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BIOLOGICAL REVIEWS Cambridge Philosophical Society 245 Biol. Rev. (2009), 84, pp. 245–257. doi:10.1111/j.1469-185X.2008.00071.x To what extent did Neanderthals and modern humans interact? Kristian J. Herrera1, Jason A. Somarelli1,2, Robert K. Lowery1,2 and Rene J. Herrera1* 1 Department of Human and Molecular Genetics, College of Medicine, Florida International University, 11200 SW 8th Street, Miami, FL 33199, USA 2 Department of Biological Sciences, Florida International University, 11200 SW 8th Street, Miami, FL 33199, USA (Received: 14 May 2008; revised: 23 October 2008; accepted: 28 November 2008) ABSTRACT Neanderthals represent an extinct hominid lineage that existed in Europe and Asia for nearly 400,000 years. They thrived in these regions for much of this time, but declined in numbers and went extinct around 30,000 years ago. Interestingly, their disappearance occurred subsequent to the arrival of modern humans into these areas, which has prompted some to argue that Neanderthals were displaced by better suited and more adaptable modern humans. Still others have postulated that Neanderthals were assimilated into the gene pool of modern humans by admixture. Until relatively recently, conclusions about the relationships between Neanderthals and contemporary humans were based solely upon evidence left behind in the fossil and archaeological records. However, in the last decade, we have witnessed the introduction of metagenomic analyses, which have provided novel tools with which to study the levels of genetic interactions between this fascinating Homo lineage and modern humans. Were Neanderthals replaced by contemporary humans through dramatic extinction resulting from competition and/or hostility or through admixture? Were Neanderthals and modern humans two independent, genetically unique species or were they a single species, capable of producing fertile offspring? Here, we review the current anthropological, archaeological and genetic data, which shed some light on these questions and provide insight into the exact nature of the relationships between these two groups of humans. Key words: archaic Homo lineages, extinction, admixture, acculturation, hybridization, ancient DNA, introgression. CONTENTS I. Introduction ...................................................................................................................................... II. Anthropological data ........................................................................................................................ (1) Archaeological results ................................................................................................................. (2) Analyses of fossil data ................................................................................................................. III. Investigations using modern human dna ......................................................................................... IV. Evidence from the neanderthal mitochondrial genome .................................................................. (1) The first mtDNA sequences from Neanderthals ....................................................................... (2) Older, more diverse Neanderthal mtDNA sequences ............................................................... (3) Issues involving the mtDNA sequences ..................................................................................... V. Evidence from the neanderthal nuclear genome ............................................................................. (1) The earliest large-scale sequencing efforts ................................................................................. (2) Issues relating to the genomic studies ........................................................................................ (3) Searches for genes ....................................................................................................................... VI. Conclusions ....................................................................................................................................... VII. References ......................................................................................................................................... 246 248 248 248 249 250 250 251 251 251 251 252 252 253 253 * Address for correspondence: Tel: 305 348 1258; Fax: 305 348 1259; E-mail: [email protected] Biological Reviews 84 (2009) 245–257 Ó 2009 The Authors Journal compilation Ó 2009 Cambridge Philosophical Society 246 I. INTRODUCTION Homo sapiens neanderthalensis was one of the closest relatives of modern humans. This extinct group of hominids first appeared in Europe about 400,000 years before present (YBP) (Stringer & Hublin, 1999). Fossil evidence indicates that approximately 150,000 YBP (Bar-Yosef, 1998; Grün & Stringer, 2000) Neanderthals extended their range into the Middle East and Asia, spreading as far east as Teshik-Tash, Uzbekistan (Debetz, 1940) and the Altai Republic in Russia (Krause et al., 2007a) (Fig. 1). They proliferated to the greatest extent in Europe, with sites from Gibraltar in the Iberian Peninsula (Finlayson et al., 2006) to the Caucasus Mountains in Russia (Golovanova et al., 1999) (Fig. 1). The vast majority of Neanderthal locations have been detected south of the Alps, France and the Iberian Peninsula; while several additional sites are situated further north in Germany and even into Wales (Green & Walker, 1991) (Fig. 1). For nearly 400,000 years, Neanderthals were the dominant hominid group in Europe and West Asia. Yet, fossil evidence indicates that approximately 50,000 YBP Neanderthals may have gone extinct in Asia (Klein, 2003), and about 34,000 YBP suffered a dramatic decline in number in Europe (Svoboda, 2005). About 2,000 years later, much of the Neanderthal population throughout Europe had disappeared (van Andel, Davies & Weninger, 2003). The remaining Neanderthals are thought to have survived in isolated refugia, including the Iberian Peninsula, for another few millennia (Finlayson et al., 2006). One finding