Praise for Biomolecular Archaeology

“This book is a perfect introduction into biomolecular archaeology not only for students interested in the field but also for experienced archaeologists, palaeontologists and archaeobiologists who engage in interdisciplinary research involving the analysis of biomolecules. It is written by one of the most prominent genomic textbook authors, Terry Brown, a pioneer in ancient DNA research and the origins of plant domestication. In this book, his qualities as both an excellent textbook writer as well as a brilliant molecular biologist merge to explain even the most advanced sequencing methods used in palaeogenomics in a way that is understandable for non-experts. The contribution of Keri Brown ensures that the book is relevant to researchers working in the field. Biomolecular Archaeology makes for an ideal manual for archaeologists and students eager to exploit the newest scientific developments to answer typical archaeological questions and better interpret the information buried in the archaeological sites they are working on.”

Eva-Maria Geigl, Université Paris Diderot

“The study of ancient and extant biomolecules has revolutionized archaeological methodologies. This textbook is an excellent, user-friendly introduction to biomolecular techniques and applications for beginning students in archaeology and physical anthropology.”

Linda Stone, Professor Emeritus of Anthropology, Washington State University

“This is a timely and welcome contribution to the rapidly developing field of biomolecular archaeology, covering the basic science as well as an introduction to the applications. It will become essential reading.”

A.M. Pollard, University of Oxford

“There are fewer and fewer areas of archaeology which are immune to biomolecular analysis. Technological innovation combined with a greater understanding of molecular survival has increased reliability of analyses and interpretation, making biomolecular research amongst the fastest moving and most exciting areas in modern archaeology. This book, helped by its easy and accessible style, leads the reader in a logical progression from the molecules themselves to their application in the study of demography, diet, innovation and migration; it should be recommended reading for all new students of archaeology.”

Matthew Collins, University of York

List of Figures

1.1 The four types of ancient biomolecule studied in biomolecular archaeology

2.1 Nucleotide structure

2.2 A short DNA polynucleotide

2.3 The double helix structure for DNA

2.4 Structures of the adenine–thymine and guanine–cytosine base pairs

2.5 Two identical copies of a DNA double helix can be made by separating the two strands

2.6 The human globin genes

2.7 The structure of the human β-globin gene

2.8 Transcription of RNA on a DNA template

2.9 Using an STR to infer kinship between a set of skeletons

2.10 The human mitochondrial genome

2.11 Purification of DNA by silica binding in the presence of guanidinium thiocyanate (GuSCN)

2.12 The first three cycles of a PCR

2.13 Agarose gel electrophoresis

2.14 A mismatched hybrid

2.15 Chain termination DNA sequencing

2.16 DNA cloning

2.17 Multiple alignment of the sequences of 10 clones from a PCR directed at part of the human mitochondrial genome

2.18 One approach to next generation sequencing

2.19 Pyrosequencing

2.20 Various methods for studying the relationships between DNA sequences or genotypes

3.1 The general structure of an amino acid

3.2 The R groups of the 20 amino acids found in proteins

3.3 The structure of a tripeptide

3.4 Schematic of the tertiary structure of a simple protein

3.5 The genetic code

3.6 The role of tRNA as an adaptor molecule in protein synthesis

3.7 O- and N-linked glycosylation

3.8 One version of the Ouchterlony technique

3.9 Crossover immunoelectrophoresis

3.10 Direct and indirect ELISA

3.11 Two-dimensional gel electrophoresis

4.1 The general structure of a carboxylic acid (A), and the structures of two fatty acids (B)

4.2 The general structure of a triglyceride

4.3 Soaps

4.4 Reaction of palmitic acid with triacontanol to form triacontanylpalmitate, the major component of beeswax

4.5 Glycerophospholipids

4.6 The bilayer structure formed by glycerophospholipids

4.7 The general structure of a sphingolipid

4.8 Isoprene and terpenoids

4.9 Important resin terpenoids

4.10 Cholesterol

4.11 BSTFA

4.12 An illustration of a gas chromatogram

4.13 Configurations of the magnetic sector and quadrupole mass analyzers

5.1 The flat structure of glyceraldehyde

5.2 (A) The arrangement of atoms around a central carbon. (B) The two enantiomers of glyceraldehyde

