Gene 528 (2013) 27–32

Contents lists available at ScienceDirect

Gene
journal homepage: www.elsevier.com/locate/gene

Review

Evolutionary anthropology and genes: Investigating the genetics of human evolution

from excavated skeletal remains
Evilena Anastasiou, Piers D. Mitchell ⁎
Division of Biological Anthropology, Department of Archaeology and Anthropology, University of Cambridge, The Henry Wellcome Building, Fitzwilliam Street, Cambridge CB2 1QH, UK

a r t i c l e i n f o a b s t r a c t

Available online 18 June 2013 The development of molecular tools for the extraction, analysis and interpretation of DNA from the remains of
ancient organisms (paleogenetics) has revolutionised a range of disciplines as diverse as the fields of human
Keywords: evolution, bioarchaeology, epidemiology, microbiology, taxonomy and population genetics. The paper draws
Human evolution attention to some of the challenges associated with the extraction and interpretation of ancient DNA from
Paleogenetics archaeological material, and then reviews the influence of paleogenetics on the field of human evolution. It
Ancient DNA
discusses the main contributions of molecular studies to reconstructing the evolutionary and phylogenetic
Human remains
relationships between extinct hominins (human ancestors) and anatomically modern humans. It also explores
the evidence for evolutionary changes in the genetic structure of anatomically modern humans in recent
millennia. This breadth of research has led to discoveries that would never have been possible using traditional
approaches to human evolution.
© 2013 Elsevier B.V. All rights reserved.

Contents

1. Introduction to paleogenetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
2. Problems with the analysis of ancient DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
3. Human evolution and paleogenetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
3.1. Extinct hominins and aDNA studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
3.2. Anatomically modern humans and aDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

1. Introduction to paleogenetics
The origins of paleogenetics can be traced back to 1984, when a team
led by Higuchi (Higuchi et al., 1984) became the first to extract and clone
ancient DNA (aDNA) from the dried muscles of a 140 year old quagga, an
extinct species of zebra. A year later, Pääbo (1985), then a PhD student at
the University of Uppsala, reported the extraction of aDNA from the
mummy of an Egyptian child, dated to 2430 ± 120 BP. With these key
papers, the field of paleogenetics began. This review does not intend to
refer to every single publication in the field, but does attempt to discuss
Abbreviations: aDNA, ancient DNA; Asp, Aspartic Acid; bp, base pair; HVR, Hyper-
variable Region; NGS, Next Generation Sequencing; PCR, Polymerase Chain Reaction;
RFLP, Restriction Length Fragment Polymorphism.
⁎ Corresponding author.
E-mail address: pdm39@cam.ac.uk (P.D. Mitchell).
a representative selection of work to give an overview of how genetic ma-
terial can be extracted from excavated human remains, and what can be
deduced from the resulting DNA sequences.
Both of these early studies relied upon the extraction of aDNA
fragments and their cloning into a vector, which then allowed the
sequencing of the cloned fragments (O'Rourke et al., 2000). This
method however, was not without problems and thus, despite the
interest of the scientific community in the analysis of aDNA, the prog-
ress in the field of paleogenetics was hindered by the difficulties in
analysing the small, fragmented and degraded ancient DNA. Neverthe-
less, the development of the Polymerase Chain Reaction (PCR) by Kary
Mullis (Mullis and Faloona, 1987; Saiki et al., 1985) in the late 1980s
allowed the analysis of small quantities of DNA and provided a solid
methodological tool for the analysis of aDNA fragments. Indeed, since
1988 when Pääbo et al. (1988) employed PCR to extract ancient DNA
from the brain of a 7000 year old individual from Little Salt Spring in

0378-1119/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
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28 E. Anastasiou, P.D. Mitchell / Gene 528 (2013) 27–32
Florida, PCR became the cornerstone of the methodologies employed in
paleogenetics. This is because PCR permits the amplification of nucleo-
tide sequences from very small quantities of DNA, even if some copies
were degraded. Such an approach allows the copying of a specific frag-
ment of DNA in vitro, through the use of a DNA polymerase, a pair of
oligonucleotide primers and the four deoxynucleotides (Brown and
Brown, 1992;).