in Gibraltar, for instance, suggests that Neanderthals may have existed in southern Iberia as late as 28,000 to 24,000 YBP (Finlayson et al., 2006). There are several interesting theories that offer explanations for the disappearance of Neanderthals from the fossil record. Some have speculated that sudden or repeated global climate change drastically reduced their habitats and limited the land that was suitable for hunting (Hublin, 1998; van Andel & Davies, 2003; Musil, 2003; Finlayson, 2004; Steudel-Numbers & Tilkens, 2004; Finlayson et al., 2006; Jimenez-Espejo et al., 2007; Finlayson & Carrion, 2007). However, recent advances in palaeoclimatology seem to suggest that the generally accepted dates for the Neanderthal extinction between 32,000 and 28,000 YBP fail to coincide with any severe climatic event (Tzedakis et al., 2007). This implies that one or more other factors instead of or in addition to climate change may have been involved in the Neanderthal extinction (Tzedakis et al., 2007). Hypotheses of these additional factors have included cave-associated contaminants such as smoke (Stormer & Mysterud, 2006). Consistent exposure to pollutants released from fires contained in caves would possibly lead to various health issues, including increased sterility among males (Stormer & Mysterud, 2006). Yet, it is not clear why this would be an issue 30,000 YBP and not at any other time. Cannibalism among Neanderthals has also been suggested (Defleur et al., 2006; Underdown, 2008). According to Underdown (2008), cannibalism may have led to the spread of lethal transmissible spongiform encephalopathies (TSEs), which could also be transmitted by the sharing of tools Kristian J. Herrera and others between sick and healthy individuals. Considering that infections are usually triggered by a number of circumstances and are subject to de novo genesis, the appearance of a novel deleterious or lethal strain at any given place and time would not be unlikely. Such an epidemic would have greatly weakened the Neanderthal population in a manner similar to that observed in the Fore of Papua New Guinea (Farquhar & Gajdusek, 1981). Along these lines, it is also possible that just as Native Americans were impacted and in some instances decimated by infections transmitted by European settlers, Neanderthals could have been the recipients of a number of deadly plagues from invading modern humans that spread through immunologically vulnerable individuals. Given that mass-scale death is facilitated at high population densities, it is possible that seasonal gatherings and subsequent dissemination of individuals may have negatively impacted a significant portion of the Neanderthal population in some locations. The decline and eventual disappearance of Neanderthals throughout Asia and Europe occurred subsequent to the arrival of Homo sapiens in these areas, approximately 100,000 and 40,000 years ago, respectively (Valladas et al., 1988; Mellars, 1992; Tishkoff et al., 1996). This has led to the postulation that modern humans may have contributed to the extinction of this archaic hominid (Finlayson, 2004). Some have proposed that modern humans were more adaptive, with greater ecological fitness than Neanderthals (Finlayson, 2004; Kuhn & Stiner, 2006). The appearance of another hominid group in Western Asia and Europe may have increased competition within the same ecological niche, with the more successful and adaptable modern humans out-competing and eventually replacing the Neanderthals (Flores, 1998). A lack of evidence for natural selection favouring modern human morphology over that of Neanderthals seems to suggest that behavioural and technological differences rather than anatomical adaptations gave humans their advantage (Weaver, Roseman & Stringer, 2007). It has been suggested that modern humans developed superior hunting techniques and tools, giving them a greater advantage in procuring adequate nutrition (Mellars, 1998). One of these possible advantages may have been a division of labour, a strategy seemingly lacking in Neanderthals, whose females joined males in hunting (Kuhn & Stiner, 2006). Not only would this have put child-bearing female Neanderthals at risk of injury, but may also have prevented females from gathering vegetation, thereby restricting the Neanderthal diet (Kuhn & Stiner, 2006). The idea of a restricted diet is further supported by isotope analyses of Neanderthal fossils and nearby fauna (Richards et al., 2000). These investigations indicate that although marine resources were used around the Mediterranean, the majority of the Neanderthal diet was meat based (Richards et al., 2000). Additionally, the development of more technically advanced weaponry by modern humans, including long-range projectiles combined with a higher distal-limb/body ratio might have made modern humans more successful hunters in steppe environments, allowing them to spread further north (Finlayson, 2004). It is also possible to imagine that competition from Biological Reviews 84 (2009) 245–257 Ó 2009 The Authors Journal compilation Ó 2009 Cambridge Philosophical Society Neanderthal/modern Human interactions 247 Fig. 1. Range expansion of Neanderthals and modern humans. Neanderthals existed throughout much of Europe for nearly 400,000 years. Their range is indicated in pink. Red dots are representative sites delineating the range of Neanderthals. Blue arrows represent the migration of modern humans out of Africa and into Europe (Luis et al., 2004; Rowold et al., 2007; Underhill & Kivisild, 2007). expanding modern human groups combined with any of the previously mentioned factors of climate fluctuation, cave pollutants, cannibalism and/or disease, may have led to the decline of the Neanderthals. Another explanation for the disappearance of Neanderthals is that they were not driven to extinction through the mechanisms described above, but rather by assimilation into early modern human populations. It has been suggested that some Neanderthals might have hybridized with the invading modern humans, and became integrated into the modern human population through introgression and