Neandertal Evolutionary Genetics: Mitochondrial DNA Data from the
Iberian Peninsula
Carles Lalueza-Fox,*1 Marı́a Lourdes Sampietro,* David Caramelli, Yvonne Puder,*2
Martina Lari, Francesc Calafell,* Cayetana Martı́nez-Maza,à Markus Bastir,à Javier Fortea,§
Marco de la Rasilla,§ Jaume Bertranpetit,* and Antonio Rosasà
*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; àDepartamento de Paleobiologia,
Museo Nacional de Ciencias Naturales, CSIC, Madrid, Spain; and §Área de Prehistoria, Departamento de Historia, Universidad de
Oviedo, Oviedo, Spain
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.
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
(Piloña, Asturias, North of Spain) represents the largest
Neandertal sample in the Iberian Peninsula. Human remains
come from the Galerı́a del Osario (43°23#01$N,
5°19#44$W), which constitutes a small lateral gallery
1
Present address: Unitat d’Antropologia, Departament de Biologia
Animal, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain.
2
Present address: Institut für Zoologie und Anthropologie, GeorgAugust-Universität Göttingen, Göttingen, Germany.
Key words: human evolution, Neandertals, ancient DNA.
E-mail: [email protected].
Mol. Biol. Evol. 22(4):1077–1081. 2005
doi:10.1093/molbev/msi094
Advance Access publication February 2, 2005
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 (Galerı́a 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 6 1,200 years ago; sample 2 (bone) (Beta 192066)
an age of 37,300 6 830 years ago; sample 3 (tooth) (Beta
192067) an age of 38,240 6 890 years ago. Calibrated with
CalPal program (by O. Jöris and B. Weninger, University
of Cologne, Cologne, Germany) the dates obtained are
44,310 6 978 years ago, 42,320 6 367 years ago, and
42,757 6 464 years ago, respectively; the average calibrated age is 43,129 6 129 years ago.
Molecular Biology and Evolution vol. 22 no. 4 Ó Society for Molecular Biology and Evolution 2005; all rights reserved.
1078 Lalueza-Fox et al.
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 highperformance 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 ll 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 5 Neandertals), on the one hand, and the short fragment between positions 16230–16262 (N 5 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 h 5 Nel 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.
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 Pääbo 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
Neandertal Mitochondrial DNA from the Iberian Peninsula 1079
Table 1
DNA Sequences of the Clones Used to Reconstruct the El Sidrón 441 mtDNA HVR-1 Between Positions 16231 and 16277
Reference
B.1.1
B.2.1
B.2.2
B.2.3
B.2.4
B.4.1
B.4.2
B.4.3
B.4.4
El Sidrón 441
Feldhofer 1
Mezmaiskaya
Vindija 75
Feldhofer 2
Vindija 80
Vindija 77
Engis 2
La Chapelle-aux-Saints
T
.
.
.
.
.
.
.
.
.
.
.
.
.
.
C
.
.
.
.
.
.
.
.
.
.
.
.
.
.
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
1
6
2
3
4
C
T
T
T
T
T
T
T
T
T
T
T
T
T
T
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
C
.
.
.
.
.
.
.
.
.
.
.
.
.
.
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
T
.
.
.
.
.
.
.
.
.
.
.
.
.
.
C
.
.
.
.
.
.
.
.
.
.
.
.
.
.
A
.
G
.
.
.
.
.
.
.
.
.
.
.
.
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
C
.
.
.
.
.
.
.
.
.
.
.
.
.
.
T
.
.
.
.
.
.
.
.
.
.
.
.
.
.
1
6
2
4
4
G
.
A
A
A
A
A
A
A
A
A
A
A
A
A
C
.
.
.
.
.
.
.
.
.
.
.
.
.
.
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
C
.
.
.
.
.
.
.
.
.
.
.
.
.
.
T
.
.
.
.
.
.
.
.
.
.
.
.
.
.
C
.
.
.
.
.
.
.
.
.
.
.
.
.
.
C
.
.
.
.
.
.
.
.
.
.
.
.
.
.
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
G
.
.
.
.
.
.
.
.
.
.
.
.
.
.
1
6
2
5
6
C
.
A
A
A
A
A
A
A
A
A
A
A
A
A
1
6
2
5
8
CA
. .
. G
. G
. G
. G
-G
-G
-G
-G
. G
. G
. .
. G
. .
. G
. G
. .
. .
C
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
C
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
1
6
2
6
2
CCT
.
.
.
.
.
. T .
. T .
. T .
. T .
. T .
. T .
. T .
. T .
. T .
. T .
.
.
.
1
6
2
6
3B
- CACCCACTAGGATA
A
A
A
A
A
A
A
A
A
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
NOTE.—B: Barcelona amplification; first digit, number of amplification, second digit, number of clone. Reference refers to the human reference sequence (Anderson et al.
1981). The B.1.1 clone might be a jumping PCR effect; the single-nucleotide substitution in Clone B.2.1 is attributable to a cloning artifact or to a chemical damage in the
original template; the single-nucleotide loss in Clones B.4.1 to B.4.4 could be due to a jumping PCR event. Sequences from the other eight Neandertals are also displayed.
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 6 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 6 0.006), 16189 (0.186 6 0.006), 16311
(0.167 6 0.006), and 16223 (0.126 6 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.
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 6 43,000 years ago. A separate
estimate of the age of the 16258 polymorphism produced a
date of 145,000 6 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 6 41,700 years ago and 92,000 6 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 6 108,000 years
ago for the TMRCA and 153,000 6 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,
1080 Lalueza-Fox et al.
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.
Acknowledgments
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.