A further turning point in the development of ancient DNA as
applied to the fields of human evolution came in 1989 when Hagelberg
et al. (1989) demonstrated that DNA can be retrieved not only from
mummified remains but also from skeletal material. In contrast to mum-
mified remains that are only found in certain regions of the world due to
the natural environment or past embalming practices, skeletal remains
are much more abundant in archaeological contexts. Therefore, the
extraction of aDNA from skeletal remains allows ancient DNA studies
on a much wider temporal and geographical scale. Today, ancient DNA
can be extracted not only from skeletal and mummified remains but
also from formalin-fixed paraffin embedded tissues, from teeth, hair,
coprolites, soil, plants and seeds (Anastasiou and Mitchell, 2013;
O'Rourke et al., 2000).
The development of Next Generation Sequencing (NGS) in 2005
(Margulies et al., 2005) has revolutionised the field of ancient DNA
research even more than was the case for PCR. With the use of NGS
approaches the amount of available aDNA significantly increases,
allowing what has been termed the observation of evolution in real
time (Knapp and Hofreiter, 2010). The use of NGS has enabled the
reliable sequencing of complete genomes of extinct Pleistocene species
(Briggs et al., 2007), and thus the field of paleogenetics has now moved
beyond the limited focus of single genetic loci (e.g. mtDNA) to genome-
wide data (Stoneking and Krause, 2011). For example, the analysis of
the nuclear genome of the extinct woolly mammoth (Miller et al.,
2008), of the genome of Neanderthals (Green et al., 2008, 2010) and
of a Paleo-Eskimo (Rasmussen et al., 2010) became possible with the
advancement of Next Generation Sequencing methods. Next generation
sequencing or high-throughput approaches permit a more rapid
sequencing template production, since no targeted amplification steps
are now necessary. This is because the extracted DNA is turned into a
sequencing library by adding artificial adaptor sequences to both ends
of each DNA fragment (Stoneking and Krause, 2011). This in turn has
led to lower costs in DNA sequencing and for the extraction of complete
ancient genomes from less than 50 mg of ancient material, in compari-
son to almost 1 g of material needed when using previous methods
(Stoneking and Krause, 2011).
The use of NGS techniques has also allowed a more detailed identi-
fication of aDNA damage patterns, improving our understanding of
DNA diagenesis and optimising the available means for evaluating the
degree of human contamination of ancient remains (Ginolhac et al.,
2011; Green et al., 2006, 2008; Stoneking and Krause, 2011). This is
especially important when extracting anatomically modern human
DNA from ancient remains since NGS technologies allow us for the
first time to distinguish between ancient and present day DNA. In con-
clusion, NGS techniques make it increasingly more feasible to achieve
a far greater and quantifiable depth-of-coverage of highly accurate
sequence data from single aDNA target strands (Brotherton et al., 2007).
2. Problems with the analysis of ancient DNA
Before discussing the impact of paleogenetics on the field of
human evolution, it is necessary to consider the problems and method-
ological challenges associated with the extraction and amplification of
DNA from ancient remains.
One of the main difficulties that ancient DNA research has to over-
come is that the genetic material undergoes modification soon after
death, which results in its fragmentation and damage. The damage to
the structure of DNA includes single strand DNA breaks, base modifica-
tions due to oxidation and hydrolysis, and base loss (Cipollaro et al.,
2005). During hydrolysis (breakdown of bonds by water) the N-
glycosyl bond between the base and the sugar can break down and may
cause depurination (guanine and adenine removal) or depyrimidination
(cytosine or thymine removal) whilst during oxidation water derived
hydroxyl or superoxide radicals modify bases or distort the helix of the
DNA (Hofreiter et al., 2001; Marota and Rollo, 2002; O'Rourke et al.,
2000). The most common base modification in degraded aDNA is deam-
ination of cytosine to form uracil, which is read by some polymerases as
thymine, therefore causing C → T and G → A changes. This in turn pro-
duces artefacts during PCR.
Besides the problems introduced by the fragmentary nature of
aDNA, its analysis becomes more complicated by the presence of en-
zymatic inhibitors such as humic acids, tannins, fulvic acids and
Maillard products in the aDNA extracts (O'Rourke et al., 2000; Pääbo
et al., 2004). These inhibitors may interact with DNA (e.g. Taq polymer-
ase) and may result in false negative results.