admixture, gradually reducing the number of pure Neanderthals by diluting their DNA in an overwhelmingly modern human gene pool. This idea necessitates that modern humans and Neanderthals were capable of reproducing successfully and giving birth to fertile offspring, thereby allowing for gene flow (Duarte et al., 1999; Smith, 1999; Smith, Jankovic & Karavanic, 2005; Trinkaus, 2007). It is possible, however, that any putative matings between the two groups would have been incapable of yielding fertile offspring, rendering such hybrids of Neanderthals and modern humans evolutionary dead-ends. Given the long period of coexistence between modern humans and Neanderthals in Europe (approximately 10,000–15,000 years) and Western Asia (as much as 40,000 years), it seems reasonable to assume that a number of interactions might have occurred between the two groups (Shreeve, 1995). As illustrated by Shreeve (1995), the presence of a similar hominid group in an area being colonised by modern humans and prolonged cohabitation would almost certainly have led to interactions between the two groups; however, the nature and significance of these encounters remains debatable. Some prehistoric population estimates have human and/or Neanderthal densities at 65 persons per 10,000 km2, which would make Biological Reviews 84 (2009) 245–257 Ó 2009 The Authors Journal compilation Ó 2009 Cambridge Philosophical Society 248 potential encounters rare (Shea, 2007). Considering the geographic expanses as well as the diversity of terrain and habitat within the ranges of modern humans and Neanderthals, it would be expected that the nature of any interaction would have been mosaic-like in time and space. Potential modern-human/Neanderthal admixture has implications for understanding both the evolution of contemporary human populations as well as Neanderthal extinction. The occurrence of such events would imply that an archaic hominid group may have played a role in the shaping of current human populations, and that possibly some selectively advantageous traits were acquired from this group. For over a century, investigators in the fields of anthropology and archaeology have generated a substantial body of knowledge on Neanderthals. Unfortunately, the fossil record is far from complete, and much of the evidence can be interpreted in various ways. Considering the recent advances in molecular biology and genomic analyses, perhaps more concrete evidence for or against introgression can be uncovered by comparing Neanderthal and modern human DNA. With the advent of in vitro DNA amplification by the polymerase chain reaction (PCR) as well as recent advances in molecular biology techniques, researchers are capable of probing the genomes of both groups in search of genetic evidence to answer the question of Neanderthal/ modern human admixture. Here, we review the available evidence regarding introgression between these two hominid groups. II. ANTHROPOLOGICAL DATA (1) Archaeological results Although modern humans and Neanderthals may have had different ecological needs and exhibited relatively low population densities that kept them from direct constant competition, it is difficult to envision the complete lack of at least sporadic interactions. According to evidence produced by several archaeological studies, interactions between the two groups may have been frequent enough to allow the transfer of technology, particularly from modern humans to Neanderthals (Mellars, 2004). For the majority of their existence, Neanderthals practiced the Mousterian toolculture. This culture almost exclusively involved the use of stone in crafting weapons and other instruments (Bordes, 1961). The most advanced non-stone implements they created were instruments crafted from splinters of bone (Bordes, 1961) and probably spears crafted from wood. Although it has been suggested that the Aurignacian culture may not necessarily be of modern human origin (HenryGambier, Maureille & White, 2004), modern humans are known to have developed more sophisticated cultures in Europe and Asia (Stringer, Hublin & Vandermeersch, 1984). The Aurignacian culture featured the earliest known widespread use of bone, teeth, and ivory for jewellery and tools (Bar-Yosef, 2004). Around the time that modern humans arrived in Europe, Neanderthal technologies in some areas of Europe experienced a change from the Kristian J. Herrera and others Mousterian tool-culture to technologies such as the Châtelperronian, which featured the usage of bone, ivory, and body ornaments similar to that of the modern human Aurignacian culture (Hublin et al., 1996). This seemingly sudden revolution in implement design and ornamentation within several Neanderthal populations taking place with the arrival of modern humans has led to the suggestion that an acculturation event may have spurred the transformation (Hublin et al., 1996). The possibility that Neanderthals independently developed the Châtelperronian technology, though, cannot yet be rejected (d’Errico et al., 1998). If the Châtelperronian tool-culture was influenced by the invading modern humans, then it would not be surprising if the differences between it and the Aurignacian were minimal (d’Errico et al., 1998). Yet, this is far from the case. This has suggested to some (d’Errico et al., 1998) that Neanderthals independently created Châtelperronian utensils. However, modern-human-inspired implements do not have to be close replicas of those from the original Aurignacians. It is also revealing that Neanderthals began fabricating more advanced instruments around the time of the dispersal of modern humans into Europe, after over 300,000 years of relatively unchanged usage of Mousterian tools. Thus, it seems more reasonable to expect that this quantum-leap in technology was not coincidental but instead the result of Neanderthals enlightened from contact with modern humans. Assuming that one or more acculturation events did occur between the two groups, it appears to have been largely unidirectional, with technology passing from modern humans to Neanderthals (Mellars, 1992). In addition to technology