of tens of thousands of the Neanderthal suggest that major demo- years ago (Hofreiter et al., 2004). It will be graphic changes occurred in Europe around interesting to see if this result holds when the time when Neanderthals became extinct. more species contemporary with the Further investigations in this direction might Neanderthals are analyzed. This preliminary lead to a better understanding of the context in result might indicate that many species under- which this disappearance occurred and per- went major demographic rearrangements haps to its causes. Analyses of the DNA mol- around the time that Neanderthals became ecules preserved in Pleistocene human bones extinct. This observation is of particular inter- are tedious and, unfortunately, still require the est as any event that affected the environment destruction of a small amount of material. so drastically must have affected the human Nonetheless, these analyses provide informa- populations as well, if not directly, at least tion that cannot be obtained by looking at the through the changes of the dietary resource current genetic diversity or through morpho- availability. An understanding of the dynamics logical/archeological studies. Eight years after of animal populations in the Pleistocene might the publication of the first Neanderthal therefore lead to major breakthroughs in our mtDNA sequence we have shifted the research understanding of Neanderthal extinction. focus towards understanding of the Neanderthal population history, and we are only beginning to reveal this fascinating Conclusion period of human evolution. The conclusions are still limited, but future analyses of addi- We have shown here that genetic analyses of tional individuals will allow us to verify (or Neanderthal and early modern human remains contradict) our preliminary results and offer can provide information about the relationship an exciting challenge for the coming years. and dynamics of these two populations. Neanderthals, at least those living in the last interglacial period, constitute a homogenous Notes genetic population different from the early modern humans that followed them in Europe. 1. Throughout this paper we use the term “human population” to describe both Neanderthals and Recent analyses of Paleolithic human remains early modern humans. All the results presented found no evidence of gene flow between the here deal with the population history of “modern two populations in either direction, and we humans” and “Neanderthals” and can be explained can show that, if any, the genetic contribution by demographic processes that do not necessitate from the Neanderthals to the modern human reproductive isolation or any other biological crite- gene pool must have been limited. We are also rion that can be used to define species. 2. The concept of genetic ancestry, as used throughout beginning to obtain some information con- this paper, is not identical to the popular meaning of cerning the demography of the Neanderthals. ancestry. In its most common meaning, the ances- Their low genetic diversity relative to that of tors of a particular individual are his/her parents, the 218 D. SERRE & S. PÄÄBO

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