Due to the heavily fragmented nature of ancient DNA, several
screening methods have been proposed to evaluate the level of aDNA
preservation prior the employment of any extraction method. The
level of degradation of nucleic acids can be predicted through the rate
of aspartic acid (Asp) racemisation (i.e. the conversion of the protein
L-amino acids into D-enantiomers), which is approximately equal to
that of DNA depurination (O'Rourke et al., 2000). Therefore, some labo-
ratories evaluate for DNA degradation based upon amino-acid analyses
to calculate the amount of amino-acids preserved in a specimen, to
evaluate the amino-acid composition and the racemisation extent of
several amino acids (Chelomina, 2006; Hofreiter et al., 2001; Marota
and Rollo, 2002; Poinar et al., 1996). Nevertheless, it must be noted
here that it is questionable whether racemisation is actually informative
of DNA preservation (Collins et al., 2009).
Interestingly, the degree of aDNA degradation correlates better with
the taphonomic and environmental conditions of the archaeological
stratum in which the remains were deposited, rather than with their
archaeological age (Allentoft et al., 2012; Donoghue et al., 2004; Gamba
et al., 2011). Chemical, biological and physical stress can affect the pres-
ervation of aDNA (Burger et al., 1999; Cipollaro et al., 2005; Lindahl,
1993), such that the degradation of the nucleic acids is slower under
anaerobic conditions, at low temperatures (ideally in permafrost), at
low acidity environments and in environments with no humidity
(Chelomina, 2006; Marota and Rollo, 2002). In particular, temperature
has been found to significantly affect preservation of aDNA, (Hoss et al.,
1996; Poinar et al., 1996). It has therefore been estimated that assuming
neutral pH and a temperature of 15 °C it would take about 100,000 years
for hydrolytic damage to degrade all DNA to such a degree that it would
become irretrievable, whilst certain conditions such as higher tempera-
tures significantly reduce this time limit (Hofreiter et al., 2001; Lindahl,
1993; Paabo and Wilson, 1991). As a result, despite the evidence that
DNA sequences have been extracted from a Siberian mammoth older
than 50,000 years old whose remains were preserved in permafrost
(Hagelberg et al., 1994; Hoss et al., 1994), some paleogeneticists doubt
that it is possible to retrieve aDNA from Egyptian remains even less
than 2000 years old (Gilbert et al., 2005a; Marota and Rollo, 2002). The
recent study of extinct moa skeletal remains from New Zealand suggests
that mtDNA may survive perhaps twice as long as nuclear DNA (Allentoft
et al., 2012).
Although multiple studies have been published reporting the
extraction and analysis of aDNA from Egyptian mummies (Crubézy
et al., 1998; Donoghue et al., 2010; Hawass et al., 2010; Nerlich et al.,
1997; Zink and Nerlich, 2003; Zink et al., 2001, 2003, 2006), many scep-
tics refute the possibility of extracting and amplifying DNA from ancient
material preserved in high temperature areas such as Egypt (Gilbert
et al., 2005b; Marota and Rollo, 2002; Marota et al., 2002). In this
ongoing debate (Marchant, 2011), the study by Marota et al. (2002)
on the preservation of aDNA from Egyptian papyri has a focal position,
since it demonstrates that the last DNA fragments on modern papyri
(0–100 years of age) will vanish within 500–700 years from when the
 E. Anastasiou, P.D. Mitchell / Gene 528 (2013) 27–32
papyri were manufactured (Marota and Rollo, 2002). Marota et al.
(2002, 2002) therefore argue that the persistent high temperatures in
Egypt are prohibitive for the preservation of aDNA and that their
study provides a solid argument against the reliability of studies that
recovered ancient DNA from Egyptian material.
Besides the degradation of ancient DNA, another major challenge
for studies of paleogenetics is the contamination of the aDNA by
extraneous genetic material. Every study that extracts and analyses
DNA from archaeological remains has to consider the possibility
that instead of extracting and amplifying ancient DNA, it may instead
be extracting modern DNA that has contaminated the ancient sample.
The possibility of contamination of the ancient material with modern
DNA is much higher when human aDNA is extracted due to the abun-
dance of modern human genetic material in the environment of labora-
tories and due to the difficulty in distinguishing it from the endogenous
DNA of ancient human remains (Hofreiter et al., 2001). Studies investi-
gating the aDNA from pathogens and microorganisms are also at poten-
tial risk of contamination, as Gilbert et al. (2005a) underline, since the
DNA of their modern counterparts might also be ubiquitous in the
environment.