This study has been developed in the framework of
the Convenio Consejerı́a de Cultura del Principado de
Asturias-Universidad de Oviedo (CN-00-184-D3; CN-01132,133,134-B1; CN-04-152), as well as Ficyt-Consejerı́a
de Cultura (FC-02-PC-SPV01-27), Dirección General de
Investigación, Ministerio de Ciencia y Tecnologı́a 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.
Literature Cited
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).
Anderson, S., A. T. Bankier, B. G. Barrell et al. (14 co-authors).
1981. Sequence and organization of the human mitochondrial
genome. Nature 290:457–465.
Cabrera Valdés, V., and J. L. Bischoff. 1989. Accelerator 14C
dates for early Upper Palaeolithic (basal Aurignacian) at El
Castillo Cave (Spain). J. Arch. Sci. 16 (6):577–584.
Caramelli, D., C. Lalueza-Fox, C. Vernesi et al. (11 co-authors).
2003. Evidence for a genetic discontinuity between Neandertals and 24,000-year-old anatomically modern Europeans.
Proc. Natl. Acad. Sci. USA 100:6593–6597.
Cooper, A., A. J. Drummond, and E. Willerslev. 2004. Ancient
DNA: would the real Neandertal please stand up? Curr. Biol.
14:R431–R433.
Duarte, C., J. Mauricio, P. B. Pettitt, P. Souto, E. Trinkaus, H. van
der Plicht, and J. Zilhao. 1999. The early Upper Paleolithic
human skeleton from the Abrigo do Lagar Velho (Portugal)
and modern human emergence in Iberia. Proc. Natl. Acad.
Sci. USA 96:7604–7609.
Fortea, J., M. de la Rasilla, E. Martinez et al. (15 co-authors).
2003. La Cueva de El Sidrón (Borines, Piloña, Asturias):
Primeros resultados. Estud. Geol. 59:159–179.
Griffiths, R. C., and S. Tavaré. 1994. Ancestral inference in population genetics. Stat. Sci. 9:307–319.
Hublin, J. J., C. Barroso Ruiz, P. Medina Lara, M. Fontugne, and
J.-L. Reyss.1995. The Mousterian site of Zafarraya (Andalucia, Spain): dating and implications on the palaeolithic peopling processes of Western Europe. C. R. Acad. Sci. Paris
IIa 321:931–937.
Krings, M., C. Capelli, F. Tschentscher, H. Geisert, S. Meyer,
A. von Haeseler, K. Grossschmidt, G. Possnert, M. Paunovic,
and S. Pääbo. 2000. A view of Neandertal genetic diversity.
Nat. Genet. 26:144–146.
Krings, M., A. Stone, R. W. Schmitz, H. Krainitzki, M. Stoneking,
and S. Pääbo. 1997. DNA sequences and the origin of modern
humans. Cell 90:19–30.
Meyer, S., G. Weiss, and A. von Haeseler. 1999. Pattern of nucleotide substitution and rate heterogeneity in the hypervariable
regions I and II of human mtDNA. Genetics 152:
1103–1110.
Neandertal Mitochondrial DNA from the Iberian Peninsula 1081
Ovchinnikov, I. V., A. Götherström, G. P. Romanova, V. M.
Kharitonov, K. Lidén, and W. Goodwin. 2000. Molecular analysis of Neandertal DNA from the northern Caucasus. Nature
404:490–493.
Petit, J. R., J. Jouzel, D. Raynaud et al. (19 co-authors). 1999. Climate and atmospheric history of the past 420,000 years from
the Vostok ice core, Antarctica. Nature 399:429–436.
Poinar, H. N., M. Hoss, J. L. Bada, and S. Pääbo. 1996. Amino
acid racemization and the preservation of ancient DNA.
Science 272:864–866.
Pusch, C. M., and L. Bachmann. 2004. Spiking of contemporary
human template DNA with ancient DNA extracts induces
mutations under PCR and generates non-authentic mitochondrial sequences. Mol. Biol. Evol. 21:957–964.
Ramirez Rozzi, F. V., and J. M. Bermudez de Castro. 2004.
Surprisingly rapid growth in Neanderthals. Nature 428:
936–939.
Richards, M., V. Macaulay, E. Hickey et al. (37 co-authors). 2000.
Tracing European founder lineages in the Near Eastern
mtDNA pool. Am. J. Hum. Genet. 67:1251–1276.
Rightmire, G. P. 2001. Patterns of hominid evolution and dispersal
in the Middle Pleistocene. Quat. Int. 75:77–84.
Rosas, A., and E. Aguirre. 1999. Restos humanos neandertales
de la cueva del Sidrón, Piloña, Asturias. Nota preliminar.
Estud. Geol. 55:181–190.
Schmitz, R. W., D. Serre, G. Bonani, S. Feine, F. Hillgruber,
H. Krainitzki, S. Pääbo, and F. H. Smith. 2002. The Neandertal
type site revisited; interdisciplinary investigations of skeletal
remains from the Neander Valley, Germany. Proc. Natl. Acad.
Sci. USA 99:13342–13347.
Serre, D., M. Hofreiter, and S. Pääbo. 2004. Mutations induced by
ancient DNA extracts? Mol. Biol. Evol. 21:1463–1467.
Serre, D., A. Langaney, M. Chech, M. Teschler-Nicola, M.
Paunovic, P. Mennecier,M. Hofreiter, G. G. Possnert, and
S. Pääbo. 2004. No evidence of Neandertal mtDNA contribution to early modern humans. PLOS Biol. 2:313–317.
Tattersall, I., and J. H. Schwartz. 1999. Hominids and hybrids: the
place of Neanderthals in human evolution. Proc. Natl. Acad.
Sci. USA 96:7117–7119.
David Goldstein, Associate Editor
Accepted January 25, 2005