Biomolecular Archaeology

Author(s): Keri A. Brown and Terence A. Brown

Source: Annual Review of Anthropology , 2013, Vol. 42 (2013), pp. 159-174
Published by: Annual Reviews

Stable URL: http://www.jstor.com/stable/43049296

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Annu. Rev. Anthropol. 2013. 42:159-74
First published online as a Review in Advance on
July 24, 2013
The Annual Review of Anthropology is online at
anthro.annualreviews.org
This article's doi:
10.11 46/annurev-anthro-092 412-155455
Copyright © 2013 by Annual Reviews.
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Biomolecular Archaeology*
Keri A. Brown and Terence A. Brown
Manchester Institute of Biotechnology, Faculty of Life Sciences, University of Manchester,
Manchester Ml 7DN, United Kingdom; email: keri.brown@manchester.ac.uk,
terry.brown@manchester.ac.uk
Keywords
ancient DNA, ancient lipid, ancient protein, human skeletons, paleodiet,
paleopathology
Abstract
Ancient biomolecules including DNA, proteins, and lipids are often pre-
served in archaeological skeletons or artifacts such as potsherds from cook-
ing vessels. Techniques for analyzing these molecules have improved dra-
matically in recent years, though challenges remain in ensuring that results
are authentic and not confused by the presence of contaminating modern
biomolecules. Ancient DNA (aDNA) can be used to identify the sex, kinship
relationships, and population affinities of human skeletons, and also to detect
the presence of disease-causing organisms such as the plague and tuberculosis
bacteria. Stable isotope ratios in collagen and other skeletal proteins enable
past diets to be studied, and similar work with lipids from potsherds have
revealed that dairying in Europe began 2,000 years earlier than previously
thought. Biomolecular archaeology has therefore developed into a mature
discipline that is making a significant contribution to different aspects of our
understanding of the human past.
l59
 INTRODUCTION

Biomolecular archaeology is the study of the ancient biomolecules that are sometim
Ancient biomolecule: in the archaeological record. Three types of biomolecules are particularly import
a biomolecule is DNA, which is present in many human and animal skeletons and can also be re
preserved in ancient various types of plant remains and from sediments. DNA specifies the biological cha
biological material
living organisms, so analysis of ancient DNA (aDNA) can reveal some of the charact
aDNA: ancient DNA
archaeological specimen, such as the sex of a human skeleton. DNA is also a record o
and so can be used to deduce whether two human skeletons are related, to study the
affinities between different hominins, and to explore the relationships between dom
mals and plants and their wild progenitors. DNA of different species can be distinguish
DNA from a pathogen such as Mycobacterium tuberculosis to be detected in human b
Protein is the second type of biomolecule that is important in archaeology. Prote
same way as DNA, specify biological characteristics and can be used to map evolutiona
but in biomolecular archaeology proteins are more commonly used as biomarkers. E
casein, which is found only in milk and can therefore be used to detect milk resid
or storage vessels, and hemoglobin, which is a biomarker for blood. Lipids, the
biomolecule, are also used as biomarkers. Their analysis in residues from cookin
identify the type of food that was being prepared, and similar studies of storage ve
whether these were used to hold, for example, a particular type of oil.
The purpose of this review is threefold: first, to summarize the analytical tec
to study ancient biomolecules; second, to consider the technical challenges of bio
chaeology; and third, to explore the contributions that biomolecular studies are m
examination of human archaeological remains, paleopathology, and studies of past d

STUDYING ANCIENT BIOMOLECULES

Biomolecular archaeology is one of a related set of disciplines that make use of an

biomolecules. Forensic scientists use information from aDNA in samples such as hair and
stains, collected at crime scenes years or decades ago, to solve what are popularly called co
(Marks 2009). Zoologists use aDNA and proteins from animal fossils to study the taxonom
tinct species (Huynen et al. 2012) and to reconstruct paleoecologies (de Bruyn et al. 20 1 1 , H
et al. 2012). Organic geochemists have detected the remains of plant lipids in sediments
tens of millions of years old (Speelman et al. 2009). This breadth is important because it
to a cross-fertilization of ideas between researchers in different disciplines, which has con
greatly to the development of the technical strategies used in biomolecular archaeology.