Contamination of human remains with extraneous human DNA can
be introduced either at the time of death of the individual, due to the
handling of the body by others in the burial rituals, or during the exca-
vation and post excavation period (Brown and Brown, 1992). Even
though the former source of contamination (contamination prior to
excavation) cannot be controlled or eliminated, for the latter (contam-
ination during analysis) many guidelines have been published in order
to decrease or control for the possibility of contamination with modern
DNA. Over time these have evolved into a detailed and extensive list
of requirements (Handt et al., 1994a; Lindahl, 1993; Paabo, 1989),
standardised in the nine key criteria published by Cooper and Poinar
(2000).
Among the most frequently cited precautions for avoiding the
contamination of ancient DNA with modern, is the performance of
aDNA analyses in laboratories where no modern DNA is analysed.
Besides having a dedicated lab, the use of multiple negative controls
(i.e. tubes with no DNA that are processed as if they contained sample)
is an essential precaution, as the presence of amplified target in a nega-
tive control is evidence for contamination. The reproduction of the
results from a second, independent extract to assess damage and con-
tamination, as well as their reproduction in a second independent lab,
provides another robust way of demonstrating the authenticity of the
results. Additionally, due to the fragmentary nature of aDNA only
the amplification of short DNA segments is possible, any long ancient
DNA sequences must be treated as likely contamination (Cooper and
Poinar, 2000; O'Rourke et al., 2000; Hofreiter et al., 2001; Pääbo et al.,
2004).
3. Human evolution and paleogenetics
Human evolution can be studied by comparing DNA sequences from
modern samples and this approach is associated with fewer methodo-
logical pitfalls than study of DNA extracted from archaeological speci-
mens. However, modern sequences provide only indirect evidence for
the ancient processes that formed them (Hofreiter et al., 2001). In
contrast, study of ancient DNA offers a more direct insight into the
processes that drove the evolution of modern DNA sequences and thus
furthers our understanding of human evolution and human migration
(Soares et al., 2010).
In earlier studies of human evolution, the extraction and amplifi-
cation of mitochondrial DNA (mtDNA) had a central position due to its
unique characteristics. mtDNA is maternally inherited and thus does
not undergo recombination, whilst it is present in hundreds of copies
per cell, therefore increasing the possibility of its positive amplification
from archaeological specimens (Chelomina, 2006; Cipollaro et al., 2005;
O'Rourke et al., 2000). Furthermore, because mtDNA has a higher
29
mutation rate than nuclear DNA, it has accumulated a number of muta-
tions that reflect different radiating maternal lineages, which diverged
as humans colonised different geographical regions (Cipollaro et al.,
2005). By sequencing the mtDNA Hypervariable Region (HVR) I and II
and Restriction Fragment Length Polymorphism (RFLP) it has been
possible to define a number of mitochondrial haplogroups. These are
groups of similar haplotypes that share a common ancestor. Based
upon the analysis of the mtDNA of extant populations, the phylogeny
of anatomically modern humans has been demonstrated and the routes
and timing of the human migrations outside Africa has been estimated
in vertical-evolutionary studies (Cann et al., 1987; Dayton, 2003; Pusch
et al., 2003; Stringer and Andrews, 1988; Wallace et al., 1999).
Besides mtDNA, nuclear markers have also been screened in
paleogenetics (Filon et al., 1995; Zierdt et al., 1966) and methods for
the determination of sex of the human remains based on ancient
DNA have also been developed (Cipollaro et al., 1998; Hummel and
Herrmann, 1991; Ovchinnikov et al., 1998).
3.1. Extinct hominins and aDNA studies
The analysis of the mtDNA hypervariable sequences of the Feldhofer
Cave Neanderthal-type specimen by Krings et al. (1997, 1999, 2000)
holds a central position among paleogenetic studies that address ques-
tions on human evolution. In their analysis, Krings et al. (1997, 1999,
2000) demonstrated that the HVRI sequence of the Neanderthal speci-
men had an average number of 27 nucleotide differences, when com-
pared to a thousand contemporary human sequences. Moreover,
based on their mtDNA analysis, the authors concluded that the average
difference between the Neanderthal and the modern human sequence
is triple that observed in modern mitochondrial lineages and about
half of that seen between modern humans and chimpanzees (Krings
et al., 1997, 1999, 2000).
