Neandertal Evolutionary Genetics: Mitochondrial DNA Data from the Iberian Peninsula
http://www.100md.com
《分子生物学进展》
* Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, Barcelona, Spain; Laboratori di Antropologia, Dipartimento di Biologia Animale e Genetica, Università degli Studi di Firenze, Firenze, Italy; Departamento de Paleobiologia, Museo Nacional de Ciencias Naturales, CSIC, Madrid, Spain; and área de Prehistoria, Departamento de Historia, Universidad de Oviedo, Oviedo, Spain
Correspondence: E-mail: clalueza@ub.edu.
Abstract
Mitochondrial DNA (mtDNA) was retrieved for the first time from a Neandertal from the Iberian Peninsula, excavated from the El Sidrón Cave (Asturias, North of Spain), and dated to ca. 43,000 years ago. The sequence suggests that Iberian Neandertals were not genetically distinct from those of other regions. An estimate of effective population size indicates that the genetic history of the Neandertals was not shaped by an extreme population bottleneck associated with the glacial maximum of 130,000 years ago. A high level of polymorphism at sequence position 16258 reflects deeply rooted mtDNA lineages, with the time to the most recent common ancestor at ca. 250,000 years ago. This coincides with the full emergence of the "classical" Neandertal morphology and fits chronologically with a proposed speciation event of Homo neanderthalensis.
Key Words: human evolution ? Neandertals ? ancient DNA
Introduction
Mitochondrial DNA (mtDNA) sequences have thus far been retrieved from eight Neandertal remains: Feldhofer 1 and 2 in Germany (Krings et al. 1997; Schmitz et al. 2002), Mezmaiskaya in Russia (Ovchinnikov et al. 2000), Vindija 75, 77, and 80 in Croatia (Krings et al. 2000; Serre et al. 2004), Engis 2 in Belgium (Serre et al. 2004), and La Chapelle-aux-Saints in France (Serre et al. 2004). In addition, mtDNA sequences have been retrieved, following high authentication standards, from Late Upper Paleolithic modern humans from Paglicci cave, in Italy (Caramelli et al. 2003). These studies provide support to the hypothesis that Neandertals did not significantly contribute to the mtDNA pool of the early modern humans and indicate that they had a low genetic diversity similar to that of modern humans (Krings et al. 1997, 2000; Caramelli et al. 2003; Cooper, Drummond, and Willerslev 2004; Serre et al. 2004).
The Iberian Peninsula represents both the Western and the Southern European edge of the Neandertal distribution. It is furthermore the place where Neandertals coexisted longest with modern humans and where it has been suggested that hybridization between these species may have taken place (Duarte et al. 1999). Consequently, the retrieval of mtDNA sequences from an Iberian Neandertal represents an important step in our understanding of the evolutionary history of this species and its past interaction with Homo sapiens.
The human fossil collection from El Sidrón cave (Pilo?a, Asturias, North of Spain) represents the largest Neandertal sample in the Iberian Peninsula. Human remains come from the Galería del Osario (43°23'01''N, 5°19'44''W), which constitutes a small lateral gallery around 250 m deep into the El Sidrón karst system. The accidental unearthing in 1994 of an outstanding set of human fossils in the El Sidrón cave led to the current archaeological excavation and interdisciplinary study of the site (Fortea et al. 2003). The result is an extensive archaeopaleontological record, dominated by well-preserved and notably robust human remains of the species Homo neanderthalensis (Rosas and Aguirre 1999). In this study, one upper left first incisor (El Sidrón 441) was used for DNA extraction in the ancient DNA laboratory of the University Pompeu Fabra (Barcelona).
Materials and Methods
The Skeletal Sample
The collection of human fossils from El Sidrón (Galería del Osario) is divided into two samples. An initial sample was extracted in 1994 without methodological control, and it is composed of 120 specimens, including two mandibles (Fortea et al. 2003). The second sample has been recovered under systematic excavation since the year 2000, and it is composed of 669 remains, around 500 of which are human. At least five individuals are represented in the skeletal sample: one infant, two juveniles, and two adults. Associated faunal remains are scarce, with the presence of red deer, a large, unidentified herbivore, small mammals, and gastropods. In addition, about 30 lithic artifacts have been recovered, including scrapers, denticulates, a hand axe, and several Levalloisian stone tools.