transfer, some locations provide evidence suggesting the two species may have coexisted in close proximity (Mellars, Gravina & Ramsey, 2007). A Châtelperronian site in central France is controversially claimed to be interstratified with Aurignacian material, representing a direct indication of humans and Neanderthals living in close contact (Mellars et al., 2007). (2) Analyses of fossil data A number of anthropological findings have also provided potential indications of hybridization events occurring during recent human evolution. Discoveries of specimens with specific characteristics thought to be unique to both groups have led to increased support for the idea of admixture in some locales. Perhaps the most notable example is the 24,500 year old human fossil from the Abrigo do Lagar Velho in western Portugal, which exhibits distinct characteristics in its skull and limbs thought to be indicative of both Neanderthals and modern humans (Duarte et al., 1999). The conglomeration of features included limb proportions more akin to Neanderthals than early modern Europeans, but jaw shape and dentition exclusive to humans. Duarte et al. (1999) proposed that the Lagar Velho sample represents evidence for admixture between Neanderthals and modern humans. Early modern human fossils unearthed in Romania, the Czech Republic and China are also said to possess a combination of features Biological Reviews 84 (2009) 245–257 Ó 2009 The Authors Journal compilation Ó 2009 Cambridge Philosophical Society Neanderthal/modern Human interactions from both groups (Trinkaus et al., 2003; Soficaru, Dobos & Trinkaus, 2006; Shang et al., 2007). The samples from Romania and the Czech Republic show distinguishing human features, date to approximately 35,000 years ago and are therefore considered to be some of the oldest modern human fossils from Europe. However, not all of their morphological traits are distinctly modern human. A few of the skulls’ features follow patterns indicative of Neanderthal origin and thought to be rare or absent in most modern humans, including larger dentition and more robust bones. Similar patterns are reported in the 42– 39,000 year old fossil from Tianyuan Cave near Beijing, China (Shang et al., 2007). If these patterns did indeed rise from Neanderthal admixture, then it would be expected that interbreeding between the two groups took place not only in Europe, but also Asia and the Middle East. Additional comparisons of contemporary European skulls with Neanderthal and Middle Eastern human skulls found that the European skull was actually more similar to the Neanderthal skull than to the Middle Eastern, suggesting two different ancestors for Europeans (Wolpoff et al., 2001). However, these and other tests using morphological features to reanalyse modern human origin theories have come under methodological criticism (Bräuer, Collard & Stringer, 2004). Comparisons of a wide range of other Neanderthal and modern human mandibles, on the other hand, yield results antithetical to those from the skulls (Rak, Ginzberg & Geffen, 2002). In fact, the degree of separation between Neanderthals and modern humans based on mandibular variation is even greater than that between modern humans and Homo erectus. Although finding anatomical features specific to both species in single individual fossils would seem to suggest interbreeding, it has been argued that the supposed Neanderthal-specific traits may be unreliable and should not be immediately accepted as unequivocal evidence for admixture (Tattersall & Schwartz, 1999). This is due not only to difficulties in the assignment of certain morphological features as being unique to either species, but to the many gaps remaining in both the archaeological and fossil records. Interestingly, a clinal distribution of Neanderthal morphology is observed across Europe and the Near East (Smith, 1991; Rak, 1993; Arensburg & Belfer-Cohen, 1998; Voisin, 2006). Fossil samples from Western Europe are more distinctively Neanderthal while those from Central Europe and the Near East bear a greater resemblance to modern humans, which would be suggestive of gene flow between the two groups (Voisin, 2006). III. INVESTIGATIONS USING MODERN HUMAN DNA Prior to the sequencing of Neanderthal DNA, research focusing on DNA from contemporary human populations yielded varying conclusions with respect to archaic admixture. The vast majority of mitochondrial DNA studies on modern humans reveal a single recent African origin (Cann, Stoneking & Wilson, 1987; Vigilant et al., 249 1991; Ingman et al., 2000). These studies collectively came to the conclusion that mtDNA lineages follow a single, linear, and recent (<200,000 years ago) path from Africa and did not experience any assimilation from archaic populations, including Neanderthals (Cann et al., 1987; Vigilant et al., 1991; Ingman et al., 2000). Similarly, Y-chromosome research suggests a recent African origin for contemporary humans less than 100,000 years ago, without any genetic input from other Homo lineages (Thomson et al., 2000; Underhill et al., 2000; Underhill & Kivisild, 2007). Although the mitochondrial and Y-chromosome evidence seems conclusive with regard to female and male lineages surviving in current human populations, it lacks information on the autosomal portion of the genome. Although autosomal data are impeded by recombination, it is estimated that regions of approximately 50,000 base pairs may remain intact since the time of potential Neanderthal and modern human admixture events (Wall, 2000). In fact, several recent autosomal studies of contemporary human populations have yielded evidence supporting the assimilation of archaic DNA into the genome of modern humans (Eswaran, Harpending & Rogers, 2005; Plagnol & Wall, 2006). An extensive series of statistical simulations of autosomal loci uncovered that the majority of these markers exhibited signs of introgression, and that the data suggested a model that involved modern humans originating from Africa and subsequently assimilating genetic material from archaic hominids (Eswaran et al., 2005). Eswaran et al. (2005) stipulated that up