More information about the phylogenetic relationship between mod-
ern humans and Neanderthals derived from the study of Ovchinnikov
et al. (2000), who extracted DNA from a Neanderthal infant, excavated
in the Mezmaiskaya site in Northern Caucasus, dated to 39,700 ±
1100 BP (Pinhasi et al., 2011). The HVRI of the sample had twice
as many differences to modern human sequences as to the Feldhofer
Neanderthal. Therefore, the authors concluded that the two Neanderthal
sequences form a separate clade from the modern human HVRI, and
that the differences observed between the Mezmaiskaya and Feldhofer
individuals are equivalent to those observed among modern human
populations. Importantly, the results from the analysis carried out by
Ovchinnikov et al. (2000) and Krings et al. (1997) provided genetic
evidence to support the out of Africa model of human origins, since
they demonstrated that the Neanderthals were not directly ancestral
to modern humans.
By employing Sanger sequencing and parallel pyrosequencing
Noonan et al. (2006) were able to recover 65,250 bp material from the
genome sequence of the 38,000 year old Neanderthal examined by
Green et al. (2006). Two years later with the complete sequencing of
the mtDNA of the same Neanderthal, Green et al. (2008) proposed a di-
vergence time for mtDNA lineage of modern humans and Neanderthals
around 660,000 ± 140,000 years ago. Furthermore, in 2010 Burbano
et al. (2010), using targeted resequencing with hybridisation capture
on microassays, were able to sequence around 14,000 protein coding
positions, that are thought to have changed on the human lineage
after the divergence from the common ancestor with the chimpanzees
and to identify 88 amino acid substitutions that became fixed in humans
after their divergence from the Neanderthals.
Finally, the amplification of the draft sequence of the Neanderthal
genome in 2010, Green et al. (2010) provided evidence to address the
long-standing question of whether Neanderthals contributed parts of
their genome to anatomically modern humans through interbreeding.
Although research on the mtDNA of Neanderthals demonstrated that
their mtDNA genome falls outside of the variation of present-day
30 E. Anastasiou, P.D. Mitchell / Gene 528 (2013) 27–32
humans, the analysis of the whole Neanderthal genome suggests that 1–
4% of the genomes of people in Eurasia derived from the Neanderthals
(Green et al., 2010). Interestingly enough, even though Neanderthal
fossils have been found only in Europe and western Asia, the contribu-
tion of Neanderthals into the genome of Eurasian people is equal
between Europeans, Chinese and Papuans. This suggests that the gene
flow between Neanderthals and modern humans most likely happened
before the divergence of Europeans, Papuans and East Asians (Green
et al., 2010).
Besides examining the genome of the Neanderthals, paleogenetics
has also been employed in the analysis of the genetic material of the
Denisovans, a recently discovered hominin species. In 2008 the distal
phalanx of a hominin was uncovered at the Denisova Cave in the Altai
Mountains of Siberia, in a stratum dated to 50–100,000 BP and was
suggested to belong to a new hominin species (Krause et al., 2010).
In 2010, Reich et al. (2010) and Krause et al. (2010) used high-
throughput sequencing and primer extension capture respectively,
in order to determine the complete mtDNA genome from the phalanx
of the Denisova individual. The results of the genome analysis dem-
onstrated that the mtDNA of the Denisova individual diverged from
the common lineage leading to Neanderthals and modern humans
one million years ago, i.e. twice as early as the divergence between
Neanderthal and modern human mtDNA (Krause et al., 2010; Reich
et al., 2010). Nevertheless, the average divergence of the nuclear
genome of the Denisovans from present day humans is similar to that
of Neanderthals. Based on this observation Reich et al. (2010) concluded
that ‘the average divergence between the Neanderthals and the Denisova
individual is 9.84% of the way to the human–chimpanzee ancestor, which
is less than the 12.38% divergence of both from present-day Africans’
(Reich et al., 2010). Meyer's subsequence paper (Meyer et al., 2012)
argues that divergence between Denisovans and present-day humans
probably occurred between 700,000 and 170,000 years ago.
Furthermore, comparisons between the genomes of modern humans,
Neanderthals and of the Denisova individual demonstrated that the
Neanderthals were more closely related to the population that contrib-
uted to the genomes of the ancestors of modern Eurasians than the
Denisovas, but that 4.8% of the Melanesian genome derived from the
Denisovans (Reich et al., 2010). A year later, in order to explore further
the admixture between Denisovans and modern humans, Reich et al.