DNA

A DNA molecule is a linear, unbranched polymer made up of four chemically distinct nucleotides
that can be linked together in any order to form chains hundreds, thousands, or millions of units
in length. The biological information in a DNA molecule is specified by its nucleotide sequence,
which is interpreted as a series of As, Cs, Gs, and Ts, the abbreviations of the chemical names of the
nucleotides. DNA sequencing methodology has undergone dramatic improvement since it first
became available in the 1970s, but it is still not possible to read more than 1,000 nucleotides in a
single experiment (Brown 2010). This is a hindrance to the study of DNA from living organisms,
as most of the molecules are much longer than 1,000 nucleotides, but is less of a problem in
biomolecular archaeology because the natural decay processes mean that aDNA molecules are

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rarely more than 150 nucleotides. The technological advances in DNA sequencing have focused
on automated procedures that enable many experiments to be performed in parallel, the most
recent next-generation sequencing (NGS) methods producing literally millions of sequence reads
Next-generation
in a few hours (Knapp & Hofreiter 2010). Bioinformatics is then used to identify overlaps between sequencing (NGS):
these reads, to build up the contiguous sequence of the original DNA molecule. This is a massive recendy developed
methods for DNA
challenge when the genome of a species is being sequenced for the first time, but is less daunting
sequencing that enable
if the genome sequence is already known and can be used as a reference to direct assembly of the
millions of short
reads. This is the strategy that enables a personal human genome, which comprises 3.2 billion
sequences to be
nucleotides, to be sequenced for as little as $1,000 (Stähl & Lundeberg 2012), and it equally can obtained in a single
be applied to aDNA to identify the personal genome variations in an archaeological specimen experiment
(Rasmussen et al. 2010, 2011; Keller et al. 2012). But for many archaeological applications, such Polymerase chain
extensive sequencing is overkill, as the required information can be obtained from small regions of a reaction (PCR):
genome, perhaps just a few hundred or thousand nucleotides in length. Traditionally, these short technique that results
in exponential
regions have been accessed by the polymerase chain reaction (PCR), which generates multiple
amplification of a
copies of a targeted region within the small quantities of aDNA extracted from an archaeological
selected region of a
sample (Brown 2010). This amplified segment is then sequenced in isolation from the rest of the DNA molecule
genome. PCR underpinned aDNA research until the mid-2000s, and the majority of projects still
use this approach (Brown & Brown 2011). Its utility is limited by the short lengths of aDNA
molecules, which means that a single PCR can generate only ~100 nucleotides of sequence.
Sequencing longer regions requires multiple overlapping PCRs, which may not be possible if
the aDNA is in short supply, which is almost always the case. A more recent approach involves
direct capture of the interesting aDNA fragments (Knapp & Hofreiter 2010), which are then
sequenced by one of the NGS methods, providing reads specifically from the informative regions
of a genome.
The dynamics of DNA degradation in archaeological remains have never been fully studied
(Overballe-Petersen et al. 2012). It is presumed that there is an initial period of rapid breakdown
immediately after death, due to entry of degradative enzymes and reactive chemicals into the
cell nucleus, followed by a slower period of more gradual diagenesis within the archaeological
specimen (Lindahl 1993). There have been attempts to relate the rate of this gradual breakdown
to the microclimate within which a specimen resides (Smith et al. 2007), but the variables, including
temperature, water and oxygen availability, and microbial activity, are probably complex (Allentoft
et al. 2012). It is also clear that DNA decay in bones accelerates after excavation of a specimen
(Pruvost et al. 2007), possibly due to resumption of microbial activity stimulated by the unfortunate
habit of washing bones after they are taken out of the ground. Predicting how long DNA can
survive is therefore difficult, but based on observations, the timespan for bones and teeth is probably
>50,000 years in permafrost (Schwarz et al. 2009), and similar ages might be possible for specimens
in cool environments such as caves (Noonan et al. 2005). aDNA has also been sequenced from
hair (Rasmussen et al. 2010), natural and artificial mummies (Marchant 201 1, Keller et al. 2012),
desiccated and charred plant remains (Allaby et al. 1994, O'Donoghue et al. 1996, Bunning et al.
2012, Palmer et al. 2012), coprolites (Gilbert et al. 2008), and sediments from archaeological sites
(Hebsgaard et al. 2009). With most of these nonskeletal sources, the timespan for survival is likely
to be similar to that of bones and teeth.