Three human samples were dated by accelerator mass spectroscopy 14C at Beta Analytic, Inc (Miami, USA). Sample 1 (tooth) (Beta 192065) yielded an uncalibrated age of 40,840 ± 1,200 years ago; sample 2 (bone) (Beta 192066) an age of 37,300 ± 830 years ago; sample 3 (tooth) (Beta 192067) an age of 38,240 ± 890 years ago. Calibrated with CalPal program (by O. J?ris and B. Weninger, University of Cologne, Cologne, Germany) the dates obtained are 44,310 ± 978 years ago, 42,320 ± 367 years ago, and 42,757 ± 464 years ago, respectively; the average calibrated age is 43,129 ± 129 years ago.
Amino Acid Preservation
About 2.2 mg of dentine was removed from the root surface of the El Sidrón 441 tooth; the stereoisomers of aspartic, glutamic, and alanine amino acids were determined by A. Casoli (University of Parma) by high-performance liquid chromatography (Poinar et al. 1996). The stereoisomeric D/L [AH1] ratio observed for the three amino acids are 0.139 (Asp), 0.097 (Glu), and 0.080 (Ala). The aspartic values are close to the proposed limit of 0.10 for DNA preservation (Poinar et al. 1996). However, this sample was obtained from the external surface and is therefore likely to be in a worse condition than the internal sample used for DNA extraction. The amino acid content is 62,187 parts per million. It has been possible to amplify endogenous DNA from Pleistocene remains when the parts per million content is higher than 30,000 (Serre et al. 2004). Overall, these results are suggestive of DNA survival.
DNA Extraction, Amplification, and Sequencing
Around 20 mg of dentine powder was removed by drilling from the El Sidrón 441 tooth. DNA was extracted by using ancient DNA methods described elsewhere (Caramelli et al. 2003). Different overlapping fragments of the hypervariable region 1 (HVR-1) of the mtDNA were amplified by means of the polymerase chain reaction (PCR) with Neandertal-specific primers: NL16230, NH16262 (Serre et al. 2004), NL16256 (5'-ATCAACTACAACTCCAAAGA-3'), and NH16278 (5'-AAGGGTGGGTAGGTTTGTTGA-3'), with the latter primer set specifically designed for this study. To avoid the risk of cross contamination, every fragment was attempted in single PCR reactions: first NL16230–NH16262 and then NL16256–NH16278. Amplifications were undertaken using 1–2 μl of extract, an annealing temperature of 48°C, and 40 cycles of PCR. PCR products were cloned with a pMOSBlue cloning kit (Amersham Biosciences, Uppsala, Sweden) following the supplier's instructions. Colonies that yielded the correct size band were sequenced with an ABI 3100 DNA sequencer machine (Applied Biosystems, Foster City, Calif.). As a further methodological test, an additional sample was taken from the El Sidrón 441 root surface and sent to the University of Florence. After extensive cloning of PCR products, no endogenous sequences were obtained, thus confirming previous observations that the internal pulp cavity of the tooth is better suited for DNA preservation than the root tissue.
Time to the Most Recent Common Ancestor Estimates
The time to the most recent common ancestor (TMRCA) was calculated using a coalescence-based approach implemented in GeneTree (Griffiths and Tavaré 1994) for the complete mtDNA HVR-1 (N = 5 Neandertals), on the one hand, and the short fragment between positions 16230–16262 (N = 9 Neandertals), on the other hand. Calculations were performed assuming constant population size and 20 year generation time, with 100,000,000 iterations used to estimate the = Neμ parameter. Calculations on the complete HVR-1 were made assuming the same mutation rate for modern humans and Neandertals (Richards et al. 2000), whereas the mutation rate for the shorter fragment was obtained by averaging the relative mutation rates in modern humans of the specific 31 nucleotides included in the studied region (Meyer, Weiss, and von Haeseler 1999). We considered the analyzed Neandertals as roughly dated to 40,000 years ago.
Results and Discussion
A total of 47 bp were retrieved from the Iberian Neandertal specimen from the HVR-1 of the mtDNA between positions 16231 and 16277 of the reference sequence. As expected from previous Neandertal studies, the DNA is highly degraded. For the 16230–16262 fragment (table 1), only 4 sequences out of 80 exhibited a Neandertal-specific haplotype (substitutions at positions 16234, 16244, 16256, and 16258). For the 16256–16278 fragment, 4 out of 86 sequences yielded Neandertal diagnostic substitutions (16258, 16262, and an adenine insertion between positions 16263–16264). The remaining sequences were obvious modern contaminants. Attempts to sequence longer fragments (e.g., 16209–16278 and 16055–16095) yielded no Neandertal motifs after extensive cloning, indicating DNA degradation to <80-bp fragments. Consequently, the retrieval of the whole HVR-1 sequence of El Sidrón 441 might be technically impossible.