5.3 The D-enantiomers of the aldotetroses and aldopentoses

5.4 Three naturally occurring aldohexoses

5.5 Fructose

5.6 Conversion of the linear form of ribose into its ring structure

5.7 The anomers of D-glucose

5.8 Maltose

5.9 The structure of the branch point in a starch molecule

5.10 The amorphous and crystalline layers within a starch grain

5.11 Typical starch grains

6.1 The differences between the carbon isotope fractionations occurring during photosynthesis in C3 and C4 plants

6.2 A δ13C/δ15N plot showing the areas of the graph in which bone collagen values should fall for different types of diet

6.3 A δ13C/δ15N plot showing the isotope shifts in bone collagen values that occur along a food chain

6.4 Isotope ratio mass spectrometry

7.1 The typical appearance of well-preserved lamellar bone

7.2 The typical appearance of badly degraded lamellar bone, histology index “0”

7.3 Mercury intrusion porosimetry

7.4 Typical appearance of cremated archaeological bone

7.5 An Egyptian mummy

7.6 Graph showing a sudden change in δ13C values along a hair shaft

7.7 Desiccated plant remains

7.8 Partial gas chromatogram

7.9 A corn dolly from the Museum of English Rural Life, Reading

7.10 Charred grains of spelt wheat from Assiros, Greece

8.1 Illustration of the short lengths of ancient DNA molecules in even the best preserved archaeological specimens

8.2 Water-induced cleavage of a β-N-glycosidic bond

8.3 A double-stranded molecule that contains nicks

8.4 Outline of the key steps of the single primer extension (SPEX) method

8.5 One of the ways in which hybrid PCR products can be synthesized by template switching

8.6 Cytosine deamination and generation of a C→T sequence error

8.7 Multiple alignment of the sequences of 10 clones of a PCR product

8.8 Deamination of adenine to hypoxanthine, and of guanine to xanthine

8.9 The structures of proline and hydroxyproline

8.10 Model for collagen degradation

8.11 The two enantiomers of alanine

8.12 Conversion of a monosaturated fatty acid to a more stable dihydroxy fatty acid

8.13 Cholesterol breakdown products

8.14 Bile acids

9.1 Biomolecular archaeologist wearing the appropriate protective clothing for working with ancient DNA

9.2 The principle behind the use of uracil-N-glycosylase to prevent amplicon cross-contamination

10.1 Views of the female and male innominate bones

10.2 Female and male skulls

10.3 The human X and Y chromosomes

10.4 Agarose gel showing the results of PCR with amelogenin primers and female (left lane) and male DNA (center lane)

10.5 Digestion of the ZFX and ZFY amplicons with HaeIII gives fragments of diagnostic sizes

10.6 Drawing of a tall Anglo-Saxon skeleton from Blacknall Field of uncertain sex

11.1 Map of Grave Circle B at Mycenae, Greece, and facial reconstructions of seven of the skulls recovered from this site

11.2 STR typing

11.3 Stuttering

11.4 Sequences of TH01 alleles 9, 10, and the microvariant 9.3

11.5 The relationship between haplotypes and a haplogroup

11.6 Maternal inheritance of mitochondrial DNA haplogroups

11.7 Relationships of living individuals with the Tsar and Tsarina

11.8 Family tree of the Earls of Königsfeld

11.9 Map of the graves in the pioneer cemetery in Durham, Upper Ontario

11.10 DNA results at the pioneer cemetery

11.11 Family grave 99 at Eulau, Germany

12.1 Differences in the dental microwear patterns for grazing (upper panel) and browsing (lower panel) sheep from Gotland, Sweden

12.2 The distinction between the synthesis of C16:0 and C18:0 fatty acids in the adipose and mammary tissues of a ruminant