Proteins

Proteins are linear unbranched polymers made up of subunits called amino acids. The polymers,
or polypeptides, can be up to 2,000 units in length. There are 20 different amino acids and hence
a great variety of possible protein structures. Methods for sequencing the amino acids in a protein

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 were developed in the 1950s, but until recently the procedures were cumbersome and required
pure samples of individual proteins (Steen & Mann 2004), which can be difficult to achieve
with an ancient sample. Less direct methods of protein identification based on immunological
procedures have therefore been used. Proteins were, in fact, the first type of biomolecule studied
by archaeologists, back in the 1930s, when blood-typing methods were applied to Egyptian and
Native American mummies (Boyd & Boyd 1937). It was quickly realized that these immunological
methods, which can detect individual proteins with high specificity when modern samples are
used, lack accuracy if the proteins are partially degraded, as might be expected for ancient material
(Downs & Lowenstein 1995). In one study, blood samples were coated onto artifacts and attempts
made to deduce the species of origin using the most sophisticated modern immunoassays. Only 20
of 54 samples were correctly identified, with many of the misidentifications being positive identifi-
cation of blood protein from a different species (Leach & Mauldin 1995). Despite these problems
with immunological detection, especially of supposed blood residues, the results of uncontrolled
studies still find their way into the popular press and sometimes even the scientific literature.
In recent years, molecular biologists have devised numerous new methods for studying pro-
teomes, the complex collections of proteins present in living cells. The term paleoproteomics is
Mass spectrometry used to describe the application of these techniques in biomolecular archaeology. One approach
(MS): analytical
involves separation of the proteins in an ancient extract by electrophoresis or chromatography,
technique in which
ions are separated followed by identification of individual ones by mass spectrometry (MS) (Cappellini et al. 2012).
according to their The identification step is preceded by digestion with an enzyme such as trypsin, which cleaves
charge-to-mass ratios the polypeptide immediately after arginine or lysine amino acids. With most proteins, this results
in peptides 5-75 amino acids in length. The MS determines the mass of each of these peptides,
which is then compared with databases of all known protein structures to identify the particular
protein that has been isolated (Brown 2010). A modification of particular importance in zoology
is Zoo-MS, which focuses primarily on the bone protein collagen, and detects small changes in
protein structure that are characteristic of different species (Buckley et al. 2010). As well as identi-
fying animal bone fragments from archaeological sites, Zoo-MS can be used in taxonomie studies,
including ones involving extinct species (Buckley 2013).
Additional information can be obtained by studying the ratios of nonradioactive, stable isotopes
of carbon and nitrogen in protein extracts. Stable isotopes coexist in nature, but not in equal
amounts (Hoefs 2009). Usually, the lightest isotope is predominant and the other(s) present in
smaller amounts. This is the case with the carbon isotope 12 C, which comprises 98.93% of all the
carbon atoms in existence, and 13 C, which makes up just 1.07%. Although different isotopes of the
same element have identical chemical properties, their masses influence the ways in which they
behave during physical and chemical processes. These effects can lead to isotope fractionation,
which results in the relative proportions of the isotopes in the products of a reaction being different
from the proportions in the starting material. Isotope fractionation occurs during some biological
reactions, which means that measurements of isotope ratios in bone collagen, expressed as 6 13 C
and 615N values, can reveal certain aspects of diet (Lee-Thorp 2008). For example, the amount of
meat in a human diet can be estimated because carnivory is a higher point on the food chain than
herbivory. Fractionation occurs at each step in the chain, so a high-meat diet results in higher
613C and 615N values in skeletal protein (Bocherens & Drucker 2003).

Lipids
Lipids are a broad group of compounds that include fats, oils, waxes, steroids, and various resins.
They have diverse structures but most are relatively resistant to degradation. Organic geochemists
first recognized the ability of some lipids to survive with little structural change for long periods,

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and in the 1970s attempts were made to study ancient lipids in archaeological contexts. This work
suffered initially from the application of techniques that were not entirely suitable for analysis of the
small amounts of material that were available, but the field matured in the early 1990s, when high-
Gas chromatograp
resolution separation by gas chromatography (GC) was linked to accurate identification methods (GC): method for
based on MS (Regert et al. 2003). Modern GC-MS can detect as little as 1 pg of lipid, meaning separating compoun
that the residues in ceramics and other archaeological materials can be analyzed. Few of these according to their
differential
molecules are fully stable, so detection depends on the identification of breakdown products. A
partitioning between a
knowledge of how different lipids degrade is therefore essential for effective use of these compounds
carrier gas and a liquid
in biomolecular archaeology (Evershed 2008). stationary phase
Lipids are useful biomarkers because many are specific to a single or small group of species.
For example, vegetables such as Brassicas have characteristic leaf waxes, which can sometimes be
detected in potsherds, identifying vessels used to cook these vegetables (Evershed et al. 1991). In
some applications, additional specificity is possible if 6 13 C values are measured in individual lipid
molecules. For example, most animals synthesize the same lipids in their muscles and milk, but by
different biochemical pathways that result in different degrees of isotopie fractionation (Copley
et al. 2003). Measurements of 613C in ceramic residues can therefore identify whether these derive
from dairy products (Evershed et al. 2008).