to 80% of human loci may have been influenced by archaic admixture (Eswaran et al., 2005). In a separate study searching for patterns of linkage disequilibrium reflecting ancient admixture, it was found that at least 5% of the modern human genome was introgressed from one or more archaic species, including Neanderthals in Europe and another, unknown ancient Homo group in Africa (Plagnol & Wall, 2006). Most importantly, the statistical model created by Plagnol & Wall (2006) identified a set of modern human alleles of single nucleotide polymorphisms (SNPs) with a high probability of having originated in an archaic species. These SNP alleles may be useful as markers to investigate Neanderthal genomic sequences to confirm or negate the theoretical work of Plagnol & Wall (2006). Utilising statistical analyses of DNA data from multiple modern human populations, Fegundes et al. (2007) suggest that the data support an African replacement model with little to no admixture from archaic Homo lineages. A pattern somewhat suggestive of an introgression event emerges at the tau MAPT locus, a sequence related to susceptibility to neurological diseases (Baker et al., 1999). The MAPT region can be divided into two haplotypes, H1 and H2. Haplotype H2, in particular, strongly correlates with lower susceptibility to progressive supranuclear palsy and corticobasal degeneration (Houlden et al., 2001). These two haplotypes diverged approximately three million years ago (Stefansson et al., 2005), yet the H2 haplotype is nearly entirely restricted to European populations (Evans et al., 2004) and seems to have Biological Reviews 84 (2009) 245–257 Ó 2009 The Authors Journal compilation Ó 2009 Cambridge Philosophical Society 250 appeared in modern humans only 30,000 years ago (Hardy et al., 2005). This date suggests that the H2 haplotype originated in a population other than from Homo sapiens and was later incorporated into modern human groups in Europe, perhaps by Neanderthals (Hardy et al., 2005). Subsequent positive selection acting upon the H2 locus may have prevented it from being lost to genetic drift. The singular high prevalence of H2 in Europe argues for the integration event occurring subsequent to the penetration of modern humans into the continent, 40,000– 45,000 years ago, corroborating the recent coalescent time estimates (Stefansson et al., 2005). Perhaps even stronger evidence comes from a recent report, which postulates that an allele of the microcephalin gene that provides a selective advantage was obtained from Neanderthals by way of an introgression event (Evans et al., 2006). Microcephalin is a gene known to regulate brain size during development of the cerebral cortex (Jackson et al., 2002). A point mutation on base pair 74 of exon 2 is linked to the disease primary microcephaly, which leads to a decrease in cranial capacity from the average modern human size of approximately 1,400 cm3 to about 400–650 cm3 (Jackson et al., 2002). The microcephalin locus has been separated into two haplogroups defined by a polymorphism on exon 8, which is not involved in the onset of primary microcephaly. The D haplogroup contains the derived allele of this site relative to chimpanzee, while the non-D haplogroup possesses the corresponding ancestral state (Evans et al., 2005). Analyses of modern human DNA from an ethnically diverse panel consisting of 16 Africans, 25 Europeans, 35 Asians, 6 Pacific Islanders and 7 Andeans (the Coriell Panel), indicate that haplogroup D is present in 70% of those analysed, despite having a coalescence age of only 37,000 YBP (Evans et al., 2006). The less frequent, non-D haplogroup, on the other hand, has a much older coalescence age of approximately 990,000 YBP (Evans et al., 2006). The age difference between the D and non-D haplogroups suggests that haplogroup D may have been introgressed from an ancient Homo lineage into the modern human population by way of an admixture event, at a time period when Neanderthals and modern humans were co-habitating (Evans et al., 2006). In addition, population analyses have shown that the non-D haplogroup is most common in sub-Saharan Africa, while the D-haplogroup exhibits higher frequencies outside of Africa (Evans et al., 2006). This implies that the introgression likely took place subsequent to the modern human dispersal out of Africa (Evans et al., 2006). Strong positive selection acting on the derived allele makes it an even more likely candidate site for an introgression event (Evans et al., 2005). Strong selection in favour of a given allele reduces the chance that the haplotype would disappear due to genetic drift and would also explain its prevalence despite its recent coalescence estimations (Evans et al., 2005). Identification of haplogroup D in the DNA of multiple Neanderthal specimens may provide further support for the hypothesis that this version of the microcephalin gene is the result of admixture between modern humans and Neanderthals. Kristian J. Herrera and others IV. EVIDENCE FROM THE NEANDERTHAL MITOCHONDRIAL GENOME (1) The first mtDNA sequences from Neanderthals Due to its susceptibility to environmental damage, DNA in fossils is typically present only in very small quantities and is often highly fragmented or modified, compromising its analysis (Pääbo, Higuchi & Wilson, 1989; Hoss et al., 1996). Therefore, it was not until the advent of PCR (Mullis & Faloona, 1987) that the sequencing of ancient DNA became a possibility (Pääbo et al., 1989). Initial comparisons of mitochondrial hypervariable region I (HVRI) DNA extracted from a Neanderthal specimen in the Feldhofer cave with a standardised modern human mtDNA sequence, the Cambridge Reference Sequence (CRS) (Anderson et al., 1981), indicated a high degree of divergence between Neanderthals and modern humans (Krings et al., 1997). In this study, Krings et al. (1997) reported an average of 27.2 ]/[ 2.2 substitutions over 360 base pairs between the Neanderthal and individuals from various modern human populations; more than three times the average number of substitutions detected between individuals from the same contemporary human