Table 1 DNA Sequences of the Clones Used to Reconstruct the El Sidrón 441 mtDNA HVR-1 Between Positions 16231 and 16277
Recently, Pusch and Bachmann (2004) reported an unknown mutagenic factor in ancient DNA extracts that could produce sequences containing numerous substitutions, some of them Neandertal specific. Other authors (Serre, Hofreiter, and P??bo 2004) failed to reproduce these results in other ancient samples. We sequenced about 70 clones from the 16209–16278 fragment of the El Sidrón 441 sample that included the previously amplified fragment with Neandertal motifs. All of the clones obtained could be attributed to the Cambridge Reference Sequence (Anderson et al. 1981) that matches most of the sequences of the researchers involved in the study. Therefore, we find no evidence in our sample of the putative mutagenic factor reported by Pusch and Bachmann (2004); most likely, the absence of Neandertal sequences in this fragment is a consequence of the excessive length of the fragment attempted.
The El Sidrón 441 specimen was superficially handled by only six people (J.F., M. de la R., A.R., M.B., C.M.-M., and T. Torres [Universidad Politécnica de Madrid]) prior to DNA extraction. Even so, the endogenous DNA constituted only 5% of the total retrieved sequences, thus confirming that exogenous DNA easily permeates throughout the dentine. Some of the modern sequences obtained (e.g., a C to T substitution at position 16069 in the 16055–16095 fragment) could be assigned to the aforementioned researchers, making this the first ancient DNA study to trace pre-laboratory contamination to its primary source. We propose this as a potential guideline for DNA studies of European Cro-Magnon specimens, at least in cases of recent excavations such as El Sidrón.
The mtDNA sequence from the Iberian specimen is found in other European Neandertals, suggesting that populations in this region were closely related to other Neandertals, at least in the female line. The Iberian Peninsula was home to some of the last surviving Neandertals (e.g., until 27,000–28,000 years ago at the Zafarraya site [Hublin et al. 1995]). In addition, the Lagar Velho skeleton in Portugal has been interpreted, albeit controversially (Tattersall and Schwartz 1999), as the product of hybridization between modern humans and Neandertals (Duarte et al. 1999). Furthermore, one of the oldest Upper Paleolithic sites with Aurignacian technology, El Castillo cave (Cabrera Valdés and Bischoff 1989), is located in the Cantabrian mountain range, not far from the El Sidrón site. Our results show that a Neandertal from this crucial region has a standard Neandertal sequence.
Among the nine Neandertal sequences studied so far, there is one highly polymorphic genetic marker, an A to G transition at position 16258 (with the former nucleotide being the ancestral state). This transition is found in three Vindija specimens (although, due to the state of fragmentation, Vindija 75 and 80 could correspond to the same individual [Serre et al. 2004]), in Feldhofer 1, and in El Sidrón 441, but it is absent in the other four Neandertals analyzed (fig. 1). The frequency of the ancestral A allele can thus be estimated as 0.44 ± 0.17. The level of polymorphism at this position in Neandertals has no parallel among modern European mtDNA, where the most polymorphic positions in a database of 4,414 individuals (Richards et al. 2000) are 16126 (0.196 ± 0.006), 16189 (0.186 ± 0.006), 16311 (0.167 ± 0.006), and 16223 (0.126 ± 0.005). The possibility of a recurrent mutation at this position cannot be ruled out, but we find this unlikely, because the 16258 position is quite stable in modern humans, with an estimated mutation rate that is 43% the average HVR-1 mutation rate (Meyer, Weiss, and von Haeseler 1999). Consequently, it is more parsimonious to assume that a unique mutational event underlies the variation at this position and that the frequency is an indication of its age.
FIG. 1.— Geographic localization and name of the Neandertals sites with genetic data available, displaying the highly polymorphic mtDNA 16258 position. Filled circles: G nucleotide in mtDNA position 16258; blank circles: A nucleotide in mtDNA position 16258; dashed lines: coastline during the glacial periods. 1: El Sidrón; 2: La Chapelle-aux-Saints; 3: Engis; 4: Feldhofer; 5: Vindija, 6: Mezmaiskaya. The box shows the localization of the El Sidrón site in a map of the Asturias region.