(2011) examined the Denisova admixture in 33 populations from Asia
and Oceania and found that admixture between Denisovans and mod-
ern humans occurred in the common ancestors of Australians, New
Guineans and Mamanwa (Philippines), but not to the ancestors of
Jehai (Malaysia) and Onge (Andaman Islands). This indicates that the
ancestors of modern day East Asians were not in Southeast Asia when
the Denisovas contributed to the gene flow of anatomically modern
humans. Based on this observation, the authors argue that the Denisova
gene flow must have occurred in Southeast Asia and not in mainland
Asia (Reich et al., 2011).
3.2. Anatomically modern humans and aDNA
Although the paleogenetic studies of extinct hominins provide
important information about our evolution and divergence from our
hominin ancestors, mtDNA and nuclear DNA studies on modern
human remains from archaeological strata are equally important. Stud-
ies of past human populations illuminate aspects of past migration
events and further our understanding of the paleophylogeography of
our species, providing direct evidence for events that explain present
day phylogeography. The Tyrolean Iceman (Otzi), dated to the Late
Neolithic (3350–3100 BC) has received considerable attention both in
archaeological and paleogenetic debate due to the unique preservation
of its soft tissues. Nevertheless, despite the excellent preservation of
Iceman's soft tissues, his DNA was severely degraded and no nuclear
amplifications were successful in early analysis in the 1990s. However,
the sequence variants of the HVRI region of the specimen were
extracted and amplified and found to be consistent with modern popu-
lations living north of the Alps (Handt et al., 1994b). More specifically,
the Iceman was found to be closely related to present day central and
eastern European populations. Whilst his DNA has regions homologous
with Europeans from the Alps, northern Europe and the Mediterranean
region, it has no regions homologous with Africans, Siberians or
Americans (Handt et al., 1994b). The complete sequencing of Otzi's
mtDNA was generated in 2008 by Ermini et al. (2008) and was found
to belong to the K1subhaplogroup, which is a subclade of the major
west Eurasia haplogroup U. Nevertheless, the mtDNA lineage of the
Iceman does not fit any of the known branches into which the K1
subhaplogroup is divided, but to a novel branch defined by transitions
at the 3513 and 8137 nucleotide positions, thus providing insights into
the genetic changes that happened in Europe in the last 6000 years
(Ermini et al., 2008). With modern whole genome sequencing technol-
ogy, the entire genome has recently been published (Keller et al., 2012).
This has shown that he probably had brown eyes, was lactose intolerant,
blood group O and was closely related to modern inhabitants of the
Tyrrhenian Sea region.
Beside the Iceman, the complete mtDNA genome of a Paleo-Eskimo
dated to 4500–3400 BP was characterised by Gilbert et al. (2008), using
a pyrosequencing approach. Their study demonstrated that the mtDNA
lineage of the Paleo-Eskimo belongs into the D2a1 haplogroup and was
distinct from that of the Neo-Eskimos and modern Native Americans.
The results from the analysis of the mtDNA of the Iceman and Paleo-
Eskimo demonstrate the value of complete ancient mtDNA sequencing
in illuminating aspects of phylogeography and population genetic
histories that are not accessible through the analysis of modern samples
(Ermini et al., 2008).
Paleogenetic studies of multiple members of ancient populations
of anatomically modern humans have also been carried out. One of
the first studies to use molecular tools to examine the genetic history
of a past population was carried out by Pääbo (1985) using 23 Egyptian
mummies dating from the Fourth dynasty (4500–4000 BP) to the late
Roman period. Pääbo cloned a region of the AluI sequence, which had
a 77% homology to the sequence exhibited in modern populations.
However, this study has been criticized due to concerns about the
survivability of DNA in warm temperatures as is found in Egypt
(Marota and Rollo, 2002).
In America, Stone and Stoneking (1998, 1999) assessed the mtDNA
haplogroup diversity of skeletons that belonged in the Oneota archaeo-
logical complex of western Illinois, Kaestle (1997) characterised the
mtDNA haplogroup of skeletal samples (6000–300 BP) from the
Pyramid Lake and Stillwater Marsh in the Western Great Basin, whilst
Parr et al. (1996) assessed the variation in the Northern Fremont of
Utah (2000–1000 BP) and Carlyle et al. (2000) studied the haplogroup
variation in Anasazi of the US southwest (2000–1000 BP). These studies
demonstrated that the examined specimens exhibited the same
geographic structure as that seen in modern North Amerindian mtDNA
variation, which is strongly geographically patterned (O'Rourke et al.,
2000).