THE TECHNICAL CHALLENGES OF BIOMOLECULAR

ARCHAEOLOGY

All areas of research present a technical challenge and biomolecular archaeology is no dif
in this regard. The study of ancient biomolecules has, however, thrown up a greater than
number of controversies. Some have been prompted by a desire to discover the oldest pr
biomolecules, as was the case with aDNA in the early 1990s. During this period, there were
reports of DNA in > 1 -Ma-old fossils (Golenberg et al. 1990, Cano et al. 1993, Poinar et al
Woodward et al. 1994), despite a clear realization that the kinetics of DNA decay provided
opportunity for survival of intact polynucleotides in specimens of more than ~ 100,000 y
age (Lindahl 1993). The quest for the oldest in the world has been a peculiar feature of
biomolecules research and not restricted just to DNA. There have been reports of pr
proteins in dinosaur bones, which have been heavily criticized as being unproven and
energetically defended as being authentic (Asara et al. 2007, Buckley et al. 2008).
The multimillion-year-old DNA sequences are generally perceived as artifacts resulting
contamination with modern EÌNA. Contamination presents a major problem when PCR is
amplify aDNA prior to sequencing. An undegraded modern molecule will act as a better t
for PCR than will a partially degraded ancient one, so the resulting amplification product
to be dominated by copies of the contaminant, even if there are very few of these in the
Humans leave their DNA everywhere, including on the surfaces of bones when handled w
gloves (Bouwman et al. 2006), which is still frequently the case despite longstanding attem
biomolecular archaeologists to educate their colleagues not to contaminate their specimens
& Brown 1992). Contamination is also possible within the laboratory, if care is not taken to p
PCR products from one experiment from being transferred to a second PCR, via aerosols gen
by opening the flip-caps of the plastic tubes in which these PCRs are performed (Tetzne
The problems posed by contamination were first recognized in the 1990s when mo
more incredible claims of multimillion-year-old DNA were being made (Stoneking 1995).
aimed at minimizing contamination and enabling it to be recognized were established
& Poinar 2000), based around physically separated work areas, including dedicated cle
used only for handling aDNA extracts, accompanied by a full suite of negative controls

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 as other controls aimed at assessing the degree of general biomolecular preservation within a
specimen. Unfortunately, only a minority of aDNA projects have attempted to meet all of the
established criteria (Bandelt 2005, Roberts & Ingham 2008). In some cases, this is understandable:
For example, biomolecular preservation cannot be assessed if only limited amounts of material
are available for study. Worryingly, though, there is often an assumption that these measures are
really necessary only when studying human specimens. This assumption is based on the mistaken
belief that handling is the only cause of contamination, whereas in reality PCR crossover is equally
likely and more difficult to detect (Wilbur et al. 2009).
The recent introduction of NGS has provided an alternative to the conventional PCR approach
to aDNA sequencing. As the PCR step underlies the contamination problem, it is believed by some
that a complete switch to NGS methods will solve the contamination issues. This is a dangerous
assumption because it is possible that the capture methods used to preselect interesting aDNA
molecules will preferentially select undamaged modern contaminants. Unfortunately, even though
aDNA research is about to celebrate its thirtieth anniversary (Higuchi et al. 1984), the problems
posed by contamination have by no means been solved, and many biomolecular archaeologists
remain skeptical of the authenticity of much of the work that is published. The onus remains on
authors to describe not only their results, but also the reasons why they believe their results to be
genuine, either because of strict adherence to the accepted criteria of authenticity or through use
of some other self-critical approach (Gilbert et al. 2005).

APPLICATIONS OF BIOMOLECULAR ARCHAEOLOGY

Biomolecular archaeology is an interdisciplinary subject, and biomolecular research is of no

unless it is carried out within an archaeological context. This may seem obvious, but project
reach high biomolecular standards sometimes fail to interest archaeologists because the resu
not relevant to the issues that they deem important. The problem arises because until recently
few biomolecular scientists possessed more than a rudimentary understanding of archaeolog
few archaeologists had substantial training in the biomolecular sciences. Successful biomole
archaeology has therefore required collaboration between archaeologists and biomolecul
entists. Here, we review some of the areas in which biomolecular studies have been prod
within archaeology as a whole.