The presence of the 16258 G substitution in individuals who are likely to trace their ancestry to two different glacial refugia, the Iberian Peninsula and the Balkans, suggests that genetic variation at this position existed among European Neandertals prior to their retreat into these Southern refugia, at the onset of the dramatic glacial maximum of ca. 130,000 years ago (Petit et al. 1999). Estimates of the time to the Neandertal most recent common ancestor (based on the five specimens with complete HVR-1 sequences) yielded a date of 195,000 ± 43,000 years ago. A separate estimate of the age of the 16258 polymorphism produced a date of 145,000 ± 36,000 years ago. These estimates included four nucleotide positions in the Feldhofer 1 sequence (16107, 16108, 16111, and 16112) that could be artifacts (Schmitz et al. 2002). Dropping these positions, the TMRCA date and the 16258 mutation age were reduced to 162,000 ± 41,700 years ago and 92,000 ± 25,000 years ago, respectively. Estimates based on the shorter 16230–16262 fragment and nine Neandertals yielded, as expected from the low number of variable positions, somewhat older dates and larger standard errors: 245,500 ± 108,000 years ago for the TMRCA and 153,000 ± 81,000 years ago for the age of the 16258 polymorphism. Although the standard errors are large, all these estimates are compatible with the hypothesis that Neandertal mtDNA variation predates the 130,000 years ago glacial maximum.
Estimates of the female effective population size (Nfe) varied between 5,000 and 9,000. Interestingly, these figures are similar to those obtained for modern humans with present-day mtDNA data, which could suggest that the evolutionary history of Neandertals and modern humans were characterized by similar demographic parameters. The long-term Nfe is approximately equal to the harmonic mean of past numbers of contemporaneous breeding females and therefore disproportionately influenced by small values, such as those resulting from a population bottleneck. However, the relatively high values of Nfe obtained for Neandertals are inconsistent with a dramatic bottleneck in Neandertals prior to their extinction. In conclusion, the TMRCA and the effective population size estimates indicate that the genetic history of the Neandertals was not shaped by a dramatic population bottleneck associated with the 130,000 years ago glacial episode. We note, however, that the coalescent ages obtained for the mtDNA variation are roughly coincident with the full emergence of the specialized Neandertal morphology, around 250,000 years ago (Rightmire 2001).
Neandertals are considered to be an evolutionary lineage rooted in the European Middle Pleistocene fossil record. This lineage includes no less than two paleontological species, Homo heidelbergensis and H. neanderthalensis. Current opinion is split on the tempo and mode of evolutionary events within this lineage. One scheme assumes a gradual emergence of distinctive Neandertal features through chronospecies continuity, while a second view sees the emergence of Neandertals as the result of a clearly defined speciation event, occurring around 250,000–300,000 years ago (Rightmire 2001).
The present genetic data support the latter hypothesis that H. neanderthalensis emerged as a distinct biological entity after a speciation event, ca. 250,000 years ago. This event not only coincides with the TMRCA estimates of the Neandertal mtDNA variation but also with the appearance in Europe of the cultural Mode 3 industry and a decrease in the morphological variation observed in H. heidelbergensis. Additional supporting evidence is provided by recent studies on dental growth histology, which have shown that H. heidelbergensis and H. neanderthalensis differed in tooth growth rates, a likely reflection of distinct ontogenetic patterns (Ramirez Rozzi and Bermudez de Castro 2004).
Additional Neandertal mtDNA sequences will no doubt continue to clarify the genetic history of these archaic humans. Despite the limited sample and problems of poor DNA preservation in regions with warm climates, our study of an El Sidrón Neandertal demonstrates that sites in Southern Europe can provide important additional specimens for ancient DNA analysis.
Acknowledgements
This study has been developed in the framework of the Convenio Consejería de Cultura del Principado de Asturias-Universidad de Oviedo (CN-00-184-D3; CN-01-132,133,134-B1; CN-04-152), as well as Ficyt-Consejería de Cultura (FC-02-PC-SPV01-27), Dirección General de Investigación, Ministerio de Ciencia y Tecnología of Spain (BMC2001-0772), the Departament d'Universitats, Recerca i Societat de la Informació, Generalitat de Catalunya (2001SGR00285), and by a fellowship to M.L.S. (AP2002-1065). We are grateful to Agnar Helgason (deCode Genetics, Iceland) who provided useful comments to this manuscript and to Antonella Casoli (Università degli Studi, Parma, Italy) who obtained the amino acid data.