12.3 Stable isotope measurements from collagen for Neanderthals and early modern humans, compared with those for carnivores and herbivores

12.4 Stable isotope data obtained from the Cahokia skeletons

12.5 Identification of fatty acids from milk in potsherd extracts

12.6 Seal myoglobin peptides identified in an Inuit potsherd

13.1 Locations of four of the primary centers for the origins of agriculture

13.2 Phylogenetic tree of domesticated rice accessions constructed from STR genotype data

13.3 The distinction between linear descent and cross-hybridization in evolution of a domesticated population

13.4 Identifying the wild origins of indica and japonica rice

13.5 Relationships between the haplogroups of domestic cattle and wild aurochsen

13.6 Trajectories of the spread of agriculture through Europe

13.7 The basis to (A) coalescence analysis, and (B) founder analysis

13.8 Graph showing the results of founder analysis of the 11 main European mitochondrial haplogroups

13.9 Sudden shift in δ13C values for collagen from British human skeletons after 5200 BP

13.10 STR genotypes of indigenous landraces and archaeological maize from South America

13.11 Alleles of tb1, pbf, and su1 found in maize cobs from Ocampo Cave, Mexico, and Tularosa Cave, New Mexico

14.1 Temperature stabilities of various derivatives of pimaric and abietic acid formed during the heating of pine resin

14.2 Coprostanol and epicoprostanol

15.1 Portion of the spine showing Pott’s disease

15.2 Locations of some of the many mutations in the human β-globin gene that result in thalassemia

15.3 Evolutionary relationships between members of the M. tuberculosis complex as revealed by typing variable loci in 100 isolates

15.4 Mycolic acids

15.5 Reverse phase HPLC separation of mycolic acids

15.6 Reverse transcriptase PCR

16.1 Timelines for some of the important pre-Homo hominins

16.2 Timeline for members of the genus Homo

16.3 The multiregional and Out of Africa hypotheses for the origins of modern humans

16.4 Using the date of the human–orangutan split to calibrate the molecular clock

16.5 Synonymous and non-synonymous substitutions

16.6 A restriction fragment length polymorphism

16.7 Tree depicting the evolutionary relationships between the mitochondrial DNAs of 147 humans from various parts of the world

16.8 Evolutionary tree showing relationships between modern humans, Neanderthals, and the Denisova hominin

16.9 Network of the most frequent human mitochondrial haplogroups, showing their geographical associations

16.10 One possibility for the route taken by the first migration of modern humans out of Africa

List of Tables

2.1 The nucleotides found in DNA molecules

2.2 The nuclear genomes of various organisms relevant to biomolecular archaeology

2.3 Some of the various functions of proteins

3.1 The 20 amino acids found in proteins

4.1 Fatty acids

4.2 Triglycerides

5.1 Examples of monosaccharides

5.2 Examples of disaccharides

6.1 Naturally occurring isotopes of elements relevant to biomolecular archaeology

7.1 The Oxford Histological Index of archaeological bone decay

7.2 Examples of natural mummification

7.3 Examples of artificial mummification

9.1 Stages in the history of an archaeological specimen when contamination with non-endogenous DNA could occur

9.2 The original “criteria of authenticity” for ancient DNA research

10.1 Targets for DNA-based sex identification

10.2 Examples of sex reversal syndromes and similar chromosomal anomalies in humans

11.1 Important post-marital residence patterns

11.2 The CODIS set of STRs

11.3 STR genotypes obtained from the skeletons thought to include the Romanovs

11.4 Results obtained with skeletons from the Canadian pioneer cemetery

12.1 Results of a “blind” test for detection of camel milk absorbed into a potsherd

13.1 Distinctive phenotypes of domesticated plants and animals

13.2 FST values for rice populations

14.1 Presence of neutral derivatives of pimaric and abietic acid at different temperatures during the heating of pine resin

15.1 Main classes of infectious disease

15.2 Some of the commonest monogenic inherited diseases

16.1 Pre-Clovis sites in the Americas


This book originated in an MSc course in Biomolecular Archaeology that was taught jointly by the University of Manchester and University of Sheffield for 10 years up to 2006. Our experience as teachers was that there are many students, from both biology and archaeology backgrounds, who are interested in biomolecular archaeology, and that the great challenge is bringing together the two sides of the subject so that the student becomes an expert in both. The objective of this book is therefore to teach the fundamentals of biomolecular archaeology within a context that emphasizes both the biomolecules and the archaeology.