Examination of Human Archaeological Remains

Probably the most important application of aDNA in archaeology is in the study of human rem
typically bones or teeth recovered during excavation. Because of the difficulty in distingu
genuine human aDNA from modern contamination, this is the most challenging area of bio
ular archaeology. If the work is done competently then it is possible to identify the sex of a ske
assess the kinship and broader population relationships between groups of skeletons, and e
the genomes of extinct hominins.
Sex identification is the most fundamental contribution that biomolecular techniques can
in studies of human remains (Brown 1998). Sex can sometimes be identified from the struc
a skeleton, but this requires that the skeleton is reasonably complete and the individual con
did not die before puberty (Mays & Cox 2000). If the osteology is inconclusive, then aDNA an
is the only alternative. The simplest DNA tests for sex identification use PCRs that are di
specifically at the Y chromosome (Lo et al. 1990); these tests are relatively easy to design b
the X and Y chromosomes are quite different. With modern DNA, for example in a prenatal
(Devaney et al. 2011), a Y-specific PCR is sufficient for unambiguous sex identification,
positive result indicating male and a negative indicating female. With aDNA there is a possibilit

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negatives occurring because of experimental failure - there is no aDNA in an extract or the DNA
is too damaged to amplify. The standard aDNA test therefore targets a sequence that is present
on both the X and Y chromosomes, but in slightly different forms. This is the amelogenin gene,
which codes for a protein that is involved in the deposition of enamel during tooth development.
In the standard test, the PCR product from the X chromosome is 106 nucleotides and that from
the Y is 1 12 nucleotides (Sullivan et al. 1993). Sex can therefore be identified simply by examining
the sizes of the PCR products. The process has been miniaturized and can be carried out using
lab-on-a-chip technology (Parton et al. 2013), which could form the basis for a portable system
for on-site use at archaeological excavations.
In addition to its value in cataloging archaeological sites, DNA-based sex identification has
been particularly useful in studies of infanticide, which of necessity involve infant remains that
are difficult to sex by osteological methods (Mays 1993, Mays & Faerman 2001). An interesting
example concerns a Roman bathhouse at Ashkelon in Israel, dating back to the fourth-sixth
centuries AD (Faerman et al. 1998). A sewer under this building contained the remains of 100
newborn babies, most less than two days old. Amelogenin PCRs were successful with 19 of 43
skeletons that were tested, 14 of these being male and 5 female. This skewed ratio was interpreted
as sex-specific infanticide. However, it is generally assumed that if infanticide were practiced in
the ancient world then female babies were more likely to be killed, because of the greater value
placed on males and the need to pay a dowry when a daughter married (Pomeroy 1983). The male
bias at Ashkelon is explained by the babies being the offspring of prostitutes who worked at the
baths, for whom there would have been more economic sense in keeping the female infants who
might subsequently take up their mothers' careers or be sold as slaves.
Kinship relationships are notoriously difficult to assign by osteological methods (Tyrrell 2000),
and here aDNA analysis can make a significant novel contribution to site interpretation. Male- and
female-specific lineages can be compared by sequencing parts of the Y chromosome and mitochon-
drial DNA (mtDNA), respectively. The latter is a DNA molecule that resides not in the nucleus but
in the mitochondria of a cell (Brown 2006). Because only the nucleus of a spermatozoon passes into
an egg cell during fertilization, the father's mtDNA is not transmitted to his offspring, who inherit
this part of their genome only from their mother. More general family relationships are examined
by typing sequences called short tandem repeats, which are also used by forensic scientists to con-
struct genetic profiles (Jobling & Gill 2004). Pioneering kinship studies using aDNA included the
identification of the remains of Tsar Nicholas II and his family (Gill et al. 1994, Coble et al. 2009);
untangling the relationships between seven generations of the Earls of Königsfeld interred in St.
Margaretha^ Church in Reichersdorf, Lower Bavaria during the sixteenth-eighteenth centuries
(Gerstenberger et al. 1999); and interpretation of the burial arrangements within a pioneer ceme-
tery in Durham, Upper Ontario (Dudar et al. 2003). DNA studies have revealed what are claimed
to be the oldest family groups identified in prehistory, from a 4,600-year-old cemetery near Eulau,
central Germany (Haak et al. 2008). The strontium isotopes 87 Sr and 86 Sr were also measured in
the teeth of these individuals. The ratio between these isotopes reflects the underlying geology of
the place where a person spends his or her childhood, when strontium from the diet is incorpo-
rated into the developing adult teeth (Bentley 2006). The adult males and all of the children had
strontium signatures similar to those in the sediments surrounding the graves, but three of the
adult females had ratios more typical of the Harz Mountains, 60 km from Eulau. The implication
is that in this society marriages were exogamous and patrilocal, and in the case of these particular
burials, some of the women moved from the Harz Mountains to join their husbands' community
(see sidebar, The Cladh Hallan Mummies, for another use of aDNA to study kinship; Figure 1).
MtDNA can similarly be used to identify groups of burials that do not have family relationships,
at least in the maternal line. Such was the conclusion for skeletons retrieved during excavation of