Interestingly, the geographic and temporal structure that was noted
in the mtDNA studies in North America does not seem to extend to
Central and South America. Merriwether et al. (1994) examined the
haplogroup diversity of the Chinchorro and Inca of Northern Chile and
the Copan Maya of Honduras, and found that the deletion marker was
absent from the Chilean samples although it is frequently found in the
modern populations of the region. Furthermore, they observed that
although modern Mayans have high frequencies of haplogroups A and
B, the Copan Mayan samples were of haplogroups C or D.
In order to demonstrate the patterns of genetic variation in prehis-
toric China, Oota et al. (1999) analysed fifty-eight 2000 year old dental
and bone samples, from the Yixi site in Shandong Province, China and
obtained data for the HRVI and II. When compared to a variety of
modern Asian sequences, their data indicate closest similarities
between the ancient Yixi samples and the modern Taiwan Han Chinese.
 E. Anastasiou, P.D. Mitchell / Gene 528 (2013) 27–32
Wang et al.(2000) studied the genetic structure of 19 human popula-
tions dated to 2500 and 2000 BP from Linzi, Shandong province. Frag-
ments of the mtDNA of 63 individuals were obtained. Comparative
analysis with present-day samples demonstrated that the 2500 BP
samples had a greater similarity with modern European than with
modern East Asian populations, whilst the 2000 BP samples had charac-
teristics that placed them between modern East Asian and European
populations. The authors concluded that these results demonstrate a
change in the genetics of the Chinese in a spatiotemporal scale that
can be attributed in part to the historical events during the Qi Qin and
Han dynasties (Wang et al., 2000).
A study of early farmers in Europe compared their mtDNA with
those of hunter-gatherers and modern people from the same region
(Bramanti et al., 2009). There were large genetic differences between
each time period, interpreted as indicating considerable migration
between each period under study. This suggests that Europe's early
farmers were not the descendants of European hunter-gatherers who
had learned to farm, but they were migrants from elsewhere who
moved to Europe to plant the fertile land.
4. Conclusions
This paper has brought together the discoveries of many of the key
paleogenetic research projects undertaken in recent years in order
to assess the impact of this emerging field. This demonstrates the
degree to which paleogenetics can contribute to our understanding
of human evolution, whilst also highlighting some of the methodo-
logical challenges faced by those who extract and interpret ancient
DNA.
The analysis of the genetic material of extinct Homo species, as
well as of ancient anatomically modern humans populations, has
revolutionised the field of human evolution. Although important
information about the biological relationship of different Homo species
can be inferred through the study of the fossil record, paleogenetics
provides direct information regarding the evolution and phylogenetic
relationship of different hominins, and provides the methodological
tools to address detailed questions about the origins and evolutionary
adaptive strategies of our ancestors. For example, the extraction and
analysis of genetic information from the Neanderthals and the
Denisovan individual, allowed the reconstruction of their phylogenetic
relationship with modern humans. This has in turn furthered under-
standing of our biological evolution and provided the first evidence
with which to address the long-standing question of how much inter-
breeding there may have been between different Homo species in the
past.
This evidence has been complimented by paleogenetic studies of
ancient remains of anatomically modern humans, further illuminating
aspects of our more recent evolution. Analysis of the genetic differences
shown by modern populations can only tell us some elements of how
the modern populations in different regions of our planet came to
exist as they do. The paleophylogeographical distribution of different
human populations and the association with modern phylogeography,
as well as the contribution of past migration events to the genetics
of modern populations are all aspects of our evolution that become
apparent through the analysis of the mtDNA and nuclear DNA of past
populations. Whilst those undertaking the analysis of ancient DNA
have to face many different methodological challenges, paleogenetic
studies provide one of the most powerful and most precise tools with
which to address questions about our biological evolution. Some of
the most exciting areas for future work we may see over the next
decade include determining the genome of other hominin species, and
further work with archaeological remains to quantify the degree to
which we may have evolved in the last 10,000 years due to the change
in lifestyle from hunter-gatherers to farmers, industrialists, and finally
technologists.
31
Conflict of interest
No conflict of interest.
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