Deciding to write the book was the easy part. Much more difficult was the actual writing. It became clear early on that we could not cover every biomolecular archaeology project that has ever been published, nor even all the important ones. Those that we describe are chosen because they illustrate key themes and important scientific approaches. We decided not to name the researchers responsible for individual pieces of work in the text, as that would be abnormal in a textbook, but instead to cite the papers describing these projects in the “Further Reading” sections. We were also conscious that as most of our own work has been with DNA we might give this part of biomolecular archaeology a greater emphasis than it deserves. We have tried hard to avoid a DNA bias, and believe that in the book as a whole the different types of biomolecule are given degrees of coverage consistent with their relative importance in biomolecular archaeology. One message that we hope comes across is that the most informative projects are those that use a range of different biomolecular techniques to address the question being asked.

A number of people have helped us in various ways, such as providing figures or permissions to use published or unpublished work, and in suggesting improvements to drafts. We would therefore like to thank Abigail Bouwman, Angela Thomas, Bettina Stoll-Tucker, Charlotte Roberts, Chris Dudar, Claudia Grimaldo, David Beresford-Jones, Diane Lister, Eva-Maria Giegl, Glynis Jones, Ingrid Mainland, John Prag, Julie Wilson, Martin Richards, Matthew Collins, Mike Richards, Mike Taylor, Peter Rowley-Conwy, Richard Allen, Richard Evershed, Robert Hedges, Robert Tykot, Rosalie David, Sandra Bunning, Susan McCouch, and Wolfgang Haak. We would also like to thank all of our research students, postdocs, and technicians, past and present, for making the last few years so interesting.

Keri Brown
Terry Brown

Brief Contents



What is Biomolecular Archaeology?

A curiosity about our past is one of the things that makes us human. Over the last century archaeology has developed into a sophisticated discipline that interprets the past through examination of the physical remains of human life, those remains often but not exclusively recovered by excavation of archaeological sites. Science has always played an important role in archaeology, increasingly so since the 1950s when techniques invented by nuclear physicists for measuring the decay of radioactive atoms were first used by scientific archaeologists to date artifacts. Biological methods have become equally important. Knowledge of human anatomy and pathology enables osteoarchaeologists to use skeletal features to identify the sex of a person, to work out an approximate age at the time of death, and to determine if the person had been suffering from diseases such as tuberculosis or anemia. Archaeobotanists are similarly adept at studying seeds and other plant remains, and from these identifying the types of plants that were grown and consumed by people in the past. By combining information from different kinds of analysis, we can address broader issues such as the development of agriculture in particular parts of the world, and how agriculture and the concomitant changes such as increases in population density affected human diet and health.

Since 1985 the way in which biological remains have been studied by scientific archaeologists has undergone a remarkable revolution. Osteology, archaeobotany, and other approaches that involve examination of the physical structure of remains are still vitally important, but they have been supplemented with techniques in which the biomolecular content of the artifact is analyzed. This is called biomolecular archaeology and the first thing we must do is understand what this term means.

1.1 The Scope of Biomolecular Archaeology

The biomolecules studied by biomolecular archaeologists are the large organic compounds found in living organisms and sometimes present, usually in a partly degraded state, in the remains of those organisms after their death ().

The four types of ancient biomolecule studied in biomolecular archaeology, with the main types of archaeological material from which each one can be obtained.