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Correspondence: E-mail: clalueza@ub.edu.
Abstract
Mitochondrial DNA (mtDNA) was retrieved for the first time from a Neandertal from the Iberian Peninsula, excavated from the El Sidrón Cave (Asturias, North of Spain), and dated to ca. 43,000 years ago. The sequence suggests that Iberian Neandertals were not genetically distinct from those of other regions. An estimate of effective population size indicates that the genetic history of the Neandertals was not shaped by an extreme population bottleneck associated with the glacial maximum of 130,000 years ago. A high level of polymorphism at sequence position 16258 reflects deeply rooted mtDNA lineages, with the time to the most recent common ancestor at ca. 250,000 years ago. This coincides with the full emergence of the "classical" Neandertal morphology and fits chronologically with a proposed speciation event of Homo neanderthalensis.
Key Words: human evolution ? Neandertals ? ancient DNA
Introduction
Mitochondrial DNA (mtDNA) sequences have thus far been retrieved from eight Neandertal remains: Feldhofer 1 and 2 in Germany (Krings et al. 1997; Schmitz et al. 2002), Mezmaiskaya in Russia (Ovchinnikov et al. 2000), Vindija 75, 77, and 80 in Croatia (Krings et al. 2000; Serre et al. 2004), Engis 2 in Belgium (Serre et al. 2004), and La Chapelle-aux-Saints in France (Serre et al. 2004). In addition, mtDNA sequences have been retrieved, following high authentication standards, from Late Upper Paleolithic modern humans from Paglicci cave, in Italy (Caramelli et al. 2003). These studies provide support to the hypothesis that Neandertals did not significantly contribute to the mtDNA pool of the early modern humans and indicate that they had a low genetic diversity similar to that of modern humans (Krings et al. 1997, 2000; Caramelli et al. 2003; Cooper, Drummond, and Willerslev 2004; Serre et al. 2004).
The Iberian Peninsula represents both the Western and the Southern European edge of the Neandertal distribution. It is furthermore the place where Neandertals coexisted longest with modern humans and where it has been suggested that hybridization between these species may have taken place (Duarte et al. 1999). Consequently, the retrieval of mtDNA sequences from an Iberian Neandertal represents an important step in our understanding of the evolutionary history of this species and its past interaction with Homo sapiens.
The human fossil collection from El Sidrón cave (Pilo?a, Asturias, North of Spain) represents the largest Neandertal sample in the Iberian Peninsula. Human remains come from the Galería del Osario (43°23'01''N, 5°19'44''W), which constitutes a small lateral gallery around 250 m deep into the El Sidrón karst system. The accidental unearthing in 1994 of an outstanding set of human fossils in the El Sidrón cave led to the current archaeological excavation and interdisciplinary study of the site (Fortea et al. 2003). The result is an extensive archaeopaleontological record, dominated by well-preserved and notably robust human remains of the species Homo neanderthalensis (Rosas and Aguirre 1999). In this study, one upper left first incisor (El Sidrón 441) was used for DNA extraction in the ancient DNA laboratory of the University Pompeu Fabra (Barcelona).
Materials and Methods
The Skeletal Sample
The collection of human fossils from El Sidrón (Galería del Osario) is divided into two samples. An initial sample was extracted in 1994 without methodological control, and it is composed of 120 specimens, including two mandibles (Fortea et al. 2003). The second sample has been recovered under systematic excavation since the year 2000, and it is composed of 669 remains, around 500 of which are human. At least five individuals are represented in the skeletal sample: one infant, two juveniles, and two adults. Associated faunal remains are scarce, with the presence of red deer, a large, unidentified herbivore, small mammals, and gastropods. In addition, about 30 lithic artifacts have been recovered, including scrapers, denticulates, a hand axe, and several Levalloisian stone tools.