populations. This supports a most recent common ancestor (MRCA) between modern humans and Neanderthals living approximately 550,000– 690,000 YBP (Krings et al., 1997). Interestingly, this date is 250,000-390,000 years older than the estimated MRCA based on some fossil and archaeological data (Foley & Lahr, 1997). However, this discrepancy may be expected, since alleles from ancient polymorphisms could be partitioned in a lineage-specific manner and separation of ancestral populations will allow clade-specific mutations to accumulate in DNA prior to large-scale anatomical differences becoming evident in the populations (Nei, 1987). The majority of subsequent mtDNA analyses (Ovchinnikov et al., 2000; Gutierrez, Sanchez & Marin, 2002; Schmitz et al., 2002; Serre et al., 2004; Beauval et al., 2005; Lalueza-Fox et al., 2006) using a total of 11 Neanderthal specimens ranging from the Iberian Peninsula to the Caucasus Mountains, corroborate the results of Krings et al. (1997). These Neanderthal samples contain a net total of 40 base pair differences from the modern human CRS over a sequence length of 363 base pairs. Of these differences, 18 are non-polymorphic among Neanderthals, six are identical in all but one or two specimens, six are found only in the Feldhofer 1 sample (Krings et al., 1997), and three are unique to the Mezmaiskaya sample from the Caucasus mountains (Ovchinnikov et al., 2000). Two additional sequences extracted from fossils in Feldhofer and Vindija were obtained from the Hypervariable Region II (HVRII) (Krings et al., 1999, 2000). These two studies confirmed the results obtained by the HVRI sequences, yielding data that corroborate that the number of differences between Neanderthal and modern human HVRII sequences is three times higher than the highest number of differences between any two modern human HVRII fragments (Krings et al., 1999, 2000). Collectively, the above investigations on both HVRI and HVRII indicate that this limited number of Neanderthal specimens possess Biological Reviews 84 (2009) 245–257 Ó 2009 The Authors Journal compilation Ó 2009 Cambridge Philosophical Society Neanderthal/modern Human interactions mtDNA highly divergent and distant from extant humans, arguing against maternally driven introgression in either direction. (2) Older, more diverse Neanderthal mtDNA sequences It is noteworthy that many of these early studies examined samples that seemed to be constituents of a Neanderthal population that was relatively low in genetic diversity (Krings et al., 2000; Excoffier, 2006). The divergence estimates calculated from these early studies were close to those found within or between various modern human populations (Krings et al., 2000). Such restrictions may have limited the probability of finding sequences similar to modern humans (Krings et al., 2000). Therefore, it was still necessary to expand the collection of mtDNA sequences of Neanderthals before any conclusions could be drawn. This need was alleviated with the examination of mtDNA from two older Neanderthal fossils, an approximately 50,000 year old sample from Monti Lessini in Italy (Caramelli et al., 2006) and an approximately 100,000 year old fossil from Scladina in Belgium (Orlando et al., 2006). Together, the mtDNA sequences from the older specimens suggest that Neanderthals were much more genetically diverse than previously hypothesized (Excoffier, 2006). When compared with a 296 bp region from Neanderthal mtDNA HVRI sequences from prior investigations (Krings et al., 1997; Ovchinnikov et al., 2000; Serre et al., 2004), the 50,000 year old Monti Lessini specimen yielded three unique nucleotide differences, as well as two changes that were shared only with the Mezmaiskaya sample (Excoffier, 2006). A comparison of the same mtDNA sequence from the Monti Lessini sample with modern humans yielded only 23 substitutions, suggesting that the Monti Lessini specimen is more closely related to modern humans than the previously investigated Neanderthals (Caramelli et al., 2006). The 100,000 year old Scladina sequence, on the other hand, was more divergent from both the other Neanderthal samples and modern humans, exhibiting 14 differences from the Revised Cambridge Reference Sequence (R-CRS) over only a 123 bp sequence, including three never-before-identified substitutions (Orlando et al., 2006). Within the same stretch of mtDNA, the previously investigated sequences (Krings et al., 1997; Ovchinnikov et al., 2000; Serre et al., 2004; Lalueza-Fox et al., 2006) exhibit an average of only 11.6 ]/[ 0.49 substitutions from the R-CRS, with only one site being variable. Relative to the Scladina sample, the Neanderthal fossils that have been dated to more recent times are more similar to modern human sequences (Excoffier, 2006), possibly indicating that modern human and Neanderthal mtDNA converged through introgression during the arrival of modern humans in Europe. However, the dating of the Monti Lessini sample to 50,000 years ago suggests that the most human-like sequence was already present in Neanderthals prior to the arrival of modern humans in Europe (Excoffier, 2006). It is possible that the decrease in genetic diversity was the result of either a putative selective sweep that may have acted upon 251 Neanderthals 100,000–40,000 YBP (Excoffier, 2006) or a possible bottleneck occurring 60,000–70,000 YBP (van Andel & Tzedakis, 1996). Either scenario could have potentially reduced the diversity of the Neanderthal gene pool, leaving only Neanderthals that fell within the limited diversity exhibited by the more recent specimens. (3) Issues involving the mtDNA sequences The present collection of mtDNA sequences does not argue for introgression of mtDNA from Neanderthals into modern humans or vice versa (Excoffier, 2006). However, these regions of the mtDNA may be selectively neutral, and genetic drift may hamper the detection of introgressed DNA in a limited number of individuals (Nordborg, 1998). In fact, several studies have examined the probabilities of introgression events in modern human mtDNA being lost to genetic drift in extant human mtDNA (Nordborg, 1998; Currat & Excoffier, 2004; Serre et al., 2004). Both Nordborg (1998) and Serre et al. (2004) agree with a maximum possible mtDNA contribution from Neanderthals of 25%. Conversely, the statistical model of Currat & Excoffier (2004) proposes a more complex depiction of modern human expansions into Europe, and predicts that the maximum rate of admixture had to be less than 0.1%; a rate corresponding with no more than 120 specific matings over the entire course of the Neanderthal and modern human coexistence. V. EVIDENCE FROM THE NEANDERTHAL NUCLEAR GENOME (1) The earliest large-scale sequencing efforts The 16.7 kB haplotype of the mtDNA genome only represents the maternal lineages. Any sexual bias in mating patterns and/or genetic drift might mask the detection of Neanderthal and modern human admixture events as a result of Neanderthal mitochondrial lineages being lost from the modern human gene pool over time. Since mtDNA haplotypes genetically behave as a single locus, they are particularly susceptible to evolutionary drop-outs. Therefore, it has become increasingly apparent that autosomal nuclear DNA analyses are also necessary to ascertain potential introgression events (Dalton, 2006; Hebsgaard et al., 2007). Fortunately, a recent breakthrough in DNA sequencing technology has allowed researchers to isolate and sequence higher quantities of ancient DNA (Margulies et al., 2005). The new method enables highthroughput sequencing of the highly fragmented DNA that results from degradation over time. Following an extensive search for the Neanderthal fossils with the most highly preserved, unfragmented DNA, two separate efforts (Noonan et al., 2006; Green et al., 2006) led to the assessment and release of large quantities of Neanderthal DNA sequences. The study by Noonan et al. (2006) yielded approximately 65,000 base pairs of Neanderthal nuclear DNA, while Green et al. (2006) released Biological Reviews 84 (2009) 245–257 Ó 2009 The Authors Journal compilation Ó 2009 Cambridge Philosophical Society 252 roughly one million base pairs of Neanderthal nuclear DNA. Interestingly, however, the two reports produced conflicting results with regard to the question of modernhuman-Neanderthal admixture (Wall & Kim, 2007). The dataset released by Noonan et al. (2006) shows few data supporting admixture between Neanderthals and modern humans. Using the NCBI Reference Genome, Noonan et al. (2006) yielded a MRCA date for Neanderthals and modern humans of 706,000 YBP (confidence interval 468,000– 1,015,000 years ago). In addition, Noonan et al. (2006) found that 3% of the SNPs present in the Neanderthal sequences exhibit the derived state with respect to the chimpanzee genome. From this, Noonan et al. (2006) estimate 0% (confidence interval 0–20%) admixture between Neanderthals and modern humans. Conversely, Green et al. (2006) calculated a MRCA date for modern humans and Neanderthals of only 516,000 years ago (confidence interval 465,000–569,000 YBP). To establish a frame of reference for this date, Green et al. (2006) generated sequences from a contemporary modern human using the same sequencing methods used for the Neanderthal, and subsequently calculated the MRCA between those sequences and the NCBI Reference genome. Interestingly, their calculated date of 459,000 YBP (confidence interval 419,000–498,000) overlaps considerably with that of the Neanderthal sequences. Furthermore, the study performed by Green et al. (2006) demonstrated that Neanderthals possessed the derived allele (in reference to the chimpanzee ancestral allele) for 30% of all human SNPs within the dataset. Such a high number of SNPs exhibiting the derived state implies either that the majority of the polymorphisms were in place as common-ancestral polymorphisms prior to the split of Neanderthals and modern humans or were introgressed at a much later time, suggesting that there was a considerable amount of gene flow between the two lineages. (2) Issues relating to the genomic studies The discrepancies between the sequencing results of Noonan et al. (2006) and Green et al. (2006) have led to a reanalysis of both datasets using the methodology of Noonan et al. (2006) (Wall & Kim, 2007). Wall & Kim (2007) found that the Noonan et al. (2006) study supported a Neanderthal and modern European population split occurring approximately 325,000 YBP with limited admixture between the two groups. However, reanalysis of the Green et al. (2006) data suggests a Neanderthal-European human population divergence around 35,000 YBP and up to 95% introgression. These numbers are in direct contradiction with nearly every human-Neanderthal comparison to date (Wall & Kim, 2007). The high amount of introgression estimated from the Green et al. (2006) data seems to indicate that one or more errors occurred in the process of generating the Green et al. (2006) sequences such as modern human DNA contamination, alignment errors, and/or sequencing misreads (Wall & Kim, 2007). Under the assumption that longer autosomal DNA fragments are more likely to represent modern human Kristian J. Herrera and others contaminants, Wall & Kim (2007) estimated the divergence times of three groups of sequences from the Green et al. (2006) dataset based on size. They observed that while short sequences agreed with the divergence time obtained by Noonan et al. (2006), the long sequences from the Green et al. (2006) data yielded a much younger date. Furthermore, in each of these three groups, they found that the percentage of modern human SNPs for which the derived state appears in Neanderthals is highest in the longer fragments (Wall & Kim, 2007). Wall & Kim (2007) also noted that the sequences generated by Noonan et al. (2006) contained a higher rate of substitutions that are more likely to be caused by post-mortem damage (C/T and G/A) (Hofreiter et al., 2001) than those of Green et al. (2006). Such errors may have affected calculations of divergence and admixture rates (Wall & Kim, 2007). The authors indicate that the most likely scenario is that approximately 73% of the sequences produced by Green et al. (2006) were derived from modern human DNA