There are four categories of these macromolecules:

In studying these four types of macromolecule, biomolecular archaeologists use a variety of methods and analytical techniques, as will be described in –. Most of these techniques are applicable to just a single type of macromolecule, but one method has greater breadth and is of such general importance that it is often looked on as a distinct area of biomolecular archaeology. This is stable isotope analysis, in which ratios of different isotopes of certain elements (primarily carbon and nitrogen) are analyzed in proteins and lipids (). The natural ratios of these elements are constant, but variations can be introduced by biological and environmental processes. These variations can be exploited in studies of diet, as the stable isotope ratios present in bone proteins, or in hair, reflect the types of organism consumed by that individual (Section 12.2). A diet rich in marine resources can be distinguished from, for example, a diet largely made up of terrestrial animal protein, and the presence of maize in the diet can also be detected because this plant has a different isotope ratio to many other cereals and vegetables. In a particularly clever application of the technique, stable isotope analysis has been used to identify lipids derived from dairy products (Section 12.3).

1.2 Ancient and Modern Biomolecules

It will already be clear that research in biomolecular archaeology largely involves the analysis of the compounds that are preserved in archaeological remains. We call these ancient biomolecules and archaeologists are not the only scientists interested in their study. Forensic scientists are increasingly using information from preserved biomolecules, especially ancient DNA, in samples such as hair, bloodstains, and other bodily fluids collected at crime scenes years or decades ago in order to solve what are popularly called “cold cases.” Zoologists also use ancient DNA from animal fossils to study extinct species such as mammoths and moas, and to follow changes in genetic diversity over time in populations of animals such as bison, whose numbers have been affected by climate change and human predation. DNA is rarely preserved for more than a few tens of thousands of years, but other biomolecules are present in much older materials. Proteins have been extracted from dinosaur bones and lipids and carbohydrates from the remains of plants and insects in sediments that are tens of millions of years in age.

The breadth of ancient biomolecules research is important because it means that biomolecular archaeologists have scientific colleagues who have very different interests but who use the same techniques and face the same challenges in planning and interpreting their experiments. Over the years there has been a large amount of cross-fertilization of ideas between researchers working with ancient biomolecules in different disciplines, and this has contributed greatly to the development of biomolecular archaeology. Indeed many biomolecular archaeologists also study ancient biomolecules in non-archaeological material, hence making direct contributions to forensic science, zoology, or paleontology, and the boundaries between the these disciplines and biomolecular archaeology often become blurred.

Although most research in biomolecular archaeology involves the study of ancient biomolecules, this is not exclusively the case. Studies of one biomolecule – DNA – in living organisms can contribute greatly to our understanding of certain archaeological issues. This is because DNA contains a record of the ancestry of individuals and the past evolution of populations and species. We can therefore study the relationships between different human populations by typing DNA taken from living representatives of those populations and using techniques from molecular phylogenetics and population genetics to analyze the data. This approach, sometimes called archaeogenetics, has been particularly informative in understanding the timing and trajectories followed by the migrations of modern humans out of Africa into Asia, Europe, Australasia, and the New World (). Using similar approaches with DNA from living crop plants and domestic animals, information is being obtained on the origins and spread of agriculture (). These studies overlap with evolutionary biology and crop and animal genetics, broadening still further the range of researchers who can be looked on as the scientific partners of biomolecular archaeologists.

1.3 The Challenges of Biomolecular Archaeology

All scientists work at the frontiers of their discipline – that is one of the characteristics of research – and all disciplines provide challenges that must be met and overcome if research is to progress. Biomolecular archaeology is no different, the challenges coming in two guises: technical and intellectual.

The technical challenges are posed by the degradation of ancient biomolecules and by contamination of specimens with modern biomolecules. All biomolecules begin to decay when the organism that contains them dies. Some, especially the nucleic acids, are relatively unstable and may completely degrade within a few years. Others, such as carbohydrates, are more stable and their decay products might still be detectable tens of millions of years after death (). These are not precise comparisons, because the environmental conditions, in particular the temperature and water content, greatly affect the rate at which a biomolecule decays, but the outcome is always the same. Almost every biomolecular archaeology project requires analysis of very small quantities of biomolecules that have undergone a greater or lesser degree of chemical degradation. The small quantities of ancient biomolecules present in archaeological specimens mean that detection techniques have to be pushed to their very limits, and often this affects the amount of information that can be obtained by biomolecular analysis of a specimen. Frequently, results are frustratingly incomplete, sometimes tempting the unwary researcher to make speculations that are not entirely warranted by their data, a problem that seems particularly prevalent in some areas of ancient DNA research.