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THE CLADH HALLAN MUMMIES

Excavations at Cladh Hallan, a Bronze Age-Iron Age settlement on South Uist in the Outer Hebrides off the w
coast of Scotland, revealed the skeletons of two adults, a subadult, and a child buried beneath the foundation
three roundhouses. The bone microstructure of the two adults suggested that the bodies had been mumm
shortly after death, probably by immersion in a peat bog for a few months, before being dried and kept ab
ground. Osteological and isotopie evidence showed that the male adult skeleton was a composite of parts of a
three different individuals. To test the hypothesis that the female skeleton (Figure 1) was also a composite,
was sequenced from the skull, mandible, right humerus, and right femur. The results showed that the man
humerus, and femur came from different individuals (Hanna et al. 2012). Insufficient data were obtained to
conclusions regarding the origin of the skull. The presence of two composite skeletons at Cladh Hallan m
indicate a deliberate merging of identities, perhaps designed to amalgamate different ancestries into a single li

three houses in Xaltocan, Mexico, dating to 1 240-1 52 1 AD. A clear difference was revealed between
the mtDNA lineages present before and after the Aztec annexation of the Xaltocan city-state dur-
ing the fifteenth century (Mata-Miguez et al. 2012). The results indicate that the expansion of the
Aztec people into Xaltocan displaced the previous Otomi population, contrary to archaeological
evidence suggesting that some of the original inhabitants remained in the settlement.
aDNA sequences can also provide information on the broader population affinities of hu-
man skeletons. The genetics of many modern populations correlate with geography (Sokal 1988,
Novembre et al. 2008, Wang et al. 2012), suggesting that the population structure reflects past
migrations (Oppenheimer 2012, Pinhasi et al. 2012). Studies of aDNA are particularly informa-
tive with regards to the Mesolithic-Neolithic transition in Europe, revealing a genetic distinction
between late hunter-gatherer and early farming populations (Bramanti et al. 2009, Malmström
et al. 2009, Gamba et al. 2012, Sánchez-Quinto et al. 2012, Skoglund et al. 2012), consistent with
the hypothesis that the first farmers in Europe were migrants from the southeast (Rowley-Conwy
2009). This research benefits greatly from the use of NGS to obtain complete or near-complete
genome sequences. The genomes of the Tyrolean iceman (Keller et al. 2012), a 4,500-year-old
Paleo-Eskimo (Rasmussen et al. 2010), and an aboriginal Australian from the early twentieth cen-
tury (Rasmussen et al. 2011) have been completely sequenced, and partial sequences are known
for four Neolithic Scandinavians (Skoglund et al. 2012). These sequences represent several orders
of magnitude more information than has previously been accessible, and hence provide much
greater detail to studies of population relationships. The Australian sequence, for example, even
though from a single person, stimulated a reappraisal of the migrations leading to the initial colo-
nization of Australasia (Rasmussen et al. 201 1). Complete genome sequences are likely to increase
in number in exponential fashion in coming years, and our understanding of prehistoric human
demographics will undoubtedly undergo a revolution of equivalent dimensions. This is already
happening with regards to the deeper aspects of human evolution. A new form of hominin - the
Denisovans - was discovered by aDNA analysis of morphologically uninformative bone fragments
(Krause et al. 2010). The Denisovan genome séquence suggests that this hominin is a sister group
of Neanderthals, and a comparison of the Denisovan, Neanderthal, and modern human genomes
has revealed unsuspected admixture between the three types (Meyer et al. 2012).