contaminants. (3) Searches for genes In addition to random, large-scale sequencing of Neanderthal autosomal DNA, there have also been efforts to examine specific genes such as Forkhead Box P2 (FOXP2) (Krause et al., 2007b). FOXP2 is one of the most highly conserved genes in the mammalian genome (Enard et al., 2002) and is essential for the proper development of speech (MacDermot et al., 2005). A defective form of the gene, related to a missense mutation at position 328 of exon 7, is known to cause developmental verbal dyspraxia (MacDermot et al., 2005). Since the divergence of humans and chimpanzees, two independent single base pair substitutions have accumulated in the FOXP2 gene, both of which are located in exon 7, at positions 911 and 977 (Enard et al., 2002). Using population data from the flanking sequences surrounding exon 7, a coalescence simulation concluded that a selective sweep ending approximately 200,000 years ago may have acted upon the gene (Enard et al., 2002). Retrieval of the FOXP2 diagnostic sites in two distinct Neanderthal samples demonstrated that both specimens contain the derived allele. The presence of the derived state implies that either these two substitutions occurred prior to the split of Neanderthals and humans, or that the derived FOXP2 allele was introgressed from Neanderthals to modern humans or vice versa (Krause et al., 2007b). The coalescence age of the gene to approximately 200,000 years ago in modern humans makes it logical to consider that FOXP2 was introgressed from modern humans into Neanderthals, as opposed to the other way round, which would necessitate an older coalescence date (Coop et al., 2008). In addition, recent reanalysis of the FOXP2 data suggests that low rates of gene flow between modern humans and Neanderthals was a likely scenario (Coop et al., 2008). Krause et al. (2007b) also generated information with regard to paternally inherited Y-chromosome data in Neanderthals. These Y-chromosome sequences were utilised as controls to assess the level of modern human DNA Biological Reviews 84 (2009) 245–257 Ó 2009 The Authors Journal compilation Ó 2009 Cambridge Philosophical Society Neanderthal/modern Human interactions contamination in the FOXP2 sequences. The dating of the modern human Y-chromosomal MRCA to 90,000 YBP (Thomson et al., 2000) allowed Krause et al. (2007b) to test the level of modern human contamination since this date is well below that of the expected Neanderthal and modern human MRCA. In turn, it was expected that the Neanderthal Y-chromosome divergence from modern humans would be far greater than that between any two modern humans. This would only hold true, however, if introgression between the two groups was limited or nonexistent and not detectable. The results of Krause et al. (2007b) indicate that all five sequenced Y-SNPs contained the ancestral allele in both of the Neanderthals that were further analysed for the FOXP2 gene. Although these conclusions are only based on a limited number of individuals and a single haplotype, these data do not argue for introgression (Krause et al., 2007b). It is worth mentioning that Krause et al. (2007b) employed the Y2 (M42) marker to assess the level of contamination from extant humans in Neanderthal samples. If the fossils were ancestral for M42, Krause et al. (2007b) assumed that the sample was genuinely Neanderthal. However, testing of M42 alone without assessing the basal M91/M13 markers does not rule out the possibility of individuals from the two other sister clades of haplogroup A (A1 and A3) contributing to contamination. In addition, if the M91/M13 markers are derived for a given Neanderthal specimen, it may represent an ancestral polymorphism or an introgression event. Although the A haplogroup is currently restricted to Sub-Saharan Africa, it has been detected in the Levant (Underhill et al., 2000; Richards et al., 2003; Luis et al., 2004), Syria (Richards et al., 2003) and Sardinia (Underhill et al., 2000) at low frequencies, where Neanderthals and modern humans once co-existed. Furthermore, the present distribution of this haplotype mainly in Africa does not preclude a much wider distribution at the time of Neanderthal/modern human interaction. Therefore, these issues should be considered when assessing admixture events. VI. CONCLUSIONS (1) At present, incontrovertible evidence for or against Neanderthal and modern human admixture has yet to be identified. Reports from varying sources seem to provide contradictory results. In support of admixture are the genetic studies of Hardy et al. (2005), Green et al. (2006), Evans et al. (2006) and Plagnol & Wall (2006), as well a number of anthropological findings (Duarte et al., 1999; Trinkaus et al., 2003; Soficaru et al., 2006; Shang et al., 2007). (2) However, most of these reports have been criticised for various reasons, and it is clear that further work is required. For instance, the sequences produced by Green et al. (2006) may suffer from modern human DNA contamination (Wall & Kim, 2007). Similarly, many anthropologists disagree (e.g. Tattersall & Schwartz, 1999) with resounding statements such as those by Duarte et al. (1999) regarding the 253 identification of hybrids or modern humans with Neanderthallike traits. (3) The modern human haplotypes that possess signals of introgression, including the microcephalin D-haplogroup (Evans et al., 2006) and the H2 haplotype of the MAPT locus (Hardy et al., 2005) as well as the set of Neanderthalspecific SNPs identified by Plagnol & Wall (2006) are in need of empirical testing and verification in multiple Neanderthal samples. Widespread distribution in Neanderthals of either the microcephalin D-haplogroup or the MAPT H2 haplotype would be highly indicative of successful interbreeding between modern humans and Neanderthals. (4) On the other hand, repeated mtDNA studies and the work by Noonan et al. (2006) seem to suggest that little or no gene flow took place between Neanderthals and modern humans. Studies of mtDNA from both Neanderthal DNA and modern humans come to this conclusion. 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