The changes in chemical structure that occur during diagenesis can also confuse the detection processes, so that precise identification of an ancient biomolecule becomes difficult. For example, a process specific for the detection of human hemoglobin in modern bloodstains, when applied to archaeological material, might also give positive results with the partially degraded hemoglobins from other animals. Because of these problems, studies of biomolecular degradation form an essential adjunct to biomolecular archaeology, as it is only by understanding the decay processes for particular biomolecules that misidentifications can be avoided.

The small quantities of ancient biomolecules present in even the best preserved archaeological specimens leads to the second major technical problem, the possibility that modern contaminating molecules swamp the detection process, again leading to erroneous results. This issue is most clearly recognized in ancient DNA studies, because the exquisite sensitivity of the polymerase chain reaction (PCR), the primary detection method for DNA (Section 2.5), enables samples containing just a few hundred ancient DNA molecules to be examined. Similar or greater numbers of modern DNA molecules are present in human sweat, droplets expelled from the mouth and nose by sneezing, and in aerosols derived from previous PCR experiments that adhere to the clothes and skin of laboratory workers. Ancient and modern human DNA are very difficult to tell apart, and it is very easy to mistakenly assign to an archaeological specimen the genetic attributes of one or a mixture of the people who have handled the specimen. The problem is so acute that ancient DNA researchers carry out their experiments in ultraclean laboratories, wearing overalls that cover their entire body and face, a regime more commonly associated with research on deadly virus pathogens. The aim is not, however, to prevent escape of a pathogen from the test tube, but to prevent entry into the test tube of modern DNA from the researcher. Such practices are possible within the confines of a modern laboratory, but less feasible in the field, so it is almost inevitable that human bones become contaminated with DNA from the excavators who first uncover them. Solving these conundrums has greatly exercised not only ancient DNA researchers but all biomolecular archaeologists, as we will see in .

In addition to these technical issues, biomolecular archaeology poses a major intellectual challenge. Biomolecular archaeology is an interdisciplinary subject, and biomolecular research is of no value if it is not carried out within an archaeological context. This may seem obvious, but frequently projects that reach high standards as far as the biomolecular aspect is concerned fail to interest archaeologists because the results are not relevant to the issues that are important in archaeology. The problem arises because, until recently, very few biomolecular scientists possessed anything more than a rudimentary understanding of archaeology, and few archaeologists had a strong training in the biomolecular sciences. Successful biomolecular archaeology therefore requires collaboration between archaeologists and biomolecular scientists, and meaningful collaboration is often difficult to achieve. It is easy to assemble a “team,” but much less easy to reach the mutual intellectual understanding that is required for interdisciplinary research to flourish. It is difficult to become an expert in both biomolecular research and archaeology – both are complex subjects with their own languages and ways of thinking – but such dual expertise has to be the goal of anyone who wishes to become a biomolecular archaeologist. The aim of this book is to help you achieve that goal.

Further Reading

Brothwell, D.R. & Pollard, A.M. (eds.) (2001) Handbook of Archaeological Sciences. Wiley, Chichester. [Covers all areas of archaeological science.]

Cox, M. & Nelson, D.L. (2008) Lehninger Principles of Biochemistry, 4th edn. Palgrave Macmillan, New York. [One of the best student textbooks in biochemistry.]

Jones, M.K. (2003) The Molecule Hunt: Archaeology and the Search for Ancient DNA. Arcade, New York. [A popular account of the early days of biomolecular archaeology.]

Renfrew, C. & Bahn, P. (2008) Archaeology: Theories, Methods and Practice, 5th edn. Thames and Hudson, London. [One of the best student textbooks in archaeology.]