Paleopathology
Biomolecular archaeology offers possibilities for studying both genetic and infectious disease.
Genetic diseases are caused by mutations in the human genome and as such can be detected by

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Figure 1
The adult female skeleton from Cladh Hallan. She was placed on her left side, with her left knee (upper tibia
and lower femur) buried separately from her body in a pit associated with a cremation pyre, and her upper
lateral incisor teeth placed in each hand, the left in the left hand and the right in the right hand. Reprinted
from Hanna et al. 2012, with permission from Elsevier.

sequencing aDNA from the mutation-containing regions. An example is thalassemia, the inherited
form of anemia or iron deficiency. Both inherited and dietary anemia result in porotic hyperostosis,
a pitting of the skull bones around the eye sockets (cribra orbitalia) and thickening of other bones
(Oxenham & Cavili 2010). These skeletal features are common in some groups of human remains,
but whether they result from dietary or genetic anemia cannot be determined by osteological
examination (Lagia et al. 2007). aDNA could answer this question by showing whether mutations
in the globin genes, responsible for thalassemia, are present in these individuals. This approach
was used with the skeleton of an eight-year-old child from an Ottoman grave at Akhziv, Israel,
dating back to the sixteenth-nineteenth century AD (Filon et al. 1995). Detection of a mutation

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 for ß -thalassemia showed that the child suffered from genetic anemia. This particular defect was
so severe that the child should not have survived long after birth, but other features of the aDNA
sequence suggested that a second globin mutation was also present, this one resulting in the fetal
version of hemoglobin being synthesized for an unusually long time after birth.
Any genetic disease could be typed by aDNA sequencing, but progress has been limited because
of the huge number of possible disease-causing mutations and the need to perform a separate PCR
to test for each one. The introduction of NGS promises to overcome this limitation, because
with this approach many different mutations can be examined at once. For example, the genome
sequence of the iceman shows that he had a genetic predisposition to coronary heart disease and
atherosclerosis (Keller et al. 2012), and the 4,500-year-old Paleo-Eskimo, whose genome has been
sequenced, displayed disease risk factors similar to those of modern Koreans (Rasmussen et al.
2010). NGS is also having an impact on the study of infectious disease. In theory, any infectious
disease whose causative agent is present in the bones, teeth, or bloodstream (which permeates
bone) should leave a biomolecular trace in a skeleton. In practice, searching for pathogen aDNA
is practical only if there are other indications of disease, so that aDNA extracts are not wasted
on PCRs directed at pathogens that are absent. For this reason, most attention has focused on
tuberculosis and leprosy, which sometimes leave characteristic (though not definitive) skeletal
lesions (Andersen et al. 1994, Roberts & Buikstra 2008), and plague, which does not leave an
osteological signature but for which there are historic records indicating the location of burial sites.
For tuberculosis and leprosy, there is the added advantage that in addition to aDNA, characteristic
mycolic lipids from the cell wall of the causative bacterium are sometimes preserved (Minnikin
et al. 1984, Lee et al. 2012). There has been good work using PCR to detect these diseases in
human remains (Salo et al. 1994; Taylor et al. 1999, 2005, 2007), but this area has attracted criticism
(Willerslev & Cooper 2005, Achtman 2008, Wilbur et al. 2009). Because the target DNA is not
human, and it is unlikely that any excavator, curator, or aDNA researcher studying the material
suffers from the disease under study, there has been a tendency to pay less attention to the dangers
of contamination, even though this can still occur if specimens are not rigorously protected from
PCR products generated in earlier experiments (Tetzner 2009, Wilbur et al. 2009). The advent of
NGS promises to bring the field to a new, more productive level, because the larger amounts of
sequence data make it possible not only to detect the disease-causing organism, but also to study
its relationships with modern strains of the disease. The genome sequence of a Yersinia pestis strain
from a fourteenth-century AD plague victim (Bos et al. 2011) indicated that the medieval Black
Death was the first exposure of humans to the Y pestis strains known today, suggesting that the
Justinian plague of the eighth century AD was caused either by Y. pestis strains that are now extinct,
or by a different pathogen. NGS has also provided detailed information on the genetic features
of the M. tuberculosis strain carried by an adolescent female buried in the nineteenth-century St
George's Crypt, in Leeds, England (Bouwman et al. 2012). This bacterium belonged to a group
that is uncommon today but which was present in North America in the early twentieth century.