Three human samples were dated by accelerator mass spectroscopy 14C at Beta Analytic, Inc (Miami, USA). Sample 1 (tooth) (Beta 192065) yielded an uncalibrated age of 40,840 ± 1,200 years ago; sample 2 (bone) (Beta 192066) an age of 37,300 ± 830 years ago; sample 3 (tooth) (Beta 192067) an age of 38,240 ± 890 years ago. Calibrated with CalPal program (by O. J?ris and B. Weninger, University of Cologne, Cologne, Germany) the dates obtained are 44,310 ± 978 years ago, 42,320 ± 367 years ago, and 42,757 ± 464 years ago, respectively; the average calibrated age is 43,129 ± 129 years ago.
Amino Acid Preservation
About 2.2 mg of dentine was removed from the root surface of the El Sidrón 441 tooth; the stereoisomers of aspartic, glutamic, and alanine amino acids were determined by A. Casoli (University of Parma) by high-performance liquid chromatography (Poinar et al. 1996). The stereoisomeric D/L [AH1] ratio observed for the three amino acids are 0.139 (Asp), 0.097 (Glu), and 0.080 (Ala). The aspartic values are close to the proposed limit of 0.10 for DNA preservation (Poinar et al. 1996). However, this sample was obtained from the external surface and is therefore likely to be in a worse condition than the internal sample used for DNA extraction. The amino acid content is 62,187 parts per million. It has been possible to amplify endogenous DNA from Pleistocene remains when the parts per million content is higher than 30,000 (Serre et al. 2004). Overall, these results are suggestive of DNA survival.
DNA Extraction, Amplification, and Sequencing
Around 20 mg of dentine powder was removed by drilling from the El Sidrón 441 tooth. DNA was extracted by using ancient DNA methods described elsewhere (Caramelli et al. 2003). Different overlapping fragments of the hypervariable region 1 (HVR-1) of the mtDNA were amplified by means of the polymerase chain reaction (PCR) with Neandertal-specific primers: NL16230, NH16262 (Serre et al. 2004), NL16256 (5'-ATCAACTACAACTCCAAAGA-3'), and NH16278 (5'-AAGGGTGGGTAGGTTTGTTGA-3'), with the latter primer set specifically designed for this study. To avoid the risk of cross contamination, every fragment was attempted in single PCR reactions: first NL16230–NH16262 and then NL16256–NH16278. Amplifications were undertaken using 1–2 μl of extract, an annealing temperature of 48°C, and 40 cycles of PCR. PCR products were cloned with a pMOSBlue cloning kit (Amersham Biosciences, Uppsala, Sweden) following the supplier's instructions. Colonies that yielded the correct size band were sequenced with an ABI 3100 DNA sequencer machine (Applied Biosystems, Foster City, Calif.). As a further methodological test, an additional sample was taken from the El Sidrón 441 root surface and sent to the University of Florence. After extensive cloning of PCR products, no endogenous sequences were obtained, thus confirming previous observations that the internal pulp cavity of the tooth is better suited for DNA preservation than the root tissue.
Time to the Most Recent Common Ancestor Estimates
The time to the most recent common ancestor (TMRCA) was calculated using a coalescence-based approach implemented in GeneTree (Griffiths and Tavaré 1994) for the complete mtDNA HVR-1 (N = 5 Neandertals), on the one hand, and the short fragment between positions 16230–16262 (N = 9 Neandertals), on the other hand. Calculations were performed assuming constant population size and 20 year generation time, with 100,000,000 iterations used to estimate the = Neμ parameter. Calculations on the complete HVR-1 were made assuming the same mutation rate for modern humans and Neandertals (Richards et al. 2000), whereas the mutation rate for the shorter fragment was obtained by averaging the relative mutation rates in modern humans of the specific 31 nucleotides included in the studied region (Meyer, Weiss, and von Haeseler 1999). We considered the analyzed Neandertals as roughly dated to 40,000 years ago.
Results and Discussion
A total of 47 bp were retrieved from the Iberian Neandertal specimen from the HVR-1 of the mtDNA between positions 16231 and 16277 of the reference sequence. As expected from previous Neandertal studies, the DNA is highly degraded. For the 16230–16262 fragment (table 1), only 4 sequences out of 80 exhibited a Neandertal-specific haplotype (substitutions at positions 16234, 16244, 16256, and 16258). For the 16256–16278 fragment, 4 out of 86 sequences yielded Neandertal diagnostic substitutions (16258, 16262, and an adenine insertion between positions 16263–16264). The remaining sequences were obvious modern contaminants. Attempts to sequence longer fragments (e.g., 16209–16278 and 16055–16095) yielded no Neandertal motifs after extensive cloning, indicating DNA degradation to <80-bp fragments. Consequently, the retrieval of the whole HVR-1 sequence of El Sidrón 441 might be technically impossible.
Table 1 DNA Sequences of the Clones Used to Reconstruct the El Sidrón 441 mtDNA HVR-1 Between Positions 16231 and 16277
Recently, Pusch and Bachmann (2004) reported an unknown mutagenic factor in ancient DNA extracts that could produce sequences containing numerous substitutions, some of them Neandertal specific. Other authors (Serre, Hofreiter, and P??bo 2004) failed to reproduce these results in other ancient samples. We sequenced about 70 clones from the 16209–16278 fragment of the El Sidrón 441 sample that included the previously amplified fragment with Neandertal motifs. All of the clones obtained could be attributed to the Cambridge Reference Sequence (Anderson et al. 1981) that matches most of the sequences of the researchers involved in the study. Therefore, we find no evidence in our sample of the putative mutagenic factor reported by Pusch and Bachmann (2004); most likely, the absence of Neandertal sequences in this fragment is a consequence of the excessive length of the fragment attempted.
The El Sidrón 441 specimen was superficially handled by only six people (J.F., M. de la R., A.R., M.B., C.M.-M., and T. Torres [Universidad Politécnica de Madrid]) prior to DNA extraction. Even so, the endogenous DNA constituted only 5% of the total retrieved sequences, thus confirming that exogenous DNA easily permeates throughout the dentine. Some of the modern sequences obtained (e.g., a C to T substitution at position 16069 in the 16055–16095 fragment) could be assigned to the aforementioned researchers, making this the first ancient DNA study to trace pre-laboratory contamination to its primary source. We propose this as a potential guideline for DNA studies of European Cro-Magnon specimens, at least in cases of recent excavations such as El Sidrón.
The mtDNA sequence from the Iberian specimen is found in other European Neandertals, suggesting that populations in this region were closely related to other Neandertals, at least in the female line. The Iberian Peninsula was home to some of the last surviving Neandertals (e.g., until 27,000–28,000 years ago at the Zafarraya site [Hublin et al. 1995]). In addition, the Lagar Velho skeleton in Portugal has been interpreted, albeit controversially (Tattersall and Schwartz 1999), as the product of hybridization between modern humans and Neandertals (Duarte et al. 1999). Furthermore, one of the oldest Upper Paleolithic sites with Aurignacian technology, El Castillo cave (Cabrera Valdés and Bischoff 1989), is located in the Cantabrian mountain range, not far from the El Sidrón site. Our results show that a Neandertal from this crucial region has a standard Neandertal sequence.
Among the nine Neandertal sequences studied so far, there is one highly polymorphic genetic marker, an A to G transition at position 16258 (with the former nucleotide being the ancestral state). This transition is found in three Vindija specimens (although, due to the state of fragmentation, Vindija 75 and 80 could correspond to the same individual [Serre et al. 2004]), in Feldhofer 1, and in El Sidrón 441, but it is absent in the other four Neandertals analyzed (fig. 1). The frequency of the ancestral A allele can thus be estimated as 0.44 ± 0.17. The level of polymorphism at this position in Neandertals has no parallel among modern European mtDNA, where the most polymorphic positions in a database of 4,414 individuals (Richards et al. 2000) are 16126 (0.196 ± 0.006), 16189 (0.186 ± 0.006), 16311 (0.167 ± 0.006), and 16223 (0.126 ± 0.005). The possibility of a recurrent mutation at this position cannot be ruled out, but we find this unlikely, because the 16258 position is quite stable in modern humans, with an estimated mutation rate that is 43% the average HVR-1 mutation rate (Meyer, Weiss, and von Haeseler 1999). Consequently, it is more parsimonious to assume that a unique mutational event underlies the variation at this position and that the frequency is an indication of its age.
FIG. 1.— Geographic localization and name of the Neandertals sites with genetic data available, displaying the highly polymorphic mtDNA 16258 position. Filled circles: G nucleotide in mtDNA position 16258; blank circles: A nucleotide in mtDNA position 16258; dashed lines: coastline during the glacial periods. 1: El Sidrón; 2: La Chapelle-aux-Saints; 3: Engis; 4: Feldhofer; 5: Vindija, 6: Mezmaiskaya. The box shows the localization of the El Sidrón site in a map of the Asturias region.