Stable isotope Paleodietary Studies

analysis:
measurement of the
stable isotope ratios in
a material, for
example, to study the
diets of past
populations
The two previous examples of biomolecular archaeology have mainly involved aDNA. Paleodietary
studies, in contrast, make use of a broader range of approaches, including chemical analysis of lipid
and protein residues found in cooking vessels, and stable isotope analysis of preserved lipids and
bone collagen.
Residues are recovered from the matrix of a potsherd, having become absorbed into the wall of
the original vessel during cooking (Evershed et al. 1991, Copley et al. 2001). Absorbed residues are
present in ~80% or more of all archaeological pottery assemblages and have enabled a broad range

1 68 Brown • Brown

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of foodstuffs to be identified from different periods and geographical regions (Evershed 2008).
These include milk lipids, which can be distinguished from the equivalent meat fats because of
differences in their 613C values. Tracing the advent of dairying has previously been difficult,
because only a few types of artifacts, such as ceramic milk strainers, can be specifically associated
with dairying, and evidence from artistic depictions of farming activities is ambiguous (Bogucki
1984). It was thought that dairying in Eurasia did not begin until 2,000^4,000 years after cattle
and other animals were originally domesticated (Levy 1983, Sherratt 1983). Studies of potsherd
residues have now shown that these dates are too recent and that milk was being used as early as
9,000 BP, only 1,000 years after farming began (Evershed et al. 2008, Dunne et al. 2012). Residues
of fresh milk do not become tightly absorbed into ceramic surfaces, so those potsherds that yield
milk signatures are likely to come from pots that were used to process milk into products such as
cheese, or to store those products (Salque et al. 2013). This is interesting because, after weaning,
humans are naturally intolerant of milk. Those modern populations that can drink milk possess
a relatively recent genetic mutation that inactivates the intolerant response (Swallow 2003), but
the first farmers probably lacked this mutation and could not have used unprocessed milk without
experiencing digestive problems (Itan et al. 2009). The earliest dairy farmers therefore not only
recognized the nutritive value of milk; they also understood how to convert milk into what would
have been, for them, more palatable products.
Carbon and nitrogen isotopes in bone collagen can also provide valuable indications of diet
(Lee-Thorp 2008), including discrimination between terrestrial, marine, and freshwater inputs
(Schoeninger & DeNiro 1984, Dufour et al. 1999), and identification of the use of maize as
opposed to other domesticated plants (Vogel & van der Merwe 1977). The isotopie fractionation
that occurs at each step in a food chain (Bocherens & Drucker 2003) is particularly informative,
as this enables assessment of the amount of meat in a diet. Skeletal isotope measurements suggest
that Neanderthals occupied a similar trophic level to wolves and hyenas, their diets being rich in
meat with relatively little plant material, and also that the animals that they ate were particular
types of herbivores with relatively high Õ15N values (Richards et al. 2000, Richards & Trinkaus
2009). The latter include mammoths, traditionally perceived as important prey for Neanderthals,
and bears. Early modern human skeletons have even higher Ó15N values, possibly indicating a
broadened diet that included freshwater fish or birds (Richards et al. 2001, Richards & Trinkaus
2009). This is because fish and birds feeding on freshwater vegetation and algae have slightly
higher Õ15N values than animals that feed on terrestrial vegetation, because of the different ways
that nitrogen is cycled in the two environments.
The shift in isotope ratios along a food chain has one further application in biomolecular
archaeology. When infants drink their mothers' milk, they are, in effect, consuming a part of the
parents. An infant is therefore a trophic level further along the food chain than the mother, and
the infant's 613C and 615N values show an equivalent shift, which disappears once the infant is
weaned. The skeletons of infants who died while still being weaned can therefore be identified by
613C and Ó15N measurements, providing information on the length of the nursing period in past
societies (Schurr 1998, Wright & Schwarcz 1998, Pearson et al. 2010).

CONCLUSIONS

Biomolecular archaeology has gradually developed into a mature discipline that is mak
nificant contribution to different aspects of our understanding of the human pas
development of analytical methods for all three types of biomolecule is resulting in s
in the nature of the investigations that are possible. Genome sequences are clarifying t
ships between human populations and are enabling the study of disease evolution. Sta

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 in animal lipids have shed light on previously intractable questions about the origins of dairying.
Paleoproteomics promises to stimulate equally novel advances in the information obtained from
ancient proteins. Perhaps of greatest importance, however, is that many of the young researchers
currently making their way in the field are experts in both biomolecular science and archaeol-
ogy. The fusion of these initially disparate disciplines is certain to continue to drive progress in
biomolecular archaeology.

DISCLOSURE STATEMENT

The authors are not aware of any affiliations, memberships, funding, or financial h
might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS

We thank the numerous students and researchers who have worked with us over the ye
acknowledge the invaluable support of funding bodies, especially the UK Natural Envi
Research Council.

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