The presence of the 16258 G substitution in individuals who are likely to trace their ancestry to two different glacial refugia, the Iberian Peninsula and the Balkans, suggests that genetic variation at this position existed among European Neandertals prior to their retreat into these Southern refugia, at the onset of the dramatic glacial maximum of ca. 130,000 years ago (Petit et al. 1999). Estimates of the time to the Neandertal most recent common ancestor (based on the five specimens with complete HVR-1 sequences) yielded a date of 195,000 ± 43,000 years ago. A separate estimate of the age of the 16258 polymorphism produced a date of 145,000 ± 36,000 years ago. These estimates included four nucleotide positions in the Feldhofer 1 sequence (16107, 16108, 16111, and 16112) that could be artifacts (Schmitz et al. 2002). Dropping these positions, the TMRCA date and the 16258 mutation age were reduced to 162,000 ± 41,700 years ago and 92,000 ± 25,000 years ago, respectively. Estimates based on the shorter 16230–16262 fragment and nine Neandertals yielded, as expected from the low number of variable positions, somewhat older dates and larger standard errors: 245,500 ± 108,000 years ago for the TMRCA and 153,000 ± 81,000 years ago for the age of the 16258 polymorphism. Although the standard errors are large, all these estimates are compatible with the hypothesis that Neandertal mtDNA variation predates the 130,000 years ago glacial maximum.
Estimates of the female effective population size (Nfe) varied between 5,000 and 9,000. Interestingly, these figures are similar to those obtained for modern humans with present-day mtDNA data, which could suggest that the evolutionary history of Neandertals and modern humans were characterized by similar demographic parameters. The long-term Nfe is approximately equal to the harmonic mean of past numbers of contemporaneous breeding females and therefore disproportionately influenced by small values, such as those resulting from a population bottleneck. However, the relatively high values of Nfe obtained for Neandertals are inconsistent with a dramatic bottleneck in Neandertals prior to their extinction. In conclusion, the TMRCA and the effective population size estimates indicate that the genetic history of the Neandertals was not shaped by a dramatic population bottleneck associated with the 130,000 years ago glacial episode. We note, however, that the coalescent ages obtained for the mtDNA variation are roughly coincident with the full emergence of the specialized Neandertal morphology, around 250,000 years ago (Rightmire 2001).
Neandertals are considered to be an evolutionary lineage rooted in the European Middle Pleistocene fossil record. This lineage includes no less than two paleontological species, Homo heidelbergensis and H. neanderthalensis. Current opinion is split on the tempo and mode of evolutionary events within this lineage. One scheme assumes a gradual emergence of distinctive Neandertal features through chronospecies continuity, while a second view sees the emergence of Neandertals as the result of a clearly defined speciation event, occurring around 250,000–300,000 years ago (Rightmire 2001).
The present genetic data support the latter hypothesis that H. neanderthalensis emerged as a distinct biological entity after a speciation event, ca. 250,000 years ago. This event not only coincides with the TMRCA estimates of the Neandertal mtDNA variation but also with the appearance in Europe of the cultural Mode 3 industry and a decrease in the morphological variation observed in H. heidelbergensis. Additional supporting evidence is provided by recent studies on dental growth histology, which have shown that H. heidelbergensis and H. neanderthalensis differed in tooth growth rates, a likely reflection of distinct ontogenetic patterns (Ramirez Rozzi and Bermudez de Castro 2004).
Additional Neandertal mtDNA sequences will no doubt continue to clarify the genetic history of these archaic humans. Despite the limited sample and problems of poor DNA preservation in regions with warm climates, our study of an El Sidrón Neandertal demonstrates that sites in Southern Europe can provide important additional specimens for ancient DNA analysis.
Acknowledgements
This study has been developed in the framework of the Convenio Consejería de Cultura del Principado de Asturias-Universidad de Oviedo (CN-00-184-D3; CN-01-132,133,134-B1; CN-04-152), as well as Ficyt-Consejería de Cultura (FC-02-PC-SPV01-27), Dirección General de Investigación, Ministerio de Ciencia y Tecnología of Spain (BMC2001-0772), the Departament d'Universitats, Recerca i Societat de la Informació, Generalitat de Catalunya (2001SGR00285), and by a fellowship to M.L.S. (AP2002-1065). We are grateful to Agnar Helgason (deCode Genetics, Iceland) who provided useful comments to this manuscript and to Antonella Casoli (Università degli Studi, Parma, Italy) who obtained the amino acid data.
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